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Sample records for intracellular bacteria encode

  1. Intracellular Bacteria in Protozoa

    Science.gov (United States)

    Görtz, Hans-Dieter; Brigge, Theo

    Intracellular bacteria in humans are typically detrimental, and such infections are regarded by the patients as accidental and abnormal. In protozoa it seems obvious that many bacteria have coevolved with their hosts and are well adapted to the intracellular way of life. Manifold interactions between hosts and intracellular bacteria are found, and examples of antibacterial resistance of unknown mechanisms are observed. The wide diversity of intracellular bacteria in protozoa has become particularly obvious since they have begun to be classified by molecular techniques. Some of the bacteria are closely related to pathogens; others are responsible for the production of toxins.

  2. Functional genomics of intracellular bacteria.

    Science.gov (United States)

    de Barsy, Marie; Greub, Gilbert

    2013-07-01

    During the genomic era, a large amount of whole-genome sequences accumulated, which identified many hypothetical proteins of unknown function. Rapidly, functional genomics, which is the research domain that assign a function to a given gene product, has thus been developed. Functional genomics of intracellular pathogenic bacteria exhibit specific peculiarities due to the fastidious growth of most of these intracellular micro-organisms, due to the close interaction with the host cell, due to the risk of contamination of experiments with host cell proteins and, for some strict intracellular bacteria such as Chlamydia, due to the absence of simple genetic system to manipulate the bacterial genome. To identify virulence factors of intracellular pathogenic bacteria, functional genomics often rely on bioinformatic analyses compared with model organisms such as Escherichia coli and Bacillus subtilis. The use of heterologous expression is another common approach. Given the intracellular lifestyle and the many effectors that are used by the intracellular bacteria to corrupt host cell functions, functional genomics is also often targeting the identification of new effectors such as those of the T4SS of Brucella and Legionella.

  3. Real-time determination of intracellular oxygen in bacteria using a genetically encoded FRET-based biosensor

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    Potzkei Janko

    2012-03-01

    Full Text Available Abstract Background Molecular oxygen (O2 is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues. Results We developed a genetically encoded Förster resonance energy transfer (FRET-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen consists of the yellow fluorescent protein (YFP that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP. Since O2 is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O2 levels in the cytoplasm of Escherichia coli cells during batch cultivation. Conclusions FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (pathophysiological processes in living cells.

  4. Intracellular cytoskeletal elements and cytoskeletons in bacteria.

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    Madkour, Mohamed H F; Mayer, Frank

    2007-01-01

    Within a short period of time after the discovery of bacterial cytoskletons, major progress had been made in areas such as general spatial layout of cytoskeletons, their involvement in a variety of cellfunctions (shape control, cell division, chromosome segregation, cell motility). This progress was achieved by application of advanced investigation techniques. Homologs of eukaryotic actin, tubulin, and intermediate filaments were found in bacteria; cytoskeletal proteins not closely or not at all related to any of these major cytoskeletal proteins were discovered in a number of bacteria such as Mycoplasmas, Spiroplasmas, Spirochetes, Treponema, Caulobacter. A structural role for bacterial elongation factor Tu was indicated. On the basis of this new thinking, new approaches in biotechnology and new drugs are on the way.

  5. Intracellular chemical gradients: morphing principle in bacteria

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    Endres Robert G

    2012-09-01

    Full Text Available Abstract Advances in computational biology allow systematic investigations to ascertain whether internal chemical gradients can be maintained in bacteria – an open question at the resolution limit of fluorescence microscopy. While it was previously believed that the small bacterial cell size and fast diffusion in the cytoplasm effectively remove any such gradient, a new computational study published in BMC Biophysics supports the emerging view that gradients can exist. The study arose from the recent observation that phosphorylated CtrA forms a gradient prior to cell division in Caulobacter crescentus, a bacterium known for its complicated cell cycle. Tropini et al. (2012 postulate that such gradients can provide an internal chemical compass, directing protein localization, cell division and cell development. More specifically, they describe biochemical and physical constraints on the formation of such gradients and explore a number of existing bacterial cell morphologies. These chemical gradients may limit in vitro analyses, and may ensure timing control and robustness to fluctuations during critical stages in cell development.

  6. Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

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    Dhritiman Samanta

    2017-05-01

    Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

  7. Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural characterization.

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    Schröder, D; Deppisch, H; Obermayer, M; Krohne, G; Stackebrandt, E; Hôlldobler, B; Goebel, W; Gross, R

    1996-08-01

    Intracellular endosymbiotic bacteria inherent to ants of the genus Camponotus were characterized. The bacteria were localized in bacteriocytes, which are specialized cells of both workers and queen ants; these cells are intercalated between epithelial cells of the midgut. The bacteriocytes show a different morphology from the normal epithelial cells and carry a large number of the rod-shaped Gram-negative bacteria free in the cytoplasm. The bacteria were never observed in the neighbouring epithelial cells, but they were found intracellularly in oocytes, strongly indicating a maternal transmission of the bacteria. The 16S DNA encoding rrs loci of the endosymbionts of four species of the genus Camponotus derived either from Germany (C. herculeanus and C. ligniperdus), North America (C. floridanus) or South America (C. rufipes) were cloned after polymerase chain reaction (PCR) amplification using oligonucleotides complementary to all so far known eubacterial rrs sequences. The DNA sequences of the rrs loci of the four endosymbionts were determined, and, using various genus- and species-specific oligonucleotides derived from variable regions in the rrs sequences, the identity of the bacteria present in the bacteriocytes and the ovarian cells was confirmed by PCR and in situ hybridization techniques. Comparison of the 16S DNA sequences with the available database showed the endosymbiotic bacteria to be members of the gamma-subclass of Proteobacteria. They formed a distinct taxonomic group, a sister taxon of the taxons defined by the tsetse fly and aphid endosymbionts. Within the gamma-subclass, the cluster of the ant, tsetse fly and aphid endosymbionts are placed adjacent to the family of Enterobacteriaceae. The evolutionary tree of the ant endosymbionts reflects the systematic classification and geographical distribution of their host insects, indicating an early co-evolution of the symbiotic partners and a vertical transmission of the bacteria.

  8. Patho-epigenetics of Infectious Diseases Caused by Intracellular Bacteria.

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    Niller, Hans Helmut; Minarovits, Janos

    2016-01-01

    In multicellular eukaryotes including plants, animals and humans, epigenetic reprogramming may play a role in the pathogenesis of a wide variety of diseases. Recent studies revealed that in addition to viruses, pathogenic bacteria are also capable to dysregulate the epigenetic machinery of their target cells. In this chapter we focus on epigenetic alterations induced by bacteria infecting humans. Most of them are obligate or facultative intracellular bacteria that produce either bacterial toxins and surface proteins targeting the host cell membrane, or synthesise effector proteins entering the host cell nucleus. These bacterial products typically elicit histone modifications, i.e. alter the "histone code". Bacterial pathogens are capable to induce alterations of host cell DNA methylation patterns, too. Such changes in the host cell epigenotype and gene expression pattern may hinder the antibacterial immune response and create favourable conditions for bacterial colonization, growth, or spread. Epigenetic dysregulation mediated by bacterial products may also facilitate the production of inflammatory cytokines and other inflammatory mediators affecting the epigenotype of their target cells. Such indirect epigenetic changes as well as direct interference with the epigenetic machinery of the host cells may contribute to the initiation and progression of malignant tumors associated with distinct bacterial infections.

  9. A bioinformatic approach to understanding antibiotic resistance in intracellular bacteria through whole genome analysis

    OpenAIRE

    Biswas, S.(National Institute of Science Education and Research, Bhubaneswar, India); Raoult, Didier; Rolain, J. M.

    2008-01-01

    Intracellular bacteria survive within eukaryotic host cells and are difficult to kill with certain antibiotics. As a result, antibiotic resistance in intracellular bacteria is becoming commonplace in healthcare institutions. Owing to the lack of methods available for transforming these bacteria, we evaluated the mechanisms of resistance using molecular methods and in silico genome analysis. The objective of this review was to understand the molecular mechanisms of antibiotic resistance throug...

  10. Genetically encoded optical sensors for monitoring of intracellular chloride and chloride-selective channel activity

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    Piotr Bregestovski

    2009-12-01

    Full Text Available This review briefly discusses the main approaches for monitoring chloride (Cl−, the most abundant physiological anion. Noninvasive monitoring of intracellular Cl− ([Cl−]i is a challenging task owing to two main difficulties: (i the low transmembrane ratio for Cl−, approximately 10:1; and (ii the small driving force for Cl−, as the Cl− reversal potential (ECl is usually close to the resting potential of the cells. Thus, for reliable monitoring of intracellular Cl−, one has to use highly sensitive probes. From several methods for intracellular Cl− analysis, genetically encoded chloride indicators represent the most promising tools. Recent achievements in the development of genetically encoded chloride probes are based on the fact that yellow fluorescent protein (YFP exhibits Cl−-sensitivity. YFP-based probes have been successfully used for quantitative analysis of Cl− transport in different cells and for high-throughput screening of modulators of Cl−-selective channels. Development of a ratiometric genetically encoded probe, Clomeleon, has provided a tool for noninvasive estimation of intracellular Cl− concentrations. While the sensitivity of this protein to Cl− is low (EC50 about 160 mM, it has been successfully used for monitoring intracellular Cl− in different cell types. Recently a CFP–YFP-based probe with a relatively high sensitivity to Cl− (EC50 about 30 mM has been developed. This construct, termed Cl-Sensor, allows ratiometric monitoring using the fluorescence excitation ratio. Of particular interest are genetically encoded probes for monitoring of ion channel distribution and activity. A new molecular probe has been constructed by introducing into the cytoplasmic domain of the Cl−-selective glycine receptor (GlyR channel the CFP–YFP-based Cl-Sensor. This construct, termed BioSensor-GlyR, has been successfully expressed in cell lines. The new genetically encoded chloride probes offer means of screening

  11. The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes

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    Ewa Jończyk-Matysiak

    2015-01-01

    Full Text Available Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired. Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients’ peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients’ phagocytes’ ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy.

  12. The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes.

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    Jończyk-Matysiak, Ewa; Łusiak-Szelachowska, Marzanna; Kłak, Marlena; Bubak, Barbara; Międzybrodzki, Ryszard; Weber-Dąbrowska, Beata; Żaczek, Maciej; Fortuna, Wojciech; Rogóż, Paweł; Letkiewicz, Sławomir; Szufnarowski, Krzysztof; Górski, Andrzej

    2015-01-01

    Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired). Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients' peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients' phagocytes' ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy.

  13. The Effect of Bacteriophage Preparations on Intracellular Killing of Bacteria by Phagocytes

    Science.gov (United States)

    Jończyk-Matysiak, Ewa; Łusiak-Szelachowska, Marzanna; Kłak, Marlena; Bubak, Barbara; Międzybrodzki, Ryszard; Weber-Dąbrowska, Beata; Żaczek, Maciej; Fortuna, Wojciech; Rogóż, Paweł; Letkiewicz, Sławomir; Szufnarowski, Krzysztof; Górski, Andrzej

    2015-01-01

    Intracellular killing of bacteria is one of the fundamental mechanisms against invading pathogens. Impaired intracellular killing of bacteria by phagocytes may be the reason of chronic infections and may be caused by antibiotics or substances that can be produced by some bacteria. Therefore, it was of great practical importance to examine whether phage preparations may influence the process of phagocyte intracellular killing of bacteria. It may be important especially in the case of patients qualified for experimental phage therapy (approximately half of the patients with chronic bacterial infections have their immunity impaired). Our analysis included 51 patients with chronic Gram-negative and Gram-positive bacterial infections treated with phage preparations at the Phage Therapy Unit in Wroclaw. The aim of the study was to investigate the effect of experimental phage therapy on intracellular killing of bacteria by patients' peripheral blood monocytes and polymorphonuclear neutrophils. We observed that phage therapy does not reduce patients' phagocytes' ability to kill bacteria, and it does not affect the activity of phagocytes in patients with initially reduced ability to kill bacteria intracellularly. Our results suggest that experimental phage therapy has no significant adverse effects on the bactericidal properties of phagocytes, which confirms the safety of the therapy. PMID:26783541

  14. Dual-color encoded DNAzyme nanostructures for multiplexed detection of intracellular metal ions in living cells.

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    Zhou, Wenjiao; Liang, Wenbing; Li, Daxiu; Yuan, Ruo; Xiang, Yun

    2016-11-15

    The detection of intracellular metal ions is of great importance in understanding metal homeostasis in cells and related diseases, and yet it remains a significant challenge to achieve this goal. Based on a new self-assembled and dual-color encoded DNAzyme nanostructure, we describe here an approach for multiplexed sensing of UO2(2+) and Pb(2+) in living cells. The fluorescently quenched nanoprobes can be prepared by simple thermal annealing of four ssDNAs containing the metal ion-dependent enzymatic and substrate sequences. The self-assembly formation of the nanostructures are verified by native polyacrylamide gel electrophoresis. The target metal ions can cleave the substrate sequences in the DNAzyme nanostructures to recover fluorescent emissions at different wavelengths for sensitive and selective in vitro multiplexed detection of UO2(2+) and Pb(2+) with the detection limits of 0.6nM and 3.9nM, respectively. Importantly, we demonstrate that these nanoprobes are stable in cell lysates and can enter cells without the aid of any transfection agents for simultaneous imaging intracellular UO2(2+) and Pb(2+). Moreover, the nanoprobes offer excellent biocompatibility and non-cytotoxicity. With these unique features, the dual-color encoded nanostructures presented here can thus offer new opportunities for multiplexed detection of specific intracellular species. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Targeting intracellular bacteria with an extended cationic amphiphilic polyproline helix.

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    Nepal, Manish; Thangamani, Shankar; Seleem, Mohamed N; Chmielewski, Jean

    2015-06-07

    An extended cationic and amphiphilic polyproline helix (CAPH) is described with a dual mode of action: effective cell penetration of human macrophages, and potent antimicrobial activity in vitro against both Gram-positive and negative pathogens, including Acinetobacter baumannii, Escherichia coli O157 and Bacillus anthracis. This dual action was successfully combined to clear pathogenic bacteria (Brucella and Salmonella) residing within macrophages.

  16. Detection of intracellular bacteria in exfoliated urothelial cells from women with urge incontinence.

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    Cheng, Ying; Chen, Zhuoran; Gawthorne, Jayde A; Mukerjee, Chinmoy; Varettas, Kerry; Mansfield, Kylie J; Schembri, Mark A; Moore, Kate H

    2016-10-01

    The role of subclinical infection in patients with urge incontinence has been largely ignored. The aim of this study was to test for the presence of intracellular bacteria in exfoliated urothelial cells obtained from the urine of patients with detrusor overactivity or mixed incontinence +/- a history of UTI, and compare this to a control group of patients with stress incontinence and no history of infection. Bacterial cystitis was assessed by routine microbiology and compared to microscopic analysis of urine by Wright staining. Subsequent analysis of urothelial cells by confocal microscopy was performed to determine the existence of intracellular bacteria. Bacterial cystitis was seen in 13% of patients based on routine microbiology. Wright staining of concentrated urothelial cells demonstrated the presence of bacteria in 72% of samples. Filamentous bacterial cells were observed in 51% of patients and were significantly more common in patients with detrusor overactivity. Intracellular Escherichia coli were observed by confocal microscopy. This study supports the possibility that a subset of patients with urge incontinence may have unrecognised chronic bacterial colonisation, maintained via an intracellular reservoir. In patients with negative routine microbiology, application of the techniques used in this study revealed evidence of infection, providing further insights into the aetiology of urge incontinence.

  17. Intracellular killing of bacteria: is Dictyostelium a model macrophage or an alien?

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    Cosson, Pierre; Lima, Wanessa C

    2014-01-01

    Predation of bacteria by phagocytic cells was first developed during evolution by environmental amoebae. Many of the core mechanisms used by amoebae to sense, ingest and kill bacteria have also been conserved in specialized phagocytic cells in mammalian organisms. Here we focus on recent results revealing how Dictyostelium discoideum senses and kills non-pathogenic bacteria. In this model, genetic analysis of intracellular killing of bacteria has revealed a surprisingly complex array of specialized mechanisms. These results raise new questions on these processes, and challenge current models based largely on studies in mammalian phagocytes. In addition, recent studies suggest one additional level on complexity by revealing how Dictyostelium recognizes specifically various bacterial species and strains, and adapts its metabolism to process them. It remains to be seen to what extent mechanisms uncovered in Dictyostelium are also used in mammalian phagocytic cells. PMID:24628900

  18. The intracellular citrus huanglongbing bacterium, 'Candidatus Liberibacter asiaticus' encodes two novel autotransporters.

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    Guixia Hao

    Full Text Available Proteins secreted by the type V secretion system (T5SS, known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of 'Candidatus Liberibacter asiaticus' (Las, and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB, a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.

  19. The intracellular citrus huanglongbing bacterium, 'Candidatus Liberibacter asiaticus' encodes two novel autotransporters.

    Science.gov (United States)

    Hao, Guixia; Boyle, Michael; Zhou, Lijuan; Duan, Yongping

    2013-01-01

    Proteins secreted by the type V secretion system (T5SS), known as autotransporters, are large extracellular virulence proteins localized to the bacterial poles. In this study, we characterized two novel autotransporter proteins of 'Candidatus Liberibacter asiaticus' (Las), and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. As a phloem-limited, intracellular bacterial pathogen, Las has a significantly reduced genome and causes huanglongbing (HLB), a devastating disease of citrus worldwide. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of a typical signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria when expressed in the plants. These findings may lead to a better understanding of the pathogenesis of this intracellular bacterium.

  20. Development of growth rate measuring method for intracellular, parasitic acid-fast bacteria using radioisotopes

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    Nakata, Noboru; Fukutomi, Yasuo [National Inst. of Infectious Deseases, Tokyo (Japan)

    1998-02-01

    To prevent and treat infections diseases caused by pathogenic acid-fast bacteria such as Mycobacterium leprae, Tubercle bacillus, it is important to elucidate the mechanisms of intracellular proliferations of these bacteria. This research project was started to make DNA library using a new constructed shuttle vector. Development of in vitro evaluation method for intracellular proliferation of mycobacterium and its transformed cells was attempted on the basis of Buddemeyer method. This method was able to precisely determine the metabolic activities as low as those in leprae and its modified method using {sup 14}C-palmitic acid was highly sensitive and the results were obtainable in a shorter period. The generated CO{sub 2} was satisfactorily absorbed into scintillator without using a filter paper. A new culture medium from which arginine, a NO-producing compound was eliminated was used to repress the sterilizing effects of NO, but the metabolic activities of leprae was not enhanced. (M.N.)

  1. Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites.

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    Coutinho-Silva, Robson; Ojcius, David M

    2012-11-01

    Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1β (IL-1β), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection.

  2. Host factors and genetic susceptibility to infections due to intracellular bacteria and fastidious organisms.

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    Asner, S A; Morré, S A; Bochud, P-Y; Greub, G

    2014-12-01

    While genetic polymorphisms play a paramount role in tuberculosis (TB), less is known about their contribution to the severity of diseases caused by other intracellular bacteria and fastidious microorganisms. We searched electronic databases for observational studies reporting on host factors and genetic predisposition to infections caused by intracellular fastidious bacteria published up to 30 May 2014. The contribution of genetic polymorphisms was documented for TB. This includes genetic defects in the mononuclear phagocyte/T helper cell type 1 (Th1) pathway contributing to disseminated TB disease in children and genome-wide linkage analysis (GWAS) in reactivated pulmonary TB in adults. Similarly, experimental studies supported the role of host genetic factors in the clinical presentation of illnesses resulting from other fastidious intracellular bacteria. These include IL-6 -174G/C or low mannose-binding (MBL) polymorphisms, which are incriminated in chronic pulmonary conditions triggered by C. pneumoniae, type 2-like cytokine secretion polymorphisms, which are correlated with various clinical patterns of M. pneumoniae infections, and genetic variation in the NOD2 gene, which is an indicator of tubal pathology resulting from Chamydia trachomatis infections. Monocyte/macrophage migration and T lymphocyte recruitment defects are corroborated to ineffective granuloma formation observed among patients with chronic Q fever. Similar genetic polymorphisms have also been suggested for infections caused by T. whipplei although not confirmed yet. In conclusion, this review supports the paramount role of genetic factors in clinical presentations and severity of infections caused by intracellular fastidious bacteria. Genetic predisposition should be further explored through such as exome sequencing.

  3. Mutations in DDHD2, Encoding an Intracellular Phospholipase A(1), Cause a Recessive Form of Complex Hereditary Spastic Paraplegia

    NARCIS (Netherlands)

    Schuurs-Hoeijmakers, Janneke H. M.; Geraghty, Michael T.; Kamsteeg, Erik-Jan; Ben-Salem, Salma; de Bot, Susanne T.; Nijhof, Bonnie; van de Vondervoort, Ilse I. G. M.; van der Graaf, Marinette; Nobau, Anna Castells; Otte-Holler, Irene; Vermeer, Sascha; Smith, Amanda C.; Humphreys, Peter; Schwartzentruber, Jeremy; Ali, Bassam R.; Al-Yahyaee, Saeed A.; Tariq, Said; Pramathan, Thachillath; Bayoumi, Riyad; Kremer, Hubertus P. H.; van de Warrenburg, Bart P.; van den Akker, Willem M. R.; Gilissen, Christian; Veltman, Joris A.; Janssen, Irene M.; Vulto-van Silfhout, Anneke T.; van der Velde-Visser, Saskia; Lefeber, Dirk J.; Diekstra, Adinda; Eramus, Corrie E.; Willemsen, Michel A.; Vissers, Lisenka E. L. M.; Lammens, Martin; van Bokhoven, Hans; Brunner, Han G.; Wevers, Ron A.; Schenck, Annette; Al-Gazali, Lihadh; de Vries, Bert B. A.; de Brouwer, Arjan P. M.

    2012-01-01

    We report on four families affected by a clinical presentation of complex hereditary spastic paraplegia (HSP) due to recessive mutations in DDHD2, encoding one of the three mammalian intracellular phospholipases A(1) (iPLA(1)). The core phenotype of this HSP syndrome consists of very early-onset (

  4. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria.

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    Miao, Edward A; Leaf, Irina A; Treuting, Piper M; Mao, Dat P; Dors, Monica; Sarkar, Anasuya; Warren, Sarah E; Wewers, Mark D; Aderem, Alan

    2010-12-01

    Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of interleukin 1β (IL-1β) and IL-18. Although infection with wild-type Salmonella typhimurium is lethal to mice, we show here that a strain that persistently expresses flagellin was cleared by the cytosolic flagellin-detection pathway through the activation of caspase-1 by the NLRC4 inflammasome; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1-induced pyroptotic cell death released bacteria from macrophages and exposed the bacteria to uptake and killing by reactive oxygen species in neutrophils. Similarly, activation of caspase-1 cleared unmanipulated Legionella pneumophila and Burkholderia thailandensis by cytokine-independent mechanisms. This demonstrates that activation of caspase-1 clears intracellular bacteria in vivo independently of IL-1β and IL-18 and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.

  5. Inhibitory activity of the isoflavone biochanin a on intracellular bacteria of genus Chlamydia and initial development of a buccal formulation

    DEFF Research Database (Denmark)

    Hanski, Leena; Genina, Natalja; Uvell, Hanna

    2014-01-01

    Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump...

  6. Inhibitory activity of the isoflavone biochanin a on intracellular bacteria of genus Chlamydia and initial development of a buccal formulation

    DEFF Research Database (Denmark)

    Hanski, Leena; Genina, Natalja; Uvell, Hanna

    2014-01-01

    Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump...

  7. Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

    Directory of Open Access Journals (Sweden)

    Yves Briers

    Full Text Available Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

  8. Sponge-associated bacteria mineralize arsenic and barium on intracellular vesicles

    Science.gov (United States)

    Keren, Ray; Mayzel, Boaz; Lavy, Adi; Polishchuk, Iryna; Levy, Davide; Fakra, Sirine C.; Pokroy, Boaz; Ilan, Micha

    2017-01-01

    Arsenic and barium are ubiquitous environmental toxins that accumulate in higher trophic-level organisms. Whereas metazoans have detoxifying organs to cope with toxic metals, sponges lack organs but harbour a symbiotic microbiome performing various functions. Here we examine the potential roles of microorganisms in arsenic and barium cycles in the sponge Theonella swinhoei, known to accumulate high levels of these metals. We show that a single sponge symbiotic bacterium, Entotheonella sp., constitutes the arsenic- and barium-accumulating entity within the host. These bacteria mineralize both arsenic and barium on intracellular vesicles. Our results indicate that Entotheonella sp. may act as a detoxifying organ for its host. PMID:28233852

  9. Induction of Specific CD8+ T Cells against Intracellular Bacteria by CD8+ T-Cell-Oriented Immunization Approaches

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    Toshi Nagata

    2010-01-01

    Full Text Available For protection against intracellular bacteria such as Mycobacterium tuberculosis and Listeria monocytogenes, the cellular arm of adaptive immunity is necessary. A variety of immunization methods have been evaluated and are reported to induce specific CD8+ T cells against intracellular bacterial infection. Modified BCG vaccines have been examined to enhance CD8+ T-cell responses. Naked DNA vaccination is a promising strategy to induce CD8+ T cells. In addition to this strategy, live attenuated intracellular bacteria such as Shigella, Salmonella, and Listeria have been utilized as carriers of DNA vaccines in animal models. Vaccination with dendritic cells pulsed with antigenic peptides or the cells introduced antigen genes by virus vectors such as retroviruses is also a powerful strategy. Furthermore, vaccination with recombinant lentivirus has been attempted to induce specific CD8+ T cells. Combinations of these strategies (prime-boost immunization have been studied for the efficient induction of intracellular bacteria-specific CD8+ T cells.

  10. Coprinopsis cinerea intracellular lactonases hydrolyze quorum sensing molecules of Gram-negative bacteria.

    Science.gov (United States)

    Stöckli, Martina; Lin, Chia-Wei; Sieber, Ramon; Plaza, David F; Ohm, Robin A; Künzler, Markus

    2017-05-01

    Biofilm formation on fungal hyphae and production of antifungal molecules are strategies of bacteria in their competition with fungi for nutrients. Since these strategies are often coordinated and under control of quorum sensing by the bacteria, interference with this bacterial communication system can be used as a counter-strategy by the fungi in this competition. Hydrolysis of N-acyl-homoserine lactones (HSL), a quorum sensing molecule used by Gram-negative bacteria, by fungal cultures has been demonstrated. However, the enzymes that are responsible for this activity, have not been identified. In this study, we identified and characterized two paralogous HSL hydrolyzing enzymes from the coprophilous fungus Coprinopsis cinerea. The C. cinerea HSL lactonases belong to the metallo-β-lactamase family and show sequence homology to and a similar biochemical activity as the well characterized lactonase AiiA from Bacillus thuringiensis. We show that the fungal lactonases, similar to the bacterial enzymes, are kept intracellularly and act as a sink for the bacterial quorum sensing signals both in C. cinerea and in Saccharomyces cerevisiae expressing C. cinerea lactonases, due to the ability of these signal molecules to diffuse over the fungal cell wall and plasma membrane. The two isogenes coding for the C. cinerea HSL lactonases are arranged in the genome as a tandem repeat and expressed preferentially in vegetative mycelium. The occurrence of orthologous genes in genomes of other basidiomycetes appears to correlate with a saprotrophic lifestyle. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Copper isotope fractionation during surface adsorption and intracellular incorporation by bacteria.

    Science.gov (United States)

    Navarrete, Jesica U; Borrok, David M; Viveros, Marian; Ellzey, Joanne T

    2011-02-01

    Copper isotopes may prove to be a useful tool for investigating bacteria-metal interactions recorded in natural waters, soils, and rocks. However, experimental data which attempt to constrain Cu isotope fractionation in biologic systems are limited and unclear. In this study, we utilized Cu isotopes (δ(65)Cu) to investigate Cu-bacteria interactions, including surface adsorption and intracellular incorporation. Experiments were conducted with individual representative species of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria, as well as with wild-type consortia of microorganisms from several natural environments. Ph-dependent adsorption experiments were conducted with live and dead cells over the pH range 2.5-6. Surface adsorption experiments of Cu onto live bacterial cells resulted in apparent separation factors (Δ(65)Cu(solution-solid) = δ(65)Cu(solution) - δ(65)Cu(solid)) ranging from +0.3‰ to +1.4‰ for B. subtilis and +0.2‰ to +2.6‰ for E. coli. However, because heat-killed bacterial cells did not exhibit this behavior, the preference of the lighter Cu isotope by the cells is probably not related to reversible surface adsorption, but instead is a metabolically-driven phenomenon. Adsorption experiments with heat-killed cells yielded apparent separation factors ranging from +0.3‰ to -0.69‰ which likely reflects fractionation from complexation with organic acid surface functional group sites. For intracellular incorporation experiments the lab strains and natural consortia preferentially incorporated the lighter Cu isotope with an apparent Δ(65)Cu(solution-solid) ranging from ~+1.0‰ to +4.4‰. Our results indicate that live bacterial cells preferentially sequester the lighter Cu isotope regardless of the experimental conditions. The fractionation mechanisms involved are likely related to active cellular transport and regulation, including the reduction of Cu(II) to Cu(I). Because similar intracellular Cu

  12. Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator.

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    Miyamoto, Akitoshi; Bannai, Hiroko; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2013-05-03

    Monitoring the pattern of intracellular Ca(2+) signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca(2+) indicators (GECIs) are used for monitoring intracellular Ca(2+) changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca(2+) signals using GECIs. We here compared the Ca(2+) signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca(2+) responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca(2+) signals.

  13. Coxiella burnetii transcriptional analysis reveals serendipity clusters of regulation in intracellular bacteria.

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    Quentin Leroy

    Full Text Available Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is mainly transmitted to humans through an aerosol route. A spore-like form allows C. burnetii to resist different environmental conditions. Because of this, analysis of the survival strategies used by this bacterium to adapt to new environmental conditions is critical for our understanding of C. burnetii pathogenicity. Here, we report the early transcriptional response of C. burnetii under temperature stresses. Our data show that C. burnetii exhibited minor changes in gene regulation under short exposure to heat or cold shock. While small differences were observed, C. burnetii seemed to respond similarly to cold and heat shock. The expression profiles obtained using microarrays produced in-house were confirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentially expressed in at least one condition, with a fold change of up to 4. Globally, the differentially expressed genes in C. burnetii were associated with bacterial division, (pppGpp synthesis, wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycan synthesis. These findings could be associated with growth arrest and witnessed transformation of the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes were differentially expressed. These clusters do not belong to operons or genetic networks; they have no evident associated functions and are not under the control of the same promoters. We also found undescribed but comparable clusters of regulation in previously reported transcriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeria monocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stresses permits the recognition of unpredicted clusters of regulation for which the trigger mechanism remains unidentified but which may be the result of a new mechanism of epigenetic regulation.

  14. Perforin-2 is essential for intracellular defense of parenchymal cells and phagocytes against pathogenic bacteria.

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    McCormack, Ryan M; de Armas, Lesley R; Shiratsuchi, Motoaki; Fiorentino, Desiree G; Olsson, Melissa L; Lichtenheld, Mathias G; Morales, Alejo; Lyapichev, Kirill; Gonzalez, Louis E; Strbo, Natasa; Sukumar, Neelima; Stojadinovic, Olivera; Plano, Gregory V; Munson, George P; Tomic-Canic, Marjana; Kirsner, Robert S; Russell, David G; Podack, Eckhard R

    2015-09-24

    Perforin-2 (MPEG1) is a pore-forming, antibacterial protein with broad-spectrum activity. Perforin-2 is expressed constitutively in phagocytes and inducibly in parenchymal, tissue-forming cells. In vitro, Perforin-2 prevents the intracellular replication and proliferation of bacterial pathogens in these cells. Perforin-2 knockout mice are unable to control the systemic dissemination of methicillin-resistant Staphylococcus aureus (MRSA) or Salmonella typhimurium and perish shortly after epicutaneous or orogastric infection respectively. In contrast, Perforin-2-sufficient littermates clear the infection. Perforin-2 is a transmembrane protein of cytosolic vesicles -derived from multiple organelles- that translocate to and fuse with bacterium containing vesicles. Subsequently, Perforin-2 polymerizes and forms large clusters of 100 Å pores in the bacterial surface with Perforin-2 cleavage products present in bacteria. Perforin-2 is also required for the bactericidal activity of reactive oxygen and nitrogen species and hydrolytic enzymes. Perforin-2 constitutes a novel and apparently essential bactericidal effector molecule of the innate immune system.

  15. pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH

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    Robertson, J. Brian; Johnson, Carl Hirschie

    2012-01-01

    We report the development of a genetically encodable and ratiometic pH probe named “pHlash” that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor–composed of a donor luciferase that is genetically fused to a Venus fluorophore–exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H+ specific; neither Ca++, Mg++, Na+, nor K+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate. PMID:22905204

  16. pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.

    Directory of Open Access Journals (Sweden)

    Yunfei Zhang

    Full Text Available We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+ specific; neither Ca(++, Mg(++, Na(+, nor K(+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.

  17. Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors

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    Mojca Benčina

    2013-12-01

    Full Text Available Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment, or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiology. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments.

  18. Stronger T cell immunogenicity of ovalbumin expressed intracellularly in Gram-negative than in Gram-positive bacteria.

    Science.gov (United States)

    Martner, Anna; Ostman, Sofia; Lundin, Samuel; Rask, Carola; Björnsson, Viktor; Telemo, Esbjörn; Collins, L Vincent; Axelsson, Lars; Wold, Agnes E

    2013-01-01

    This study aimed to clarify whether Gram-positive (G+) and Gram-negative (G-) bacteria affect antigen-presenting cells differently and thereby influence the immunogenicity of proteins they express. Lactobacilli, lactococci and Escherichia coli strains were transformed with plasmids conferring intracellular ovalbumin (OVA) production. Murine splenic antigen presenting cells (APCs) were pulsed with washed and UV-inactivated OVA-producing bacteria, control bacteria, or soluble OVA. The ability of the APCs to activate OVA-specific DO11.10 CD4(+) T cells was assessed by measurments of T cell proliferation and cytokine (IFN-γ, IL-13, IL-17, IL-10) production. OVA expressed within E. coli was strongly immunogenic, since 500 times higher concentrations of soluble OVA were needed to achieve a similar level of OVA-specific T cell proliferation. Furthermore, T cells responding to soluble OVA produced mainly IL-13, while T cells responding to E. coli-expressed OVA produced high levels of both IFN-γ and IL-13. Compared to E. coli, G+ lactobacilli and lactococci were poor inducers of OVA-specific T cell proliferation and cytokine production, despite efficient intracellular expression and production of OVA and despite being efficiently phagocytosed. These results demonstrate a pronounced difference in immunogenicity of intracellular antigens in G+ and G- bacteria and may be relevant for the use of bacterial carriers in vaccine development.

  19. Genetically encoded fluorescent probe to visualize intracellular phosphatidylinositol 3,5-bisphosphate localization and dynamics.

    Science.gov (United States)

    Li, Xinran; Wang, Xiang; Zhang, Xiaoli; Zhao, Mingkun; Tsang, Wai Lok; Zhang, Yanling; Yau, Richard Gar Wai; Weisman, Lois S; Xu, Haoxing

    2013-12-24

    Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] is a low-abundance phosphoinositide presumed to be localized to endosomes and lysosomes, where it recruits cytoplasmic peripheral proteins and regulates endolysosome-localized membrane channel activity. Cells lacking PI(3,5)P2 exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P2-metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI(3,5)P2, however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI(3,5)P2 binds, in the low nanomolar range, to a cytoplasmic phosphoinositide-interacting domain (ML1N) to activate late endosome and lysosome (LEL)-localized transient receptor potential Mucolipin 1 (TRPML1) channels. Here, we report the generation and characterization of a PI(3,5)P2-specific probe, generated by the fusion of fluorescence tags to the tandem repeats of ML1N. The probe was mainly localized to the membranes of Lamp1-positive compartments, and the localization pattern was dynamically altered by either mutations in the probe, or by genetically or pharmacologically manipulating the cellular levels of PI(3,5)P2. Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P2 probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P2-specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.

  20. Identification of intracellular bacteria in adenoid and tonsil tissue specimens: the efficiency of culture versus fluorescent in situ hybridization (FISH).

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    Stępińska, M; Olszewska-Sosińska, O; Lau-Dworak, M; Zielnik-Jurkiewicz, B; Trafny, E A

    2014-01-01

    Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14(+) cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14(+) cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14(+) cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14(+) cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.

  1. Urinary ATP and visualization of intracellular bacteria: a superior diagnostic marker for recurrent UTI in renal transplant recipients?

    Science.gov (United States)

    Kelley, Stephen P; Courtneidge, Holly R; Birch, Rebecca E; Contreras-Sanz, Alberto; Kelly, Mark C; Durodie, Jerome; Peppiatt-Wildman, Claire M; Farmer, Christopher K; Delaney, Michael P; Malone-Lee, James; Harber, Mark A; Wildman, Scott S

    2014-01-01

    Renal transplant recipients (RTR) are highly susceptible to urinary tract infections (UTIs) with over 50% of patients having at least one UTI within the first year. Yet it is generally acknowledged that there is considerable insensitivity and inaccuracy in routine urinalysis when screening for UTIs. Thus a large number of transplant patients with genuine urine infections may go undiagnosed and develop chronic recalcitrant infections, which can be associated with graft loss and morbidity. Given a recent study demonstrating ATP is released by urothelial cells in response to bacteria exposure, possibly acting at metabotropic P2Y receptors mediating a proinflammatory response, we have investigated alternative, and possibly more appropriate, urinalysis techniques in a cohort of RTRs. Mid-stream urine (MSU) samples were collected from 53 outpatient RTRs. Conventional leukocyte esterase and nitrite dipstick tests, and microscopic pyuria counts (in 1 μl), ATP concentration measurements, and identification of intracellular bacteria in shed urothelial cells, were performed on fresh unspun samples and compared to 'gold-standard' bacterial culture results. Of the 53 RTRs, 22% were deemed to have a UTI by 'gold-standard' conventional bacteria culture, whereas 87%, 8% and 4% showed evidence of UTIs according to leukocyte esterase dipstick, nitrite dipstick, and a combination of both dipsticks, respectively. Intracellular bacteria were visualized in shed urothelial cells of 44% of RTRs, however only 1 of the 23 RTRs (44%) was deemed to have a UTI by conventional bacteria culture. A significant association of the 'gold-standard' test with urinary ATP concentration combined with visualization of intracellular bacteria in shed urothelial cells was determined using the Fisher's exact test. It is apparent that standard bedside tests for UTIs give variable results and that seemingly quiescent bacteria in urothelial cells are very common in RTRs and may represent a focus of

  2. Two plant bacteria, S. meliloti and Ca. Liberibacter asiaticus, share functional znuABC homologues that encode for a high affinity zinc uptake system.

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    Cheryl M Vahling-Armstrong

    Full Text Available The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria.

  3. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

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    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

  4. Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis

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    Langerak Ankie A

    2008-02-01

    Full Text Available Abstract Background The obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease. Results A collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. For comparison, sequences of C. abortus, C. psittaci, C. caviae, C. felis, C. pecorum (Chlamydophila, C. muridarum (Chlamydia and of Candidatus protochlamydia and Simkania negevensis were also included. Sequences of fragments (400 – 500 base pairs from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the C. trachomatis strains, while the C. pneumoniae strains formed a single group. An UPGMA tree produced from the allelic profiles resulted in three groups of sequence types. The LGV strains grouped in a single cluster, while the urogenital strains were distributed over two separated groups, one consisted solely of strains with frequent occurring serovars (E, D and F. The distribution of the different serovars over the three groups was not consistent, suggesting exchange of serovar encoding ompA sequences. In one instance, exchange of fumC sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of Candidatus Protochlamydia amoebophila and Simkania

  5. Visualizing early splenic memory CD8+ T cells reactivation against intracellular bacteria in the mouse.

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    Marc Bajénoff

    Full Text Available Memory CD8(+ T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m. Memory CD8(+ T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8(+ T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8(+ T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8(+ T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8(+ T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8(+ T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8(+ T produce inflammatory cytokines such as IFN-gamma and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8(+ T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8(+ T cells provide a local response by secreting effector molecules around infected cells.

  6. [Lignocellulose degrading bacteria and their genes encoding cellulase/hemicellulase in rumen--a review].

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    Chen, Furong; Zhu, Yaxin; Dong, Xiuzhu; Liu, Lihua; Huang, Li; Dai, Xin

    2010-08-01

    Rumen of ruminant animals is known as a natural reactor involved in highly efficient lignocelluloses degradation. Rumen fibrolytic microbes have attracted an increasing attention for their potential value in biofuel research. Studies on rumen microbes have traditionally entailed the isolation of fibrolytic bacteria and subsequent analysis of fibrolytic enzymes. Developments in genomic and metagenomic approaches have made it possible to isolate directly genes and gene clusters encoding fibrolytic activities from rumen samples, permitting a global analysis of mechanisms of degradation of lignocellulose in rumen. Research in this field shows that lignocellulose degradation in rumen is a complex process involving a number of different microbes and is effected by a huge array of hydrolytic enzymes in a concerted fashion. This review briefly summarizes results from recent studies, especially metagenomic studies, on lignocellulose degradation in rumen.

  7. Imaging intracellular Ca²⁺ signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators.

    Science.gov (United States)

    Jiang, Ruotian; Haustein, Martin D; Sofroniew, Michael V; Khakh, Baljit S

    2014-11-19

    Astrocytes display spontaneous intracellular Ca(2+) concentration fluctuations ([Ca(2+)]i) and in several settings respond to neuronal excitation with enhanced [Ca(2+)]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca(2+)]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca(2+)]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca(2+)]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca(2+)]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca(2+)]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca(2+)]i signals in the striatal microcircuitry.

  8. Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

    Science.gov (United States)

    Brezden, Anna; Mohamed, Mohamed F; Nepal, Manish; Harwood, John S; Kuriakose, Jerrin; Seleem, Mohamed N; Chmielewski, Jean

    2016-08-31

    Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

  9. The role of the cytoskeleton in the life cycle of viruses and intracellular bacteria: tracks, motors, and polymerization machines.

    Science.gov (United States)

    Bearer, E L; Satpute-Krishnan, P

    2002-09-01

    Recent advances in microbiology implicate the cytoskeleton in the life cycle of some pathogens, such as intracellular bacteria, Rickettsia and viruses. The cellular cytoskeleton provides the basis for intracellular movements such as those that transport the pathogen to and from the cell surface to the nuclear region, or those that produce cortical protrusions that project the pathogen outwards from the cell surface towards an adjacent cell. Transport in both directions within the neuron is required for pathogens such as the herpesviruses to travel to and from the nucleus and perinuclear region where replication takes place. This trafficking is likely to depend on cellular motors moving on a combination of microtubule and actin filament tracks. Recently, Bearer et al. reconstituted retrograde transport of herpes simplex virus (HSV) in the giant axon of the squid. These studies identified the tegument proteins as the viral proteins most likely to recruit retrograde motors for the transport of HSV to the neuronal nucleus. Similar microtubule-based intracellular movements are part of the biological behavior of vaccinia, a poxvirus, and of adenovirus. Pathogen-induced surface projections and motility within the cortical cytoplasm also play a role in the life cycle of intracellular pathogens. Such motility is driven by pathogen-mediated actin polymerization. Virulence depends on this actin-based motility, since virulence is reduced in Listeria ActA mutants that lack the ability to recruit Arp2/3 and polymerize actin and in vaccinia virus mutants that cannot stimulate actin polymerization. Inhibition of intracellular movements provides a potential strategy to limit pathogenicity. The host cell motors and tracks, as well as the pathogen factors that interact with them, are potential targets for novel antimicrobial therapy.

  10. Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria

    Directory of Open Access Journals (Sweden)

    Giuditta Fiorella Schiavano

    2016-01-01

    Full Text Available The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701 after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages.

  11. A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

    Directory of Open Access Journals (Sweden)

    Galperin Michael Y

    2005-06-01

    Full Text Available Abstract Background Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. Results This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. Conclusion The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the

  12. Pathoadaptation of the Intracellular Bacteria Shigella and Chlamydia: Virulence, Antivirulence, and Tissue Tropism

    Science.gov (United States)

    2015-04-27

    PMN Polymorphonuclear leukocyte PvlArgDC Pyruvoyl-dependent ArgDC RB Reticulate body SER Shigella entry region Smip Shigella minimally invasive...bacterial-mediated adhesion processes. Helicobacter pylori binds to the adhesion decoy Muc1, a mucin expressed on the surface of epithelial cells in...the gastrointestinal tract (349). Muc1 is subsequently shed from the epithelial surface along with coupled bacteria, precluding long-term adhesion

  13. The cDNA sequences encoding two components of the polymeric fraction of the intracellular hemoglobin of Glycera dibranchiata.

    Science.gov (United States)

    Zafar, R S; Chow, L H; Stern, M S; Scully, J S; Sharma, P R; Vinogradov, S N; Walz, D A

    1990-12-15

    The intracellular hemoglobin of the polychaete Glycera dibranchiata consists of several components, some of which self-associate into a "polymeric" fraction. The cDNA library constructed from the poly(A+) mRNA of Glycera erythrocytes (Simons, P. C., and Satterlee, J. D. (1989) Biochemistry 28, 8525-8530) was screened with two oligodeoxynucleotide probes corresponding to the amino acid sequences MEEKVP and AMNSKV. Each of the two probes identified a full-length positive insert; these were sequenced using the dideoxynucleotide chain termination method. One clone was 630 bases long and contained 36 bases of 5'-untranslated RNA, a reading frame of 441 bases coding for the 147 amino acids of globin P2 including the residues MEEKVP, and a 3'-untranslated region of 153 bases. The other clone was 540 bases long and contained 24 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for globin P3 including the residues AMNSKV, and a 3'-untranslated region of 75 bases. The inferred amino acid sequences of the two globins were in agreement with the partial amino acid sequences obtained by chemical methods. The P2 and P3 globin sequences, together with the previously determined P1 sequence of a complete insert and partial sequences P4, P5, and P6 obtained from partial inserts (Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) Biochim. Biophys. Acta, in press) suggest that there are at least six components in the polymeric fraction of Glycera hemoglobin, which is in agreement with the results of polyacrylamide gel electrophoresis in Tris/glycine buffer, pH 8.3, 6 M urea. Nothern and dot blot analyses of Glycera erythrocyte poly(A+) mRNA using the foregoing two cDNA probes clearly demonstrated the presence of mature messages encoding both types of globins. Comparison of the polymeric sequences P1, P2, and P3 with the "monomeric" globins M-II and M-IV using the alignment and templates of Bashford et al. (Bashford, D., Chothia, C

  14. Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii.

    Science.gov (United States)

    Abd, Hadi; Saeed, Amir; Weintraub, Andrej; Nair, G Balakrish; Sandström, Gunnar

    2007-04-01

    Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.

  15. Tuberculosis and the risk of infection with other intracellular bacteria: a population-based study.

    Science.gov (United States)

    Huaman, M A; Fiske, C T; Jones, T F; Warkentin, J; Shepherd, B E; Ingram, L A; Maruri, F; Sterling, T R

    2015-04-01

    SUMMARY Persons who develop tuberculosis (TB) may have subtle immune defects that could predispose to other intracellular bacterial infections (ICBIs). We obtained data on TB and five ICBIs (Chlamydia trachomatis, Salmonella spp., Shigella spp., Yersinia spp., Listeria monocytogenes) reported to the Tennessee Department of Health, USA, 2000-2011. Incidence rate ratios (IRRs) comparing ICBIs in persons who developed TB and ICBIs in the Tennessee population, adjusted for age, sex, race and ethnicity were estimated. IRRs were not significantly elevated for all ICBIs combined [IRR 0.87, 95% confidence interval (CI) 0.71-1.06]. C. trachomatis rate was lowest in the year post-TB diagnosis (IRR 0.17, 95% CI 0.04-0.70). More Salmonella infections occurred in extrapulmonary TB compared to pulmonary TB patients (IRR 14.3, 95% CI 1.67-122); however, this appeared to be related to HIV co-infection. TB was not associated with an increased risk of other ICBIs. In fact, fewer C. trachomatis infections occurred after recent TB diagnosis. Reasons for this association, including reduced exposure, protection conferred by anti-TB drugs or macrophage activation by Mycobacterium tuberculosis infection warrant further investigation.

  16. Dibenzocyclooctadiene lignans from Schisandra spp. selectively inhibit the growth of the intracellular bacteria Chlamydia pneumoniae and Chlamydia trachomatis.

    Science.gov (United States)

    Hakala, Elina; Hanski, Leena; Uvell, Hanna; Yrjönen, Teijo; Vuorela, Heikki; Elofsson, Mikael; Vuorela, Pia Maarit

    2015-10-01

    Lignans from Schisandra chinensis berries show various pharmacological activities, of which their antioxidative and cytoprotective properties are among the most studied ones. Here, the first report on antibacterial properties of six dibenzocyclooctadiene lignans found in Schisandra spp. is presented. The activity was shown on two related intracellular Gram-negative bacteria Chlamydia pneumoniae and Chlamydia trachomatis upon their infection in human epithelial cells. All six lignans inhibited C. pneumoniae inclusion formation and infectious progeny production. Schisandrin B inhibited C. pneumoniae inclusion formation even when administered 8 h post infection, indicating a target that occurs relatively late within the infection cycle. Upon infection, lignan-pretreated C. pneumoniae elementary bodies had impaired inclusion formation capacity. The presence and substitution pattern of methylenedioxy, methoxy and hydroxyl groups of the lignans had a profound impact on the antichlamydial activity. In addition our data suggest that the antichlamydial activity is not caused only by the antioxidative properties of the lignans. None of the compounds showed inhibition on seven other bacteria, suggesting a degree of selectivity of the antibacterial effect. Taken together, the data presented support a role of the studied lignans as interesting antichlamydial lead compounds.

  17. Development of a method to measure intracellular growth rate of parasitic acid-fast bacteria using radio-isotope and its improvement

    Energy Technology Data Exchange (ETDEWEB)

    Nakata, Noboru; Fukutomi, Yasuo [National Inst. of Infectious Diseases, Tokyo (Japan)

    1999-02-01

    Development of measurement method for intracellular growth rate was attempted using gene-transfected acid-fast bacteria and Mycobacterium leprae. M. leprae was inoculated into a well, which was filled with fetus bovine serum containing a cover slip pasted with mouse monocyte-derived malignant cell lines, J774 and P388D1 and cultured for 3-4 hours. Then, the cells on the cover slip were mobilized with 0.1 N NaOH. The metabolic activity of M. leprae was assessed based on the {beta}-oxidation activity of {sup 14}C-palmitic acid. Then, it was investigated whether TNF is produced by the cell culture added with M. leprae or LPS. J774 cells abundantly produced TNF after sensitization with LPS and its production was depending on the amount of added bacteria, whereas TNF production after sensitization with LPS or M. leprae was little in P388D1 cells. Staining for acid-fast bacteria revealed that either of these cell lines has phagocytic activity for M. leprae. To identify the bacterial factor involved to the intracellular proliferation of acid-fast bacteria, transposon insertion mutagenesis was attempted to M. avium complex (MAC) and the degrees of drug-resistance in M. avium mino, M. intracellulare JATA-52 and 8 clinically isolated M. intracellulare strains were determined. M. intracellulare JATA-52 was resistant to kanamycin and plasmid pAL8 and pYT937 were both able to transform the strain with dose-dependency. Since M. intracellulare is pathogenic to human and the strain proliferates with a generation time shorter than that of M. tuberculosis, the former strain is thought suitable for the analysis of a mutated gene. Thus, it became possible to study transposition insertion mutagenesis in M. intracellulare. (M.N.)

  18. Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Marion Le Coadic

    Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

  19. The PAM1 gene of petunia, required for intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi, encodes a homologue of VAPYRIN.

    Science.gov (United States)

    Feddermann, Nadja; Muni, Rajasekhara Reddy Duvvuru; Zeier, Tatyana; Stuurman, Jeroen; Ercolin, Flavia; Schorderet, Martine; Reinhardt, Didier

    2010-11-01

    Most terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.

  20. Genomes of rumen bacteria encode atypical pathways for fermenting hexoses to short-chain fatty acids

    KAUST Repository

    Hackmann, Timothy J.

    2017-09-11

    Bacteria have been thought to follow only a few well-recognized biochemical pathways when fermenting glucose or other hexoses. These pathways have been chiseled in the stone of textbooks for decades, with most sources rendering them as they appear in the classic 1986 text by Gottschalk. Still, it is unclear how broadly these pathways apply, given that they were established and delineated biochemically with only a few model organisms. Here we show that well-recognized pathways often cannot explain fermentation products formed by bacteria. In the most extensive analysis of its kind, we reconstructed pathways for glucose fermentation from genomes of 48 species and subspecies of bacteria from one environment (the rumen). In total, 44% of these bacteria had atypical pathways, including several that are completely unprecedented for bacteria or any organism. In detail, 8% of bacteria had an atypical pathway for acetate formation; 21% for propionate or succinate formation; 6% for butyrate formation; and 33% had an atypical or incomplete Embden-Meyerhof-Parnas pathway. This study shows that reconstruction of metabolic pathways-a common goal of omics studies-could be incorrect if well-recognized pathways are used for reference. Further, it calls for renewed efforts to delineate fermentation pathways biochemically. This article is protected by copyright. All rights reserved.

  1. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Directory of Open Access Journals (Sweden)

    Natalie R Lazar Adler

    Full Text Available Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA. Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE. A single mutant (bpaC was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA, those attenuated for virulence and net intracellular replication (BpaE, the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA. Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors

  2. Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

    Science.gov (United States)

    Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

    2015-01-01

    Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were

  3. The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages.

    Directory of Open Access Journals (Sweden)

    Auricelio A Macedo

    Full Text Available Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi. In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment.

  4. Inhibitory activity of the isoflavone biochanin A on intracellular bacteria of genus Chlamydia and initial development of a buccal formulation.

    Directory of Open Access Journals (Sweden)

    Leena Hanski

    Full Text Available Given the established role of Chlamydia spp. as causative agents of both acute and chronic diseases, search for new antimicrobial agents against these intracellular bacteria is required to promote human health. Isoflavones are naturally occurring phytoestrogens, antioxidants and efflux pump inhibitors, but their therapeutic use is limited by poor water-solubility and intense first-pass metabolism. Here, we report on effects of isoflavones against C. pneumoniae and C. trachomatis and describe buccal permeability and initial formulation development for biochanin A. Biochanin A was the most potent Chlamydia growth inhibitor among the studied isoflavones, with an IC50 = 12 µM on C. pneumoniae inclusion counts and 6.5 µM on infectious progeny production, both determined by immunofluorescent staining of infected epithelial cell cultures. Encouraged by the permeation of biochanin A across porcine buccal mucosa without detectable metabolism, oromucosal film formulations were designed and prepared by a solvent casting method. The film formulations showed improved dissolution rate of biochanin A compared to powder or a physical mixture, presumably due to the solubilizing effect of hydrophilic additives and presence of biochanin A in amorphous state. In summary, biochanin A is a potent inhibitor of Chlamydia spp., and the in vitro dissolution results support the use of a buccal formulation to potentially improve its bioavailability in antichlamydial or other pharmaceutical applications.

  5. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    Science.gov (United States)

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  6. Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto-Kakizaki rats.

    Science.gov (United States)

    Salem, K A; Adrian, T E; Qureshi, M A; Parekh, K; Oz, M; Howarth, F C

    2012-12-01

    There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0

  7. Ultrastructural characterization and multilocus sequence analysis (MLSA) of 'Candidatus Rickettsiella isopodorum', a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda).

    Science.gov (United States)

    Kleespies, Regina G; Federici, Brian A; Leclerque, Andreas

    2014-07-01

    The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation 'Candidatus Rickettsiella isopodorum' is introduced for this new lineage of isopod-associated Rickettsiella bacteria.

  8. Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers.

    Science.gov (United States)

    Cocking, Edward C; Stone, Philip J; Davey, Michael R

    2005-12-01

    It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs.

  9. Isolation of endophytic bacteria from arboreal species of the Amazon and identification by sequencing of the 16S rRNA encoding gene

    Directory of Open Access Journals (Sweden)

    Mariza M. Coêlho

    2011-01-01

    Full Text Available Endophytic bacteria from three arboreal species native to the Amazon (Carapa guianenses, Ceiba pentandra, and Swietenia macrophylla, were isolated and identified, through partial sequencing of the 16S rRNA encoding gene. From these, 16 isolates were obtained, although, when compared to sequences deposited in GenBank, only seven had produced identifiable fragments. Bacillus, Pantoea and two non-culturable samples were identified. Results obtained through sequence analysis revealed low genetic diversity across the isolates, even when analyzing different species and plant structures. This is the first report concerning the isolation and identification of endophytic bacteria in these plant species.

  10. Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

    Institute of Scientific and Technical Information of China (English)

    Edward C. Cocking; Philip J. Stone; Michael R. Davey

    2005-01-01

    It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems,and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intraceliularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers,we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus,with minimal or zero inputs.

  11. Development and improvement of measuring method for growth rate of intracellular symbiotic acid-fast bacteria using radioisotopes

    Energy Technology Data Exchange (ETDEWEB)

    Nakata, Noboru; Fukutomi, Yasuo [National Inst. for Leprosy Research, Higashimurayama, Tokyo (Japan)

    1997-02-01

    The aim of this research group was to investigate the factors which might mediate the growth of mycobacterium lepra and relate to its affinity to the nerve tissue. In this year, constructions of a mycobacterium smegmatis mutant having a high transform ability and a shuttle vector between E. coli and acid-fast bacteria was attempted. From the wild type of m. smegmatis, a highly transformable mutant was obtained and the rate of transformation of the mutant was ca. 10{sup 5} times higher than the parent. And two shuttle vectors for E. coli/acid-fast bacteria; pALKMZErO (6.2 kb) and pHSGM59 (5.4 kb) were constructed. Since the former was unstable in M. smegmatis, the latter vector was used for the following experiments. Expression of `cat` gene cloned by pHSGM59 was identified in M. smegmatis. Further, DNA library of M. leprae was prepared by the use of the vector. Approximately, 1 x 10{sup 4} transformed clones were obtained. The analysis of the plasmids recovered from the clones is under way. (M.N.)

  12. Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria

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    Muscariello Lidia

    2006-05-01

    Full Text Available Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters. Conclusion We propose that the CscA, CscB, CscC and Csc

  13. Legionella pneumophila transcriptome during intracellular multiplication in human macrophages

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    Sebastien P Faucher

    2011-04-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoa, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known L. pneumophila virulence determinant is the Icm/Dot Type IVB secretion system (TFBSS, which is used to translocate more than 150 effector proteins to host cells. While the transcriptional response of Legionella to the intracellular environment of A. castellanii has been investigated, much less is known about the Legionella transcriptional response inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth as well as during infection of human cultured macrophages. This was accomplished with microarrays and an RNA amplification procedure called SCOTS to detect small amounts of mRNA from low numbers of intracellular bacteria. Among the genes induced intracellularly are those involved in amino acid biosynthetic pathways leading to L-arginine, L-histidine and L-proline as well as many transport systems involved in amino acid and iron uptake. Gene involved in catabolism of glycerol is also induced during intracellular growth and could be used as a carbon source. The genes encoding the Icm/Dot system are not differentially expressed inside cells compared to control bacteria grown in rich broth, but the genes encoding several translocated effectors are strongly induced. Moreover, we used the transcriptome data to predict previously unrecognized Icm/Dot effector genes based on their expression pattern and confirmed translocation for three candidates. This study provides a comprehensive view of how L. pneumophila responds to the human macrophage intracellular environment.

  14. Growth performance, rumen fermentation, bacteria composition, and gene expressions involved in intracellular pH regulation of rumen epithelium in finishing Hu lambs differing in residual feed intake phenotype.

    Science.gov (United States)

    Liang, Y S; Li, G Z; Li, X Y; Lü, J Y; Li, F D; Tang, D F; Li, F; Deng, Y; Zhang, H; Wang, Z L; Weng, X X

    2017-04-01

    The objective of this study was to evaluate the effect of residual feed intake (RFI) on rumen function in finishing lambs. A total of 60 male Hu lambs (average initial BW = 25.2 ± 2.5kg) were used and were offered a pelleted high-concentrate diet, of which the forage to concentrate ratio was 25:75. Individual feed intake was recorded over a period of 42 d, then 10 lambs with the lowest RFI and the highest RFI were selected, respectively. The rumen fluid used for fermentation variables and relative abundance of bacteria measurement was obtained on d 10 and 20 after RFI measurement. At the end of this experiment, the selected lambs were slaughtered and rumen epithelium and liver tissues were collected for RNA extraction. Low-RFI lambs had lower ( 0.05). Additionally, RFI was positively ( = 0.57; 0.05) were not affected by RFI classification. Nonetheless, low-RFI group lambs had a greater ( 0.05) on gene expression associated with intracellular pH regulation (, , , , , , , and ) in rumen epithelium and β-hydroxybutyrate metabolism () in both rumen epithelium and liver tissues. In conclusion, even though low-RFI lambs had lower DMI, however, the number of was lower. Additionally, there was no difference in gene expressions level associated with intracellular pH regulation in rumen epithelium between RFI groups.

  15. Expression of genes involved in the uptake of inorganic carbon in the gill of a deep-sea vesicomyid clam harboring intracellular thioautotrophic bacteria.

    Science.gov (United States)

    Hongo, Yuki; Ikuta, Tetsuro; Takaki, Yoshihiro; Shimamura, Shigeru; Shigenobu, Shuji; Maruyama, Tadashi; Yoshida, Takao

    2016-07-10

    Deep-sea vesicomyid clams, including the genus Phreagena (formerly Calyptogena), harbor thioautotrophic bacterial symbionts in the host symbiosome, which consists of cytoplasmic vacuoles in gill epithelial cells called bacteriocytes. The symbiont requires inorganic carbon (Ci), such as CO2, HCO3(-), and CO3(2-), to synthesize organic compounds, which are utilized by the host clam. The dominant Ci in seawater is HCO3(-), which is impermeable to cell membranes. Within the bacteriocyte, cytoplasmic carbonic anhydrase (CA) from the host, which catalyzes the inter-conversion between CO2 and HCO3(-), has been shown to be abundant and is thought to supply intracellular CO2 to symbionts in the symbiosome. However, the mechanism of Ci uptake by the host gill from seawater is poorly understood. To elucidate the influx pathway of Ci into the bacteriocyte, we isolated the genes related to Ci uptake via the pyrosequencing of cDNA from the gill of Phreagena okutanii, and investigated their expression patterns. Using phylogenetic and amino acid sequence analyses, three solute carrier family 4 (SLC4) bicarbonate transporters (slc4co1, slc4co2, and slc4co4) and two membrane-associated CAs (mcaco1 and mcaco2) were identified as candidate genes for Ci uptake. In an in situ hybridization analysis of gill sections, the expression of mcaco1 and mcaco2 was detected in the bacteriocytes and asymbiotic non-ciliated cells, respectively, and the expression of slc4co1 and slc4co2 was detected in the asymbiotic cells, including the intermediate cells of the inner area and the non-ciliated cells of the external area. Although subcellular localizations of the products of these genes have not been fully elucidated, they may play an important role in the uptake of Ci into the bacteriocytes. These findings will improve our understanding of the Ci transport system in the symbiotic relationships of chemosynthetic bivalves.

  16. Evolution of intracellular compartmentalization.

    Science.gov (United States)

    Diekmann, Yoan; Pereira-Leal, José B

    2013-01-15

    Cells compartmentalize their biochemical functions in a variety of ways, notably by creating physical barriers that separate a compartment via membranes or proteins. Eukaryotes have a wide diversity of membrane-based compartments, many that are lineage- or tissue-specific. In recent years, it has become increasingly evident that membrane-based compartmentalization of the cytosolic space is observed in multiple prokaryotic lineages, giving rise to several types of distinct prokaryotic organelles. Endosymbionts, previously believed to be a hallmark of eukaryotes, have been described in several bacteria. Protein-based compartments, frequent in bacteria, are also found in eukaryotes. In the present review, we focus on selected intracellular compartments from each of these three categories, membrane-based, endosymbiotic and protein-based, in both prokaryotes and eukaryotes. We review their diversity and the current theories and controversies regarding the evolutionary origins. Furthermore, we discuss the evolutionary processes acting on the genetic basis of intracellular compartments and how those differ across the domains of life. We conclude that the distinction between eukaryotes and prokaryotes no longer lies in the existence of a compartmentalized cell plan, but rather in its complexity.

  17. Pediococcus parvulus gtf gene encoding the GTF glycosyltransferase and its application for specific PCR detection of beta-D-glucan-producing bacteria in foods and beverages.

    Science.gov (United States)

    Werning, Maria Laura; Ibarburu, Idoia; Dueñas, Maria Teresa; Irastorza, Ana; Navas, Jesús; López, Paloma

    2006-01-01

    Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, beta-D-glucan-producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize beta-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the beta-D-glucan producers Lactobacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of beta-D-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 X 10(2) CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

  18. Mechanisms of Intracellular Chlamydiae Survival

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    Lukic Ruzica

    2016-06-01

    Full Text Available Chlamydiae are Gram-negative, non-motile, obligate intracellular, and spherically shaped bacteria with a diameter of 0.2-1.5 μm. Chlamydiae are present in several different morphological forms: the elementary body, the reticular body, and in the last several years, there has been the observation of a third form known as the persistent or atypical form. The intracellular localization of Chlamydia provides a unique replication cycle that occurs inside a membrane-surrounded vacuole in the host cell cytoplasm and is significantly different from the method of multiplication of other microorganisms. Chlamydiae are capable of manipulating different signalling pathways inside the infected cell, thus avoiding the host immune response. This ensures intracellular multiplication, survival, and long-term persistence of Chlamydiae. There are two basic means of achieving this persistence: inhibition of apoptosis and manipulation of NF-κB (nuclear factor kappa B-mediated signals in the host.

  19. Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis.

    Science.gov (United States)

    Zenno, S; Saigo, K

    1994-06-01

    Genes encoding NAD(P)H-flavin oxidoreductases (flavin reductases) similar in both size and sequence to Fre, the most abundant flavin reductase in Escherichia coli, were identified in four species of luminous bacteria, Photorhabdus luminescens (ATCC 29999), Vibrio fischeri (ATCC 7744), Vibrio harveyi (ATCC 33843), and Vibrio orientalis (ATCC 33934). Nucleotide sequence analysis showed Fre-like flavin reductases in P. luminescens and V. fischeri to consist of 233 and 236 amino acids, respectively. As in E. coli Fre, Fre-like enzymes in luminous bacteria preferably used riboflavin as an electron acceptor when NADPH was used as an electron donor. These enzymes also were good suppliers of reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. In V. fischeri, the Fre-like enzyme is a minor flavin reductase representing Fre-like enzyme has no appreciable homology in amino acid sequence to the major flavin reductase in V. fischeri, FRase I, indicates that at least two different types of flavin reductases supply FMNH2 to the luminescence system in V. fischeri. Although Fre-like flavin reductases are highly similar in sequence to luxG gene products (LuxGs), Fre-like flavin reductases and LuxGs appear to constitute two separate groups of flavin-associated proteins.

  20. Responses of community structure of amoA-encoding archaea and ammonia-oxidizing bacteria in ammonia biofilter with rockwool mixtures to the gradual increases in ammonium and nitrate.

    Science.gov (United States)

    Yasuda, T; Waki, M; Kuroda, K; Hanajima, D; Fukumoto, Y; Yamagishi, T; Suwa, Y; Suzuki, K

    2013-03-01

    To investigate community shifts of amoA-encoding archaea (AEA) and ammonia-oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process. A laboratory-scale rockwool biofilter with an irrigated water circulation system was operated for 436 days with ammonia loading rates of 49-63 NH(3) g m(-3) day(-1). The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real-time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively. The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH. One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology. © 2013 The Society for Applied Microbiology.

  1. Phylogenetic and in silico functional analyses of thermostable-direct hemolysin and tdh-related encoding genes in Vibrio parahaemolyticus and other Gram-negative bacteria.

    Science.gov (United States)

    Bhowmik, Sushanta K; Pazhani, Gururaja P; Ramamurthy, Thandavarayan

    2014-01-01

    Emergence and spread of pandemic strains of Vibrio parahaemolyticus have drawn attention to make detailed study on their genomes. The pathogenicity of V. parahaemolyticus has been associated with thermostable-direct hemolysin (TDH) and/or TDH-related hemolysin (TRH). The present study evaluated characteristics of tdh and trh genes, considering the phylogenetic and in silico functional features of V. parahaemolyticus and other bacteria. Fifty-two tdh and trh genes submitted to the GenBank were analyzed for sequence similarity. The promoter sequences of these genes were also analyzed from transcription start point to -35 regions and correlated with amino acid substitution within the coding regions. The phylogenetic analysis revealed that tdh and trh are highly distinct and also differ within the V. parahaemolyticus strains that were isolated from different geographical regions. Promoter sequence analysis revealed nucleotide substitutions and deletions at -18 and -19 positions among the pandemic, prepandemic, and nonpandemic tdh sequences. Many amino acid substitutions were also found within the signal peptide and also in the matured protein region of several TDH proteins as compared to TDH-S protein of pandemic V. parahaemolyticus. Experimental evidences are needed to recognize the importance of substitutions and deletions in the tdh and trh genes.

  2. Phylogenetic and In Silico Functional Analyses of Thermostable-Direct Hemolysin and tdh-Related Encoding Genes in Vibrio parahaemolyticus and Other Gram-Negative Bacteria

    Directory of Open Access Journals (Sweden)

    Sushanta K. Bhowmik

    2014-01-01

    Full Text Available Emergence and spread of pandemic strains of Vibrio parahaemolyticus have drawn attention to make detailed study on their genomes. The pathogenicity of V. parahaemolyticus has been associated with thermostable-direct hemolysin (TDH and/or TDH-related hemolysin (TRH. The present study evaluated characteristics of tdh and trh genes, considering the phylogenetic and in silico functional features of V. parahaemolyticus and other bacteria. Fifty-two tdh and trh genes submitted to the GenBank were analyzed for sequence similarity. The promoter sequences of these genes were also analyzed from transcription start point to −35 regions and correlated with amino acid substitution within the coding regions. The phylogenetic analysis revealed that tdh and trh are highly distinct and also differ within the V. parahaemolyticus strains that were isolated from different geographical regions. Promoter sequence analysis revealed nucleotide substitutions and deletions at −18 and −19 positions among the pandemic, prepandemic, and nonpandemic tdh sequences. Many amino acid substitutions were also found within the signal peptide and also in the matured protein region of several TDH proteins as compared to TDH-S protein of pandemic V. parahaemolyticus. Experimental evidences are needed to recognize the importance of substitutions and deletions in the tdh and trh genes.

  3. Ectopic Expression in Arabidopsis thaliana of an NB-ARC Encoding Putative Disease Resistance Gene from Wild Chinese Vitis pseudoreticulata Enhances Resistance to Phytopathogenic Fungi and Bacteria

    Directory of Open Access Journals (Sweden)

    Zhifeng eWen

    2015-12-01

    Full Text Available Plant resistance proteins mediate pathogen recognition and activate innate immune responses to restrict pathogen proliferation. One common feature of these proteins is an NB-ARC domain. In this study, we characterized a gene encoding a protein with an NB-ARC domain from wild Chinese grapevine Vitis pseudoreticulata accession Baihe-35-1, which was identified in a transcriptome analysis of the leaves following inoculation with Erysiphe necator (Schw., a causal agent of powdery mildew. Transcript levels of this gene, designated VpCN (GenBank accession number KT265084, increased strongly after challenge of grapevine leaves with E. necator. The deduced amino acid sequence was predicted to contain an NB-ARC domain in the C-terminus and an RxCC-like domain similar to CC domain of Rx protein in the N-terminus. Ectopic expression of VpCN in Arabidopsis thaliana resulted in either a wild-type phenotype or a dwarf phenotype. The phenotypically normal transgenic A. thaliana showed enhance resistance to A. thaliana powdery mildew Golovinomyces cichoracearum, as well as to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Moreover, promoter::GUS (β-glucuronidase analysis revealed that powdery mildew infection induced the promoter activity of VpCN in grapevine leaves. Finally, a promoter deletion analysis showed that TC rich repeat elements likely play an important role in the response to E. necator infection. Taken together, our results suggest that VpCN contribute to powdery mildew disease resistant in grapevine.

  4. Regulation of the rplY gene encoding 5S rRNA binding protein L25 in Escherichia coli and related bacteria.

    Science.gov (United States)

    Aseev, Leonid V; Bylinkina, Natalia S; Boni, Irina V

    2015-05-01

    Ribosomal protein (r-protein) L25 is one of the three r-proteins (L25, L5, L18) that interact with 5S rRNA in eubacteria. Specific binding of L25 with a certain domain of 5S r-RNA, a so-called loop E, has been studied in detail, but information about regulation of L25 synthesis has remained totally lacking. In contrast to the rplE (L5) and rplR (L18) genes that belong to the polycistronic spc-operon and are regulated at the translation level by r-protein S8, the rplY (L25) gene forms an independent transcription unit. The main goal of this work was to study the regulation of the rplY expression in vivo. We show that the rplY promoter is down-regulated by ppGpp and its cofactor DksA in response to amino acid starvation. At the level of translation, the rplY expression is subjected to the negative feedback control. The 5'-untranslated region of the rplY mRNA comprises specific sequence/structure features, including an atypical SD-like sequence, which are highly conserved in a subset of gamma-proteobacterial families. Despite the lack of a canonical SD element, the rplY'-'lacZ single-copy reporter showed unusually high translation efficiency. Expression of the rplY gene in trans decreased the translation yield, indicating the mechanism of autogenous repression. Site-directed mutagenesis of the rplY 5' UTR revealed an important role of the conserved elements in the translation control. Thus, the rplY expression regulation represents one more example of regulatory pathways that control ribosome biogenesis in Escherichia coli and related bacteria. © 2015 Aseev et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  5. Glutathione provides a source of cysteine essential for intracellular multiplication of Francisella tularensis.

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    Khaled Alkhuder

    2009-01-01

    Full Text Available Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. Its ability to multiply and survive in macrophages is critical for its virulence. By screening a bank of HimarFT transposon mutants of the F. tularensis live vaccine strain (LVS to isolate intracellular growth-deficient mutants, we selected one mutant in a gene encoding a putative gamma-glutamyl transpeptidase (GGT. This gene (FTL_0766 was hence designated ggt. The mutant strain showed impaired intracellular multiplication and was strongly attenuated for virulence in mice. Here we present evidence that the GGT activity of F. tularensis allows utilization of glutathione (GSH, gamma-glutamyl-cysteinyl-glycine and gamma-glutamyl-cysteine dipeptide as cysteine sources to ensure intracellular growth. This is the first demonstration of the essential role of a nutrient acquisition system in the intracellular multiplication of F. tularensis. GSH is the most abundant source of cysteine in the host cytosol. Thus, the capacity this intracellular bacterial pathogen has evolved to utilize the available GSH, as a source of cysteine in the host cytosol, constitutes a paradigm of bacteria-host adaptation.

  6. The Brucella suis virB operon is induced intracellularly in macrophages

    Science.gov (United States)

    Boschiroli, Maria Laura; Ouahrani-Bettache, Safia; Foulongne, Vincent; Michaux-Charachon, Sylvie; Bourg, Gisele; Allardet-Servent, Annick; Cazevieille, Chantal; Liautard, Jean Pierre; Ramuz, Michel; O'Callaghan, David

    2002-01-01

    A type IV secretion system similar to the VirB system of the phytopathogen Agrobacterium tumefaciens is essential for the intracellular survival and multiplication of the mammalian pathogen Brucella. Reverse transcriptase–PCR showed that the 12 genes encoding the Brucella suis VirB system form an operon. Semiquantitative measurements of virB mRNA levels by slot blotting showed that transcription of the virB operon, but not the flanking genes, is regulated by environmental factors in vitro. Flow cytometry used to measure green fluorescent protein expression from the virB promoter confirmed the data from slot blots. Fluorescence-activated cell sorter analysis and fluorescence microscopy showed that the virB promoter is induced in macrophages within 3 h after infection. Induction only occurred once the bacteria were inside the cells, and phagosome acidification was shown to be the major signal inducing intracellular expression. Because phagosome acidification is essential for the intracellular multiplication of Brucella, we suggest that it is the signal that triggers the secretion of unknown effector molecules. These effector molecules play a role in the remodeling of the phagosome to create the unique intracellular compartment in which Brucella replicates. PMID:11830669

  7. Targeting intracellular Staphylococcus aureus to lower recurrence of orthopaedic infection.

    Science.gov (United States)

    Dusane, Devendra H; Kyrouac, Douglas; Petersen, Iris; Bushrow, Luke; Calhoun, Jason H; Granger, Jeffrey F; Phieffer, Laura S; Stoodley, Paul

    2017-09-08

    Staphylococcus aureus is often found in orthopaedic infections and may be protected from commonly prescribed antibiotics by forming biofilms or growing intracellularly within osteoblasts. To investigate the effect of non-antibiotic compounds in conjunction with antibiotics to clear intracellular and biofilm forming S. aureus causing osteomyelitis. SAOS-2 osteoblast-like cell lines were infected with S. aureus BB1279. Antibiotics (vancomycin, VAN; and dicloxacillin, DICLOX), bacterial efflux pump inhibitors (piperine, PIP; carbonyl cyanide m-chlorophenyl hydrazone, CCCP) and bone morphogenetic protein (BMP-2) were evaluated individually and in combination to kill intracellular bacteria. We present direct evidence that after gentamicin killed extracellular planktonic bacteria and antibiotics had been stopped, seeding from the infected osteoblasts grew as biofilms. VAN was ineffective in treating the intracellular bacteria even at 10x MIC; however in presence of PIP or CCCP the intracellular S. aureus was significantly reduced. Bacterial efflux pump inhibitors (PIP and CCCP) were effective in enhancing permeability of antibiotics within the osteoblasts and facilitated killing of intracellular S. aureus. Confocal laser scanning microscopy (CLSM) showed increased uptake of propidium iodide within osteoblasts in presence of PIP and CCCP. BMP-2 had no effect on growth of S. aureus either alone or in combination with antibiotics. Combined application of antibiotics and natural agents could help in the treatment of osteoblast infected intracellular bacteria and biofilms associated with osteomyelitis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. Identification and functional characterization of K+ transporters encoded by Legionella pneumophila kup genes

    Science.gov (United States)

    Hori, Juliana I.; Pereira, Marcelo S.F.; Roy, Craig R.; Nagai, Hiroki; Zamboni, Dario S.

    2013-01-01

    Summary Legionnaires’ disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the E. coli K+ transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K+ acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella-containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but it did not influence the replication of wild-type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K+ transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K+ transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient-limited conditions. PMID:23848378

  9. Identification and functional characterization of K(+) transporters encoded by Legionella pneumophila kup genes.

    Science.gov (United States)

    Hori, Juliana I; Pereira, Marcelo S F; Roy, Craig R; Nagai, Hiroki; Zamboni, Dario S

    2013-12-01

    Legionnaires' disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the Escherichia coli K(+) transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K(+) acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella-containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but itdid not influence the replication of wild-type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K(+) transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K(+) transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient-limited conditions.

  10. The complete genome of Teredinibacter turnerae T7901: an intracellular endosymbiont of marine wood-boring bivalves (shipworms).

    Science.gov (United States)

    Yang, Joyce C; Madupu, Ramana; Durkin, A Scott; Ekborg, Nathan A; Pedamallu, Chandra S; Hostetler, Jessica B; Radune, Diana; Toms, Bradley S; Henrissat, Bernard; Coutinho, Pedro M; Schwarz, Sandra; Field, Lauren; Trindade-Silva, Amaro E; Soares, Carlos A G; Elshahawi, Sherif; Hanora, Amro; Schmidt, Eric W; Haygood, Margo G; Posfai, Janos; Benner, Jack; Madinger, Catherine; Nove, John; Anton, Brian; Chaudhary, Kshitiz; Foster, Jeremy; Holman, Alex; Kumar, Sanjay; Lessard, Philip A; Luyten, Yvette A; Slatko, Barton; Wood, Nicole; Wu, Bo; Teplitski, Max; Mougous, Joseph D; Ward, Naomi; Eisen, Jonathan A; Badger, Jonathan H; Distel, Daniel L

    2009-07-01

    Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host's nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2-40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100). However, unlike S. degradans, which degrades a broad spectrum (>10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose

  11. The complete genome of Teredinibacter turnerae T7901: an intracellular endosymbiont of marine wood-boring bivalves (shipworms.

    Directory of Open Access Journals (Sweden)

    Joyce C Yang

    Full Text Available Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms. This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host's nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2-40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100. However, unlike S. degradans, which degrades a broad spectrum (>10 classes of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection. These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production

  12. An optimal method of iron starvation of the obligate intracellular pathogen, Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Christopher C. Thompson

    2011-02-01

    Full Text Available Iron is an essential cofactor in a number of critical biochemical reactions, and as such, its acquisition, storage, and metabolism is highly regulated in most organisms. The obligate intracellular bacterium, Chlamydia trachomatis experiences a developmental arrest when iron within the host is depleted. The nature of the iron starvation response in Chlamydia is relatively uncharacterized because of the likely inefficient method of iron depletion, which currently relies on the compound deferoxamine mesylate (DFO. Inefficient induction of the iron starvation response precludes the identification of iron-regulated genes. This report evaluated DFO with another iron chelator, 2,2’-bipyridyl (Bpdl and presented a systematic comparison of the two across a range of criteria in a single-treatment time-of-infection regimen. We demonstrate that the membrane permeable Bpdl was superior to DFO in the inhibition of chlamydia development, the induction of aberrant morphology, and the induction of an iron starvation transcriptional response in both host and bacteria. Furthermore, iron starvation using Bpdl identified the periplasmic iron binding protein-encoding ytgA gene as iron- responsive. Overall, the data present a compelling argument for the use of Bpdl, rather than DFO, in future iron starvation studies of chlamydia and other intracellular bacteria.

  13. Genome sequence of Rickettsia bellii illuminates the role of amoebae in gene exchanges between intracellular pathogens.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available The recently sequenced Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair-long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic "melting pot" where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.

  14. Advances in genetic manipulation of obligate intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Paul eBeare

    2011-05-01

    Full Text Available Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

  15. Internal affairs: investigating the Brucella intracellular lifestyle.

    Science.gov (United States)

    von Bargen, Kristine; Gorvel, Jean-Pierre; Salcedo, Suzana P

    2012-05-01

    Bacteria of the genus Brucella are Gram-negative pathogens of several animal species that cause a zoonotic disease in humans known as brucellosis or Malta fever. Within their hosts, brucellae reside within different cell types where they establish a replicative niche and remain protected from the immune response. The aim of this article is to discuss recent advances in the field in the specific context of the Brucella intracellular 'lifestyle'. We initially discuss the different host cell targets and their relevance during infection. As it represents the key to intracellular replication, the focus is then set on the maturation of the Brucella phagosome, with particular emphasis on the Brucella factors that are directly implicated in intracellular trafficking and modulation of host cell signalling pathways. Recent data on the role of the type IV secretion system are discussed, novel effector molecules identified and how some of them impact on trafficking events. Current knowledge on Brucella gene regulation and control of host cell death are summarized, as they directly affect intracellular persistence. Understanding how Brucella molecules interplay with their host cell targets to modulate cellular functions and establish the intracellular niche will help unravel how this pathogen causes disease.

  16. Chromosomal and plasmid-encoded factors of Shigella flexneri induce secretogenic activity ex vivo.

    Directory of Open Access Journals (Sweden)

    Christina S Faherty

    Full Text Available Shigella flexneri is a Gram-negative, facultative intracellular pathogen that causes millions of cases of watery or bloody diarrhea annually, resulting in significant global mortality. Watery diarrhea is thought to arise in the jejunum, and subsequent bloody diarrhea occurs as a result of invasion of the colonic epithelium. Previous literature has demonstrated that Shigella encodes enterotoxins, both chromosomally and on the 220 kilobase virulence plasmid. The ShigellaEnterotoxins 1 and 2 (ShET1 and ShET2 have been shown to increase water accumulation in the rabbit ileal loop model. In addition, these toxins increase the short circuit current in rabbit tissue mounted in Ussing chambers, which is a model for the ion exchange that occurs during watery diarrhea. In this study, we sought to validate the use of mouse jejunum in Ussing chamber as an alternative, more versatile model to study bacterial pathogenesis. In the process, we also identified enterotoxins in addition to ShET1 and ShET2 encoded by S. flexneri. Through analysis of proteins secreted from wildtype bacteria and various deletion mutants, we have identified four factors responsible for enterotoxin activity: ShET1 and Pic, which are encoded on the chromosome; ShET2 (encoded by sen or ospD3, which requires the type-III secretion system for secretion; and SepA, an additional factor encoded on the virulence plasmid. The use of mouse jejunum serves as a reliable and reproducible model to identify the enterotoxins elaborated by enteric bacteria. Moreover, the identification of all Shigella proteins responsible for enterotoxin activity is vital to our understanding of Shigella pathogenicity and to our success in developing safe and effective vaccine candidates.

  17. Intracellular drug release nanosystems

    Directory of Open Access Journals (Sweden)

    Fenghua Meng

    2012-10-01

    Full Text Available In order to elicit therapeutic effects, many drugs including small molecule anticancer drugs, proteins, siRNA, and DNA have to be delivered and released into the specific cellular compartments typically the cytoplasm or nucleus of target cells. Intracellular environment-responsive nanosystems that exhibit good extracellular stability while rapidly releasing drugs inside cancer cells have been actively pursued for effective cancer therapy. Here, we highlight novel designs of smart nanosystems that release drugs in response to an intracellular biological signal of cancer cells such as acidic pH in endo/lysosomal compartments, enzymes in lysosomes, and redox potential in cytoplasm and the cell nucleus.

  18. Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

    Science.gov (United States)

    Nasrallah, Gheyath K; Abdelhady, Hany; Tompkins, Nicholas P; Carson, Kaitlyn R; Garduño, Rafael A

    2014-07-01

    L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted.

  19. The Escherichia coli MntR Miniregulon Includes Genes Encoding a Small Protein and an Efflux Pump Required for Manganese Homeostasis ▿†

    OpenAIRE

    Waters, Lauren S.; Sandoval, Melissa; Storz, Gisela

    2011-01-01

    Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA...

  20. Biology and intracellular life of chlamydia

    Directory of Open Access Journals (Sweden)

    Ranin Lazar

    2011-01-01

    Full Text Available Introduction. Chlamydiae are Gram-negative obligate intracellular bacteria. The developmental cycle of Chlamydiae is specific and different from other bacteria. The elementary body is the infectious form of the organism, responsible for attaching to the target host cell and promoting its entry. The reticulate body is the larger, metabolically active form of the organism, synthesizing deoxyribonucleic acid, ribonucleic acid and proteins. The elementary body and reticulate body represent evolutionary adaptations to extracellular and intracellular environments. Intracellular persistence of Chlamydia. Predisposition of Chlamydia to persist within the host cell has been recognized as a major factor in the pathogenesis of chlamydial disease. The persistence implies a long-term association between chlamydiae and their host cell that may not manifest as clinically recognizable disease. The ability of chlamydia to remain within one morphological state for a long time in response to exogenous factors suggests an innate ability of these organisms to persist intracellulary in a unique developmental form. Chlamydiae induce interferon γ and exhibit growth inhibition in their presence. While the high levels of interferon γ completely restrict the development of chlamydia, its low levels induce the development of morphologically aberrant intracellular forms. The persistent forms contain reduced levels of major outer membrane protein but high levels of chlamydial heat shock protein. Conclusion. Immunopathogenesis of chlamydial infection is one of the main focal points of current research into Chlamydia. Chlamydial infections are highly prevalent, usually asymptomatic and associated with serious sequelae. Screening programmes are the most important in the prevention of a long-term sequele.

  1. Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria.

    Science.gov (United States)

    Agostoni, Marco; Waters, Christopher M; Montgomery, Beronda L

    2016-02-01

    The second messenger cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP) has been implicated in the transition between motile and sessile lifestyles in bacteria. In this study, we demonstrate that biofilm formation, cellular aggregation or flocculation, and cellular buoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 (Synechocystis) and Fremyella diplosiphon. Synechocystis is a unicellular cyanobacterium and displays lower levels of c-di-GMP; F. diplosiphon is filamentous and displays higher intracellular c-di-GMP levels. We transformed Synechocystis and F. diplosiphon with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae or Escherichia coli, respectively. These engineered strains allowed us to modulate intracellular c-di-GMP levels. Biofilm formation and cellular deposition were induced in the DGC-expressing Synechocystis strain which exhibited high intracellular levels of c-di-GMP; whereas strains expressing PDE in Synechocystis and F. diplosiphon to drive low intracellular levels of c-di-GMP exhibited enhanced cellular buoyancy. In addition, the PDE-expressing F. diplosiphon strain showed elevated chlorophyll levels. These results imply roles for coordinating c-di-GMP homeostasis in regulating native cyanobacterial phenotypes. Engineering exogenous DGC or PDE proteins to regulate intracellular c-di-GMP levels represents an effective tool for uncovering cryptic phenotypes or modulating phenotypes in cyanobacteria for practical applications in biotechnology applicable in photobioreactors and in green biotechnologies, such as energy-efficient harvesting of cellular biomass or the treatment of metal-containing wastewaters.

  2. Commensal ocular bacteria degrade mucins.

    Science.gov (United States)

    Berry, M; Harris, A; Lumb, R; Powell, K

    2002-12-01

    Antimicrobial activity in tears prevents infection while maintaining a commensal bacterial population. The relation between mucin and commensal bacteria was assessed to determine whether commensals possess mucinolytic activity, how degradation depends on mucin integrity, and whether mucins affect bacterial replication. Bacteria were sampled from healthy eyes and contact lenses from asymptomatic wearers. Intracellular mucins were extracted and purified from cadaver conjunctivas, and surface mucins from extended wear contact lenses. After exposure to bacteria, changes in mucin hydrodynamic volume (proteolytic cleavage) and subunit charge (oligosaccharide degradation) were assayed by size exclusion and ion exchange chromatography. The effect of mucin on bacterial replication was followed for up to 24 hours from the end of incubation with purified ocular mucins. Ocular bacteria decreased the hydrodynamic volume of intracellular and contact lens adherent mucins, irrespective of glycosylation density. A decrease in mucin sialylation was observed after exposure to commensal bacteria. Subunit charge distributions were generally shifted to lesser negative charge, consistent with loss of charged epitopes. Subunits with high negative charge, observed after digesting lightly adhering contact lens mucins with bacteria, suggest preferential cleavage sites in the mucin molecule. The presence of purified ocular mucin in the medium inhibited bacterial growth. Bacteria in the healthy ocular surface possess mucinolytic activity on both intact and surface processed mucins, targeted to discrete sites in the mucin molecule. Inhibition of bacterial growth by ocular mucins can be seen as part of the mucosal control of microbiota.

  3. Conjugal transfer of a virulence plasmid in the opportunistic intracellular actinomycete Rhodococcus equi.

    Science.gov (United States)

    Tripathi, V N; Harding, W C; Willingham-Lane, J M; Hondalus, M K

    2012-12-01

    Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ~81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil.

  4. Cell-penetrating antimicrobial peptides - prospectives for targeting intracellular infections

    DEFF Research Database (Denmark)

    Bahnsen, Jesper S; Franzyk, Henrik; Sayers, Edward J;

    2015-01-01

    . TPk showed the highest antibacterial activity. SA-3 exhibited selective disruption of liposomes mimicking Gram-positive and Gram-negative membranes. CONCLUSION: PK-12-KKP is an unlikely candidate for targeting intracellular bacteria, as the eukaryotic cell-penetrating ability is poor. SA-3, affected...

  5. 'Candidatus Rhabdochlamydia porcellionis', an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda).

    Science.gov (United States)

    Kostanjsek, Rok; Strus, Jasna; Drobne, Damjana; Avgustin, Gorazd

    2004-03-01

    Intracellular bacteria were observed in the hepatopancreas of the terrestrial isopod Porcellio scaber. Comparative 16S rRNA gene sequence analysis and electron microscopic observations were used to determine the taxonomic position of these intracellular bacteria. Phylogenetic analysis and a complex developmental cycle affiliate these bacteria to the order Chlamydiales, within which they form a distinctive lineage, close to the family Simkaniaceae. They share scaber.

  6. Intracellular Symbiotic Bacteria of Camponotus textor, Forel (Hymenoptera, Formicidae).

    Science.gov (United States)

    Ramalho, Manuela O; Martins, Cintia; Silva, Larissa M R; Martins, Vanderlei G; Bueno, Odair C

    2017-05-01

    This study focuses on the weaver ant, Camponotus textor, Forel which occurs in some areas of the Brazilian Cerrado and Atlantic Forest, and its symbionts: Blochmannia, an obligate symbiont of Camponotus, and Wolbachia, known for causing reproductive alterations in their hosts. The main goal of this study was to investigate the presence, frequency of occurrence, and diversity of Wolbachia and Blochmannia strains in C. textor colonies. We found high infection rates (100%) and the occurrence of at least two distinct strains of Blochmannia (H_1 or H_7) in the same species. The observed haplotype variation within a single species may result from the high mutation rate of the symbiont. Similarly, the Wolbachia was found in all colonies with different rates of infections and a new strain (supergroup A) was deposited in the MLST database. The diversity found in the present study shows that there is still much to explore to understand about these symbiotic interactions.

  7. The Escherichia coli MntR Miniregulon Includes Genes Encoding a Small Protein and an Efflux Pump Required for Manganese Homeostasis ▿†

    Science.gov (United States)

    Waters, Lauren S.; Sandoval, Melissa; Storz, Gisela

    2011-01-01

    Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA) is repressed by manganese through MntR and encodes an unannotated 42-amino-acid protein. Overproduction of MntS causes manganese sensitivity, while a lack of MntS perturbs proper manganese-dependent repression of mntH. We also provide evidence that mntP (formerly yebN), which encodes a putative efflux pump, is positively regulated by MntR. Deletion of mntP leads to profound manganese sensitivity and to elevated intracellular manganese levels. This work thus defines two new proteins involved in manganese homeostasis and suggests mechanisms for their action. PMID:21908668

  8. The Escherichia coli MntR miniregulon includes genes encoding a small protein and an efflux pump required for manganese homeostasis.

    Science.gov (United States)

    Waters, Lauren S; Sandoval, Melissa; Storz, Gisela

    2011-11-01

    Manganese is a critical micronutrient for cells, serving as an enzyme cofactor and protecting against oxidative stress. Yet, manganese is toxic in excess and little is known about its distribution in cells. Bacteria control intracellular manganese levels by the transcription regulator MntR. When this work began, the only Escherichia coli K-12 gene known to respond to manganese via MntR repression was mntH, which encodes a manganese importer. We show that mntS (formerly the small RNA gene rybA) is repressed by manganese through MntR and encodes an unannotated 42-amino-acid protein. Overproduction of MntS causes manganese sensitivity, while a lack of MntS perturbs proper manganese-dependent repression of mntH. We also provide evidence that mntP (formerly yebN), which encodes a putative efflux pump, is positively regulated by MntR. Deletion of mntP leads to profound manganese sensitivity and to elevated intracellular manganese levels. This work thus defines two new proteins involved in manganese homeostasis and suggests mechanisms for their action.

  9. Autophagic clearance of bacterial pathogens: molecular recognition of intracellular microorganisms.

    Science.gov (United States)

    Pareja, Maria Eugenia Mansilla; Colombo, Maria I

    2013-01-01

    Autophagy is involved in several physiological and pathological processes. One of the key roles of the autophagic pathway is to participate in the first line of defense against the invasion of pathogens, as part of the innate immune response. Targeting of intracellular bacteria by the autophagic machinery, either in the cytoplasm or within vacuolar compartments, helps to control bacterial proliferation in the host cell, controlling also the spreading of the infection. In this review we will describe the means used by diverse bacterial pathogens to survive intracellularly and how they are recognized by the autophagic molecular machinery, as well as the mechanisms used to avoid autophagic clearance.

  10. Intracellular Cadmium Isotope Fractionation

    Science.gov (United States)

    Horner, T. J.; Lee, R. B.; Henderson, G. M.; Rickaby, R. E.

    2011-12-01

    Recent stable isotope studies into the biological utilization of transition metals (e.g. Cu, Fe, Zn, Cd) suggest several stepwise cellular processes can fractionate isotopes in both culture and nature. However, the determination of fractionation factors is often unsatisfactory, as significant variability can exist - even between different organisms with the same cellular functions. Thus, it has not been possible to adequately understand the source and mechanisms of metal isotopic fractionation. In order to address this problem, we investigated the biological fractionation of Cd isotopes within genetically-modified bacteria (E. coli). There is currently only one known biological use or requirement of Cd, a Cd/Zn carbonic anhydrase (CdCA, from the marine diatom T. weissfloggii), which we introduce into the E. coli genome. We have also developed a cleaning procedure that allows for the treating of bacteria so as to study the isotopic composition of different cellular components. We find that whole cells always exhibit a preference for uptake of the lighter isotopes of Cd. Notably, whole cells appear to have a similar Cd isotopic composition regardless of the expression of CdCA within the E. coli. However, isotopic fractionation can occur within the genetically modified E. coli during Cd use, such that Cd bound in CdCA can display a distinct isotopic composition compared to the cell as a whole. Thus, the externally observed fractionation is independent of the internal uses of Cd, with the largest Cd isotope fractionation occurring during cross-membrane transport. A general implication of these experiments is that trace metal isotopic fractionation most likely reflects metal transport into biological cells (either actively or passively), rather than relating to expression of specific physiological function and genetic expression of different metalloenzymes.

  11. Endocytosis of Viruses and Bacteria

    Science.gov (United States)

    Cossart, Pascale; Helenius, Ari

    2014-01-01

    Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens. PMID:25085912

  12. Intracellular nitrate of marine diatoms as a driver of anaerobic nitrogen cycling in sinking aggregates

    DEFF Research Database (Denmark)

    Kamp, Anja; Stief, Peter; Bristow, Laura A.;

    2016-01-01

    Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species...... store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom......-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly...

  13. The role of autophagy in the intracellular survival of Campylobacter concisus

    Directory of Open Access Journals (Sweden)

    Jose A. Burgos-Portugal

    2014-01-01

    Full Text Available Campylobacter concisus is an emerging pathogen that has been associated with gastrointestinal diseases. Given the importance of autophagy for the elimination of intracellular bacteria and the subversion of this process by pathogenic bacteria, we investigated the role of autophagy in C. concisus intracellular survival. Gentamicin protection assays were employed to assess intracellular levels of C. concisus within Caco-2 cells, following autophagy induction and inhibition. To assess the interaction between C. concisus and autophagosomes, confocal microscopy, scanning electron microscopy, and transmission electron microscopy were employed. Expression levels of 84 genes involved in the autophagy process were measured using qPCR. Autophagy inhibition resulted in two- to four-fold increases in intracellular levels of C. concisus within Caco-2 cells, while autophagy induction resulted in a significant reduction in intracellular levels or bacterial clearance. C. concisus strains with low intracellular survival levels showed a dramatic increase in these levels upon autophagy inhibition. Confocal microscopy showed co-localization of the bacterium with autophagosomes, while transmission electron microscopy identified intracellular bacteria persisting within autophagic vesicles. Further, qPCR showed that following infection, 13 genes involved in the autophagy process were significantly regulated, and a further five showed borderline results, with an overall indication towards a dampening effect exerted by the bacterium on this process. Our data collectively indicates that while autophagy is important for the clearance of C. concisus, some strains may manipulate this process to benefit their intracellular survival.

  14. Pepsin homologues in bacteria

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    Bateman Alex

    2009-09-01

    Full Text Available Abstract Background Peptidase family A1, to which pepsin belongs, had been assumed to be restricted to eukaryotes. The tertiary structure of pepsin shows two lobes with similar folds and it has been suggested that the gene has arisen from an ancient duplication and fusion event. The only sequence similarity between the lobes is restricted to the motif around the active site aspartate and a hydrophobic-hydrophobic-Gly motif. Together, these contribute to an essential structural feature known as a psi-loop. There is one such psi-loop in each lobe, and so each lobe presents an active Asp. The human immunodeficiency virus peptidase, retropepsin, from peptidase family A2 also has a similar fold but consists of one lobe only and has to dimerize to be active. All known members of family A1 show the bilobed structure, but it is unclear if the ancestor of family A1 was similar to an A2 peptidase, or if the ancestral retropepsin was derived from a half-pepsin gene. The presence of a pepsin homologue in a prokaryote might give insights into the evolution of the pepsin family. Results Homologues of the aspartic peptidase pepsin have been found in the completed genomic sequences from seven species of bacteria. The bacterial homologues, unlike those from eukaryotes, do not possess signal peptides, and would therefore be intracellular acting at neutral pH. The bacterial homologues have Thr218 replaced by Asp, a change which in renin has been shown to confer activity at neutral pH. No pepsin homologues could be detected in any archaean genome. Conclusion The peptidase family A1 is found in some species of bacteria as well as eukaryotes. The bacterial homologues fall into two groups, one from oceanic bacteria and one from plant symbionts. The bacterial homologues are all predicted to be intracellular proteins, unlike the eukaryotic enzymes. The bacterial homologues are bilobed like pepsin, implying that if no horizontal gene transfer has occurred the duplication

  15. LEA (Late Embryogenesis Abundant proteins and their encoding genes in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Hincha Dirk K

    2008-03-01

    Full Text Available Abstract Background LEA (late embryogenesis abundant proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown. Results We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE and/or low temperature response (LTRE elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded. Conclusion The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for

  16. Big bacteria

    DEFF Research Database (Denmark)

    Schulz, HN; Jørgensen, BB

    2001-01-01

    A small number of prokaryotic species have a unique physiology or ecology related to their development of unusually large size. The biomass of bacteria varies over more than 10 orders of magnitude, from the 0.2 mum wide nanobacteria to the largest cells of the colorless sulfur bacteria......, Thiomargarita namibiensis, with a diameter of 750 mum. All bacteria, including those that swim around in the environment, obtain their food molecules by molecular diffusion. Only the fastest and largest swimmers known, Thiovulum majus, are able to significantly increase their food supply by motility...... and by actively creating an advective flow through the entire population. Diffusion limitation generally restricts the maximal size of prokaryotic cells and provides a selective advantage for mum-sized cells at the normally low substrate concentrations in the environment. The largest heterotrophic bacteria...

  17. Anaerobic bacteria

    Science.gov (United States)

    Brook I, Goldstein EJ. Diseases caused by non-spore forming anaerobic bacteria. In: Goldman L, Schafer AI, eds. Goldman's Cecil Medicine . 25th ed. Philadelphia, PA: Elsevier Saunders; 2015:chap 297. Stedman's Online ...

  18. The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" reveals common mechanisms for host cell interaction among amoeba-associated bacteria.

    Science.gov (United States)

    Schmitz-Esser, Stephan; Tischler, Patrick; Arnold, Roland; Montanaro, Jacqueline; Wagner, Michael; Rattei, Thomas; Horn, Matthias

    2010-02-01

    Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of "Candidatus Amoebophilus asiaticus," an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. "Ca. Amoebophilus asiaticus" encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of "Ca. Amoebophilus asiaticus". More generally, we show that the eukaryotic domains identified in "Ca. Amoebophilus asiaticus" are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.

  19. The destructive citrus pathogen, 'Candidatus Liberibacter asiaticus' encodes a functional flagellin characteristic of a pathogen-associated molecular pattern.

    Directory of Open Access Journals (Sweden)

    Huasong Zou

    Full Text Available Huanglongbing (HLB is presently the most devastating citrus disease worldwide. As an intracellular plant pathogen and insect symbiont, the HLB bacterium, 'Candidatus Liberibacter asiaticus' (Las, retains the entire flagellum-encoding gene cluster in its significantly reduced genome. Las encodes a flagellin and hook-associated protein (Fla of 452 amino acids that contains a conserved 22 amino acid domain (flg22 at positions 29 to 50 in the N-terminus. The phenotypic alteration in motility of a Sinorhizobium meliloti mutant lacking the fla genes was partially restored by constitutive expression of Fla(Las. Agrobacterium-mediated transient expression in planta revealed that Fla(Las induced cell death and callose deposition in Nicotiana benthamiana, and that the transcription of BAK1 and SGT1, which are associated with plant innate immunity, was upregulated. Amino acid substitution experiments revealed that residues 38 (serine and 39 (aspartate of Fla(Las were essential for callose induction. The synthetic flg22(Las peptide could not induce plant cell death but retained the ability to induce callose deposition at a concentration of 20 µM or above. This demonstrated that the pathogen-associated molecular pattern (PAMP activity of flg22 in Las was weaker than those in other well-studied plant pathogenic bacteria. These results indicate that Fla(Las acts as a PAMP and may play an important role in triggering host plant resistance to the HLB bacteria.

  20. Mucolipins: Intracellular TRPML1-3 channels.

    Science.gov (United States)

    Cheng, Xiping; Shen, Dongbiao; Samie, Mohammad; Xu, Haoxing

    2010-05-17

    The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal "recycling center" for biological "garbage", are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously overexpressed have revealed the channel properties of TRPMLs in mediating cation (Ca(2+)/Fe(2+)) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration.

  1. Ascorbate recycling in human neutrophils: Induction by bacteria

    OpenAIRE

    Wang, Yaohui; Russo, Thomas A.; Kwon, Oran; Chanock, Stephen; Rumsey, Steven C.; Levine, Mark

    1997-01-01

    Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by measuring intracellular ascorbate accumulation. Ascorbate recycling in neutrophils was induced by both Gram-positive and Gram-negative pathogenic bacteria, and the fungal pathogen C...

  2. Characterization of a Mycobacterium intracellulare Variant Strain by Molecular Techniques

    Science.gov (United States)

    Menendez, M. C.; Palenque, E.; Navarro, M. C.; Nuñez, M. C.; Rebollo, M. J.; Garcia, M. J.

    2001-01-01

    This paper describes a Mycobacterium intracellulare variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the Mycobacterium avium complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as mig (macrophage-induced gene). The results obtained identify the isolates as a variant of M. intracellulare, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members. PMID:11724827

  3. Characterization of a Mycobacterium intracellulare variant strain by molecular techniques.

    Science.gov (United States)

    Menendez, M C; Palenque, E; Navarro, M C; Nuñez, M C; Rebollo, M J; Garcia, M J

    2001-12-01

    This paper describes a Mycobacterium intracellulare variant strain causing an unusual infection. Several isolates obtained from an immunocompromised patient were identified as members of the Mycobacterium avium complex (MAC) by the commercial AccuProbe system and biochemical standard identification. Further molecular approaches were undertaken for a more accurate characterization of the bacteria. Up to seven different genomic sequences were analyzed, ranging from conserved mycobacterial genes such as 16S ribosomal DNA to MAC-specific genes such as mig (macrophage-induced gene). The results obtained identify the isolates as a variant of M. intracellulare, an example of the internal variability described for members of the MAC, particularly within that species. The application of other molecular approaches is recommended for more accurate identification of bacteria described as MAC members.

  4. Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

    Science.gov (United States)

    Lehar, Sophie M; Pillow, Thomas; Xu, Min; Staben, Leanna; Kajihara, Kimberly K; Vandlen, Richard; DePalatis, Laura; Raab, Helga; Hazenbos, Wouter L; Morisaki, J Hiroshi; Kim, Janice; Park, Summer; Darwish, Martine; Lee, Byoung-Chul; Hernandez, Hilda; Loyet, Kelly M; Lupardus, Patrick; Fong, Rina; Yan, Donghong; Chalouni, Cecile; Luis, Elizabeth; Khalfin, Yana; Plise, Emile; Cheong, Jonathan; Lyssikatos, Joseph P; Strandh, Magnus; Koefoed, Klaus; Andersen, Peter S; Flygare, John A; Wah Tan, Man; Brown, Eric J; Mariathasan, Sanjeev

    2015-11-19

    Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

  5. Identification and functional characterization of K+ transporters encoded by Legionella pneumophila kup genes

    OpenAIRE

    Hori, Juliana I.; Pereira, Marcelo S. F.; Roy, Craig R.; Nagai, Hiroki; Zamboni, Dario S.

    2013-01-01

    Legionnaires’ disease is an emerging, severe, pneumonia-like illness caused by the Gram-negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the E. coli K+ transporters. These three gen...

  6. Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga.

    Science.gov (United States)

    Lamothe, Julie; Thyssen, Sandra; Valvano, Miguel A

    2004-12-01

    We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba. In this work, we show that the clinical isolates B. vietnamiensis strain CEP040 and B. cenocepacia H111 survived but did not replicate within vacuoles of A. polyphaga. B. cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH. In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue. Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h. In contrast, Escherichia coli did not survive within amoebae after 2 h post infection. Furthermore, intracellular B. vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen. Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact. In addition, both Legionella pneumophila- and B. vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome. Thus, our observations support the notion that B. cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.

  7. Intracellular Nitrate of Marine Diatoms as a Driver of Anaerobic Nitrogen Cycling in Sinking Aggregates

    Directory of Open Access Journals (Sweden)

    Anja Kamp

    2016-11-01

    Full Text Available Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations > 60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2-3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.

  8. Big bacteria

    DEFF Research Database (Denmark)

    Schulz, HN; Jørgensen, BB

    2001-01-01

    , Thiomargarita namibiensis, with a diameter of 750 mum. All bacteria, including those that swim around in the environment, obtain their food molecules by molecular diffusion. Only the fastest and largest swimmers known, Thiovulum majus, are able to significantly increase their food supply by motility......, the 80 x 600 mum large Epulopiscium sp. from the gut of tropical fish, are presumably living in a very nutrient-rich medium. Many large bacteria contain numerous inclusions in the cells that reduce the volume of active cytoplasm. The most striking examples of competitive advantage from large cell size...

  9. Exploring anti-bacterial compounds against intracellular Legionella.

    Directory of Open Access Journals (Sweden)

    Christopher F Harrison

    Full Text Available Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.

  10. Exploring Anti-Bacterial Compounds against Intracellular Legionella

    Science.gov (United States)

    Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

    2013-01-01

    Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

  11. Iron metabolism and resistance to infection by invasive bacteria in the social amoeba Dictyostelium discoideum.

    Science.gov (United States)

    Bozzaro, Salvatore; Buracco, Simona; Peracino, Barbara

    2013-01-01

    Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. Starvation triggers a change in life style, forcing cells to gather into aggregates to form multicellular organisms capable of cell differentiation and morphogenesis. As a soil amoeba and a phagocyte that grazes on bacteria as the obligate source of food, Dictyostelium could be a natural host of pathogenic bacteria. Indeed, many pathogens that occasionally infect humans are hosted for most of their time in protozoa or free-living amoebae, where evolution of their virulence traits occurs. Due to these features and its amenability to genetic manipulation, Dictyostelium has become a valuable model organism for studying strategies of both the host to resist infection and the pathogen to escape the defense mechanisms. Similarly to higher eukaryotes, iron homeostasis is crucial for Dictyostelium resistance to invasive bacteria. Iron is essential for Dictyostelium, as both iron deficiency or overload inhibit cell growth. The Dictyostelium genome shares with mammals many genes regulating iron homeostasis. Iron transporters of the Nramp (Slc11A) family are represented with two genes, encoding Nramp1 and Nramp2. Like the mammalian ortholog, Nramp1 is recruited to phagosomes and macropinosomes, whereas Nramp2 is a membrane protein of the contractile vacuole network, which regulates osmolarity. Nramp1 and Nramp2 localization in distinct compartments suggests that both proteins synergistically regulate iron homeostasis. Rather than by absorption via membrane transporters, iron is likely gained by degradation of ingested bacteria and efflux via Nramp1 from phagosomes to the cytosol. Nramp gene disruption increases Dictyostelium sensitivity to infection, enhancing intracellular growth of Legionella or Mycobacteria. Generation of mutants in other "iron genes" will help identify genes essential for iron homeostasis and resistance to pathogens.

  12. Iron metabolism and resistance to infection by invasive bacteria in the social amoeba Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Salvatore eBozzaro

    2013-09-01

    Full Text Available Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. Starvation triggers a change in life style, forcing cells to gather into aggregates to form multicellular organisms capable of cell differentiation and morphogenesis. As a soil amoeba and a phagocyte that grazes on bacteria as the obligate source of food, Dictyostelium could be a natural host of pathogenic bacteria. Indeed, many pathogens that occasionally infect humans are hosted for most of their time in protozoa or free-living amoebae, where evolution of their virulence traits occurs. Due to these features and its amenability to genetic manipulation, Dictyostelium has become a valuable model organism for studying strategies of both the host to resist infection and the pathogen to escape the defence mechanisms. Similarly to higher eukaryotes, iron homeostasis is crucial for Dictyostelium resistance to invasive bacteria. Iron is essential for Dictyostelium, as both iron deficiency or overload inhibit cell growth. The Dictyostelium genome shares with mammals many genes regulating iron homeostasis. Iron transporters of the Nramp (Slc11A family are represented with two genes, encoding Nramp1 and Nramp2. Like the mammalian ortholog, Nramp1 is recruited to phagosomes and macropinosomes, whereas Nramp2 is a membrane protein of the contractile vacuole network, which regulates osmolarity. Nramp1 and Nramp2 localization in distinct compartments suggests that both proteins synergistically regulate iron homeostasis. Rather than by absorption via membrane transporters, iron is likely gained by degradation of ingested bacteria and efflux via Nramp1 from phagosomes to the cytosol. Nramp gene disruption increases Dictyostelium sensitivity to infection, enhancing intracellular growth of Legionella or Mycobacteria. Generation of mutants in other "iron genes" will help identify genes essential for iron homeostasis and resistance to

  13. The link between independent acquisition of intracellular gamma-endosymbionts and concerted evolution in Tremblaya princeps

    Directory of Open Access Journals (Sweden)

    Sergio eLópez-Madrigal

    2015-06-01

    Full Text Available Many insect species establish mutualistic symbiosis with intracellular bacteria that complement their unbalanced diets. The betaproteobacterium Candidatus Tremblaya maintains an ancient symbiosis with mealybugs (Hemiptera: Pseudococcidae, which are classified in subfamilies Phenacoccinae and Pseudococcinae. Most Phenacoccinae mealybugs have Candidatus Tremblaya phenacola as their unique endosymbiont, while most Pseudococcinae mealybugs show a nested symbiosis (a bacterial symbiont placed inside another one where every Candidatus Tremblaya princeps cell harbors several cells of a gammaproteobacterium. Genomic characterization of the endosymbiotic consortium from Planococcus citri, composed by Ca. Tremblaya princeps and Candidatus Moranella endobia, unveiled several atypical features of the former’s genome, including the concerted evolution of paralogous loci. Its comparison with the genome of Ca. Tremblaya phenacola PAVE, single endosymbiont of Phenacoccus avenae, suggests that the atypical reductive evolution of Ca. Tremblaya princeps could be linked to the acquisition of Ca. Moranella endobia, which possess an almost complete set of genes encoding proteins involved in homologous recombination. In order to test this hypothesis, we performed comparative genomics between Ca. Tremblaya phenacola and Ca. Tremblaya princeps and searched for the co-occurrence of concerted evolution and homologous recombination genes in endosymbiotic consortia from four unexplored mealybug species, Dysmicoccus boninsis, Planococcus ficus, Pseudococcus longispinus and Pseudococcus viburni. Our results support a link between concerted evolution and nested endosymbiosis.

  14. The link between independent acquisition of intracellular gamma-endosymbionts and concerted evolution in Tremblaya princeps

    Science.gov (United States)

    López-Madrigal, Sergio; Latorre, Amparo; Moya, Andrés; Gil, Rosario

    2015-01-01

    Many insect species establish mutualistic symbiosis with intracellular bacteria that complement their unbalanced diets. The betaproteobacterium “Candidatus Tremblaya” maintains an ancient symbiosis with mealybugs (Hemiptera: Pseudococcidae), which are classified in subfamilies Phenacoccinae and Pseudococcinae. Most Phenacoccinae mealybugs have “Candidatus Tremblaya phenacola” as their unique endosymbiont, while most Pseudococcinae mealybugs show a nested symbiosis (a bacterial symbiont placed inside another one) where every “Candidatus Tremblaya princeps” cell harbors several cells of a gammaproteobacterium. Genomic characterization of the endosymbiotic consortium from Planococcus citri, composed by “Ca. Tremblaya princeps” and “Candidatus Moranella endobia,” unveiled several atypical features of the former's genome, including the concerted evolution of paralogous loci. Its comparison with the genome of “Ca. Tremblaya phenacola” PAVE, single endosymbiont of Phenacoccus avenae, suggests that the atypical reductive evolution of “Ca. Tremblaya princeps” could be linked to the acquisition of “Ca. Moranella endobia,” which possess an almost complete set of genes encoding proteins involved in homologous recombination. In order to test this hypothesis, we performed comparative genomics between “Ca. Tremblaya phenacola” and “Ca. Tremblaya princeps” and searched for the co-occurrence of concerted evolution and homologous recombination genes in endosymbiotic consortia from four unexplored mealybug species, Dysmicoccus boninsis, Planococcus ficus, Pseudococcus longispinus, and Pseudococcus viburni. Our results support a link between concerted evolution and nested endosymbiosis. PMID:26161080

  15. The link between independent acquisition of intracellular gamma-endosymbionts and concerted evolution in Tremblaya princeps.

    Science.gov (United States)

    López-Madrigal, Sergio; Latorre, Amparo; Moya, Andrés; Gil, Rosario

    2015-01-01

    Many insect species establish mutualistic symbiosis with intracellular bacteria that complement their unbalanced diets. The betaproteobacterium "Candidatus Tremblaya" maintains an ancient symbiosis with mealybugs (Hemiptera: Pseudococcidae), which are classified in subfamilies Phenacoccinae and Pseudococcinae. Most Phenacoccinae mealybugs have "Candidatus Tremblaya phenacola" as their unique endosymbiont, while most Pseudococcinae mealybugs show a nested symbiosis (a bacterial symbiont placed inside another one) where every "Candidatus Tremblaya princeps" cell harbors several cells of a gammaproteobacterium. Genomic characterization of the endosymbiotic consortium from Planococcus citri, composed by "Ca. Tremblaya princeps" and "Candidatus Moranella endobia," unveiled several atypical features of the former's genome, including the concerted evolution of paralogous loci. Its comparison with the genome of "Ca. Tremblaya phenacola" PAVE, single endosymbiont of Phenacoccus avenae, suggests that the atypical reductive evolution of "Ca. Tremblaya princeps" could be linked to the acquisition of "Ca. Moranella endobia," which possess an almost complete set of genes encoding proteins involved in homologous recombination. In order to test this hypothesis, we performed comparative genomics between "Ca. Tremblaya phenacola" and "Ca. Tremblaya princeps" and searched for the co-occurrence of concerted evolution and homologous recombination genes in endosymbiotic consortia from four unexplored mealybug species, Dysmicoccus boninsis, Planococcus ficus, Pseudococcus longispinus, and Pseudococcus viburni. Our results support a link between concerted evolution and nested endosymbiosis.

  16. The Golden Ratio Encoder

    CERN Document Server

    Daubechies, I; Wang, Y; Yilmaz, Ö

    2008-01-01

    This paper proposes a novel Nyquist-rate analog-to-digital (A/D) conversion algorithm which achieves exponential accuracy in the bit-rate despite using imperfect components. The proposed algorithm is based on a robust implementation of a beta-encoder where the value of the base beta is equal to golden mean. It was previously shown that beta-encoders can be implemented in such a way that their exponential accuracy is robust against threshold offsets in the quantizer element. This paper extends this result by allowing for imperfect analog multipliers with imprecise gain values as well. A formal computational model for algorithmic encoders and a general test bed for evaluating their robustness is also proposed.

  17. Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection.

    Science.gov (United States)

    Robey, M; O'Connell, W; Cianciotto, N P

    2001-07-01

    In the course of characterizing a locus involved in heme utilization, we identified a Legionella pneumophila gene predicted to encode a protein with homology to the product of the Salmonella enterica serovar Typhimurium pagP gene. In Salmonella, pagP increases resistance to the bactericidal effects of cationic antimicrobial peptides (CAMPs). Mutants with insertions in the L. pneumophila pagP-like gene were generated and showed decreased resistance to different structural classes of CAMPs compared to the wild type; hence, this gene was designated rcp for resistance to cationic antimicrobial peptides. Furthermore, Legionella CAMP resistance was induced by growth in low-magnesium medium. To determine whether rcp had any role in intracellular survival, mutants were tested in the two most relevant host cells for Legionnaires' disease, i.e., amoebae and macrophages. These mutants exhibited a 1,000-fold-decreased recovery during a Hartmannella vermiformis coculture. Complementation of the infectivity defect could be achieved by introduction of a plasmid containing the intact rcp gene. Mutations in rcp consistently reduced both the numbers of bacteria recovered during intracellular infection and their cytopathic capacity for U937 macrophages. The rcp mutant was also more defective for lung colonization of A/J mice. Growth of rcp mutants in buffered yeast extract broth was identical to that of the wild type, indicating that the observed differences in numbers of bacteria recovered from host cells were not due to a generalized growth defect. However, in low-Mg(2+) medium, the rcp mutant was impaired in stationary-phase survival. This is the first demonstration of a pagP-like gene, involved in resistance to CAMPs, being required for intracellular infection and virulence.

  18. Metallochaperones regulate intracellular copper levels.

    Directory of Open Access Journals (Sweden)

    W Lee Pang

    Full Text Available Copper (Cu is an important enzyme co-factor that is also extremely toxic at high intracellular concentrations, making active efflux mechanisms essential for preventing Cu accumulation. Here, we have investigated the mechanistic role of metallochaperones in regulating Cu efflux. We have constructed a computational model of Cu trafficking and efflux based on systems analysis of the Cu stress response of Halobacterium salinarum. We have validated several model predictions via assays of transcriptional dynamics and intracellular Cu levels, discovering a completely novel function for metallochaperones. We demonstrate that in addition to trafficking Cu ions, metallochaperones also function as buffers to modulate the transcriptional responsiveness and efficacy of Cu efflux. This buffering function of metallochaperones ultimately sets the upper limit for intracellular Cu levels and provides a mechanistic explanation for previously observed Cu metallochaperone mutation phenotypes.

  19. Uniform designation for genes of the Calvin-Benson-Bassham reductive pentose phosphate pathway of bacteria

    NARCIS (Netherlands)

    Tabita, F. Robert; Gibson, Janet L.; Bowien, Botho; Dijkhuizen, Lubbert; Meijer, Wilhelmus

    1992-01-01

    Structural and regulatory genes encoding enzymes and proteins of the reductive pentose phosphate pathway have been isolated from a number of bacteria recently. In the phototroph Rhodobacter sphaeroides, and in two chemoautotrophic bacteria, Alcaligenes eutrophus and Xanthobacter flavus, these genes

  20. Genetically Encoded Sensors for Metabolites

    Science.gov (United States)

    Deuschle, Karen; Fehr, Marcus; Hilpert, Melanie; Lager, Ida; Lalonde, Sylvie; Looger, Loren L.; Okumoto, Sakiko; Persson, Jörgen; Schmidt, Anja; Frommer, Wolf B.

    2009-01-01

    Background Metabolomics, i.e., the multiparallel analysis of metabolite changes occurring in a cell or an organism, has become feasible with the development of highly efficient mass spectroscopic technologies. Functional genomics as a standard tool helped to identify the function of many of the genes that encode important transporters and metabolic enzymes over the past few years. Advanced expression systems and analysis technologies made it possible to study the biochemical properties of the corresponding proteins in great detail. We begin to understand the biological functions of the gene products by systematic analysis of mutants using systematic PTGS/RNAi, knockout and TILLING approaches. However, one crucial set of data especially relevant in the case of multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of metabolite levels at cellular and subcellular levels. Methods We therefore developed genetically encoded nanosensors for several metabolites to provide a basic set of tools for the determination of cytosolic and subcellular metabolite levels in real time by using fluorescence microscopy. Results Prototypes of these sensors were successfully used in vitro and also in vivo, i.e., to measure sugar levels in fungal and animal cells. Conclusions One of the future goals will be to expand the set of sensors to a wider spectrum of substrates by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets in conjunction with mutagenesis. This toolbox can then be applied for four-dimensional imaging of cells and tissues to elucidate the spatial and temporal distribution of metabolites as a discovery tool in functional genomics, as a tool for high-throughput, high-content screening for drugs, to test metabolic models, and to analyze the interplay of cells in a tissue or organ. PMID:15688353

  1. Intracellular calcium release modulates polycystin-2 trafficking

    Directory of Open Access Journals (Sweden)

    Miyakawa Ayako

    2013-02-01

    Full Text Available Abstract Background Polycystin-2 (PC2, encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD, functions as a calcium (Ca2+ permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells. Methods We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy. Results We demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca2+ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R inhibition with 2-aminoethoxydiphenyl borate (2-APB or Ca2+/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM. Conclusions These novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.

  2. Negative Beta Encoder

    CERN Document Server

    Kohda, Tohru; Aihara, Kazuyuki

    2008-01-01

    A new class of analog-digital (A/D), digital-analog (D/A) converters as an alternative to conventional ones, called $\\beta$-encoder, has been shown to have exponential accuracy in the bit rates while possessing self-correction property for fluctuations of amplifier factor $\\beta$ and quantizer threshold $\

  3. DNA sequences encoding erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Lin, F.K.

    1987-10-27

    A purified and isolated DNA sequence is described consisting essentially of a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of that of erythropoietin to allow possession of the biological property of causing bone marrow cells to increase production of reticulocytes and red blood cells, and to increase hemoglobin synthesis or iron uptake.

  4. Time-Encoded Imagers.

    Energy Technology Data Exchange (ETDEWEB)

    Marleau, Peter; Brubaker, Erik

    2014-11-01

    This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.

  5. Identification of flgZ as a flagellar gene encoding a PilZ domain protein that regulates swimming motility and biofilm formation in Pseudomonas.

    Science.gov (United States)

    Martínez-Granero, Francisco; Navazo, Ana; Barahona, Emma; Redondo-Nieto, Miguel; González de Heredia, Elena; Baena, Irene; Martín-Martín, Irene; Rivilla, Rafael; Martín, Marta

    2014-01-01

    Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

  6. Identification of flgZ as a flagellar gene encoding a PilZ domain protein that regulates swimming motility and biofilm formation in Pseudomonas.

    Directory of Open Access Journals (Sweden)

    Francisco Martínez-Granero

    Full Text Available Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC and one phosphodiesterase (BifA implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

  7. Detection of intracellular bacterial communities in human urinary tract infection.

    Directory of Open Access Journals (Sweden)

    David A Rosen

    2007-12-01

    Full Text Available BACKGROUND: Urinary tract infections (UTIs are one of the most common bacterial infections and are predominantly caused by uropathogenic Escherichia coli (UPEC. While UTIs are typically considered extracellular infections, it has been recently demonstrated that UPEC bind to, invade, and replicate within the murine bladder urothelium to form intracellular bacterial communities (IBCs. These IBCs dissociate and bacteria flux out of bladder facet cells, some with filamentous morphology, and ultimately establish quiescent intracellular reservoirs that can seed recurrent infection. This IBC pathogenic cycle has not yet been investigated in humans. In this study we sought to determine whether evidence of an IBC pathway could be found in urine specimens from women with acute UTI. METHODS AND FINDINGS: We collected midstream, clean-catch urine specimens from 80 young healthy women with acute uncomplicated cystitis and 20 asymptomatic women with a history of UTI. Investigators were blinded to culture results and clinical history. Samples were analyzed by light microscopy, immunofluorescence, and electron microscopy for evidence of exfoliated IBCs and filamentous bacteria. Evidence of IBCs was found in 14 of 80 (18% urines from women with UTI. Filamentous bacteria were found in 33 of 80 (41% urines from women with UTI. None of the 20 urines from the asymptomatic comparative group showed evidence of IBCs or filaments. Filamentous bacteria were present in all 14 of the urines with IBCs compared to 19 (29% of 66 samples with no evidence of IBCs (p < 0.001. Of 65 urines from patients with E. coli infections, 14 (22% had evidence of IBCs and 29 (45% had filamentous bacteria, while none of the gram-positive infections had IBCs or filamentous bacteria. CONCLUSIONS: The presence of exfoliated IBCs and filamentous bacteria in the urines of women with acute cystitis suggests that the IBC pathogenic pathway characterized in the murine model may occur in humans. The

  8. Intracellular Staphylococcus aureus Control by Virulent Bacteriophages within MAC-T Bovine Mammary Epithelial Cells.

    Science.gov (United States)

    Zhang, Lili; Sun, Lichang; Wei, Ruicheng; Gao, Qiang; He, Tao; Xu, Cunfa; Liu, Xianjin; Wang, Ran

    2017-02-01

    Bacteriophages (phages) are known to effectively kill extracellular multiplying bacteria. The present study demonstrated that phages penetrated bovine mammary epithelial cells and cleared intracellular Staphylococcus aureus in a time-dependent manner. In particular, phage vB_SauM_JS25 reached the nucleus within 3 h postincubation. The phages had an endocytotic efficiency of 12%. This ability to kill intracellular host bacteria suggests the utility of phage-based therapies and may protect patients from recurrent infection and treatment failure. Copyright © 2017 American Society for Microbiology.

  9. A first step toward liposome-mediated intracellular bacteriophage therapy.

    Science.gov (United States)

    Nieth, Anita; Verseux, Cyprien; Barnert, Sabine; Süss, Regine; Römer, Winfried

    2015-01-01

    The emergence of antibiotic-resistant bacteria presents a severe challenge to medicine and public health. While bacteriophage therapy is a promising alternative to traditional antibiotics, the general inability of bacteriophages to penetrate eukaryotic cells limits their use against resistant bacteria, causing intracellular diseases like tuberculosis. Bacterial vectors show some promise in carrying therapeutic bacteriophages into cells, but also bring a number of risks like an overload of bacterial antigens or the acquisition of virulence genes from the pathogen. As a first step in the development of a non-bacterial vector for bacteriophage delivery into pathogen-infected cells, we attempted to encapsulate bacteriophages into liposomes. Here we report effective encapsulation of the model bacteriophage λeyfp and the mycobacteriophage TM4 into giant liposomes. Furthermore, we show that liposome-associated bacteriophages are taken up into eukaryotic cells more efficiently than free bacteriophages. These are important milestones in the development of an intracellular bacteriophage therapy that might be useful in the fight against multi-drug-resistant intracellular pathogens like Mycobacterium tuberculosis.

  10. Nucleic acid compositions and the encoding proteins

    Science.gov (United States)

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  11. Mycobacterium tuberculosis RNA expression patterns in sputum bacteria indicate secreted Esx factors contributing to growth are highly expressed in active disease

    Directory of Open Access Journals (Sweden)

    Archana eBukka

    2012-01-01

    Full Text Available To identify factors contributing to the ability of tubercle bacilli to grow in the lung during active infection, we analyzed RNA expression patterns in bacteria present in patient sputum. Prominent among bacterial transcripts identified were those encoding secreted peptides of the Esat-6 subfamily that includes EsxK and EsxL (Rv1197 and Rv1198. H37Rv esxKL and esxJI transcripts were differentially expressed under different growth conditions, and disruption of these genes altered growth phase kinetics in typical laboratory batch broth cultures. These growth defects, including the reduced intracellular growth of an ΔesxKL mutant in primary human macrophages, were reversed by either low multiplicity co-infection or co-culture with wild-type bacteria, demonstrating the ability of the secreted factors to rescue isogenic mutants. Complementing either only esxL or esxI alone (Rv1198 or Rv1037c also reduced observed growth defects, indicating these genes encode factors capable of contributing to growth. Our studies indicate that the M. tuberculosis Mtb9.9 family secreted factors EsxL and EsxI can act in trans to modulate growth of intracellular bacteria, and are highly expressed during active human lung infection. EsxL (Rv1197 and Rv1198. The H37Rv genome contains 4 additional and nearly identical pairs of co-linear open reading frames designated esx JI, esx MN, esx PO, and esxWV. These ORFs show little sequence similarity to esxBA (Cfp10-Esat-6, other than encoding 2 short ~100 residue peptides with the 5’ ORF encoding a variant carboxyl-terminal 'QILSS' motif and the 3’ encoding the Mtb9.9 family of secreted T-cell antigens. All contain a central ‘WXG100’ esx family structural motif, and are thought to encode effectors of an uncharacterized ESX-5 transport system. esxKL and esxJI transcripts were differentially expressed under different growth conditions, and disruption of these genes altered different growth phase kinetics in typical

  12. Replication Restart in Bacteria.

    Science.gov (United States)

    Michel, Bénédicte; Sandler, Steven J

    2017-07-01

    In bacteria, replication forks assembled at a replication origin travel to the terminus, often a few megabases away. They may encounter obstacles that trigger replisome disassembly, rendering replication restart from abandoned forks crucial for cell viability. During the past 25 years, the genes that encode replication restart proteins have been identified and genetically characterized. In parallel, the enzymes were purified and analyzed in vitro, where they can catalyze replication initiation in a sequence-independent manner from fork-like DNA structures. This work also revealed a close link between replication and homologous recombination, as replication restart from recombination intermediates is an essential step of DNA double-strand break repair in bacteria and, conversely, arrested replication forks can be acted upon by recombination proteins and converted into various recombination substrates. In this review, we summarize this intense period of research that led to the characterization of the ubiquitous replication restart protein PriA and its partners, to the definition of several replication restart pathways in vivo, and to the description of tight links between replication and homologous recombination, responsible for the importance of replication restart in the maintenance of genome stability. Copyright © 2017 American Society for Microbiology.

  13. Activity of 10 antimicrobial agents against intracellular Rhodococcus equi.

    Science.gov (United States)

    Giguère, Steeve; Berghaus, Londa J; Lee, Elise A

    2015-08-05

    Studies with facultative intracellular bacterial pathogens have shown that evaluation of the bactericidal activity of antimicrobial agents against intracellular bacteria is more closely associated with in vivo efficacy than traditional in vitro susceptibility testing. The objective of this study was to determine the relative activity of 10 antimicrobial agents against intracellular Rhodococcus equi. Equine monocyte-derived macrophages were infected with virulent R. equi and exposed to erythromycin, clarithromycin, azithromycin, rifampin, ceftiofur, gentamicin, enrofloxacin, vancomycin, imipenem, or doxycycline at concentrations achievable in plasma at clinically recommended dosages in foals. The number of intracellular R. equi was determined 48h after infection by counting colony forming units (CFUs). The number of R. equi CFUs in untreated control wells were significantly higher than those of monolayers treated with antimicrobial agents. Numbers of R. equi were significantly lower in monolayers treated with enrofloxacin followed by those treated with gentamicin, and vancomycin, when compared to monolayers treated with other antimicrobial agents. Numbers of R. equi in monolayers treated with doxycycline were significantly higher than those of monolayers treated with other antimicrobial agents. Differences in R. equi CFUs between monolayers treated with other antimicrobial agents were not statistically significant. Enrofloxacin, gentamicin, and vancomycin are the most active drugs in equine monocyte-derived macrophages infected with R. equi. Additional studies will be needed to determine if these findings correlate with in vivo efficacy. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Dual Readout BRET/FRET Sensors for Measuring Intracellular Zinc

    Science.gov (United States)

    2016-01-01

    Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25–30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time. PMID:27547982

  15. Multi-species bacterial biofilm and intracellular infection in otitis media

    Directory of Open Access Journals (Sweden)

    Thornton Ruth B

    2011-10-01

    Full Text Available Abstract Background Bacteria which are metabolically active yet unable to be cultured and eradicated by antibiotic treatment are present in the middle ear effusion of children with chronic otitis media with effusion (COME and recurrent acute otitis media (rAOM. These observations are suggestive of biofilm presence or intracellular sequestration of bacteria and may play a role in OM pathogenesis. The aim of this project is to provide evidence for the presence of otopathogenic bacteria intracellularly or within biofilm in the middle ear mucosa of children with COME or rAOM. Methods Middle ear mucosal biopsies from 20 children with COME or rAOM were examined for otopathogenic bacteria (either in biofilm or located intracellularly using transmission electron microscopy (TEM or species specific fluorescent in situ hybridisation (FISH and confocal laser scanning microscopy (CLSM. One healthy control biopsy from a child undergoing cochlear implant surgery was also examined. Results No bacteria were observed in the healthy control sample. In 2 of the 3 biopsies imaged using TEM, bacteria were observed in mucus containing vacuoles within epithelial cells. Bacterial species within these could not be identified and biofilm was not observed. Using FISH with CLSM, bacteria were seen in 15 of the 17 otitis media mucosal specimens. In this group, 11 (65% of the 17 middle ear mucosal biopsies showed evidence of bacterial biofilm and 12 demonstrated intracellular bacteria. 52% of biopsies were positive for both biofilm and intracellular bacteria. At least one otopathogen was identified in 13 of the 15 samples where bacteria were present. No differences were observed between biopsies from children with COME and those with rAOM. Conclusion Using FISH and CLSM, bacterial biofilm and intracellular infection with known otopathogens are demonstrated on/in the middle ear mucosa of children with COME and/or rAOM. While their role in disease pathogenesis remains to be

  16. Stochastic models of intracellular transport

    KAUST Repository

    Bressloff, Paul C.

    2013-01-09

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

  17. Measuring intracellular redox conditions using GFP-based sensors

    DEFF Research Database (Denmark)

    Björnberg, Olof; Ostergaard, Henrik; Winther, Jakob R

    2006-01-01

    Recent years have seen the development of methods for analyzing the redox conditions in specific compartments in living cells. These methods are based on genetically encoded sensors comprising variants of Green Fluorescent Protein in which vicinal cysteine residues have been introduced at solvent......-exposed positions. Several mutant forms have been identified in which formation of a disulfide bond between these cysteine residues results in changes of their fluorescence properties. The redox sensors have been characterized biochemically and found to behave differently, both spectroscopically and in terms...... of redox properties. As genetically encoded sensors they can be expressed in living cells and used for analysis of intracellular redox conditions; however, which parameters are measured depends on how the sensors interact with various cellular redox components. Results of both biochemical and cell...

  18. Rhizosphere Bacteria

    Directory of Open Access Journals (Sweden)

    N.V. Feoktistova

    2016-06-01

    Full Text Available The review deals with the analysis of modern literature data on rhizosphere bacteria and their role in plant life. The structure of rhizosphere has been characterized. The role of plants as the centers of formation of microbial communities has been shown. Data on the main groups of microorganisms inhabiting the rhizosphere have been provided. The associative relationship between rhizobacteria and partner plants has been investigated. The modern concept of holobiont defined as the whole host plant organism and microorganisms associated with it has been reviewed. The role of rhizobacteria in the processes of nitrogen fixation has been discussed in detail. The mechanisms of direct stimulation of plant growth by biosynthesis of phytohormones, improvement of phosphorus and nitrogen nutrition, increase in resistance to stress, and stimulation mediated by antagonism against pathogenic microorganisms have been analyzed. The criteria for selection of rhizobacteria for practical purposes have been discussed.

  19. Characterization of Leptin Intracellular Trafficking

    Directory of Open Access Journals (Sweden)

    E Walum

    2009-12-01

    Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

  20. Adaptive Spike Threshold Enables Robust and Temporally Precise Neuronal Encoding.

    Directory of Open Access Journals (Sweden)

    Chao Huang

    2016-06-01

    Full Text Available Neural processing rests on the intracellular transformation of information as synaptic inputs are translated into action potentials. This transformation is governed by the spike threshold, which depends on the history of the membrane potential on many temporal scales. While the adaptation of the threshold after spiking activity has been addressed before both theoretically and experimentally, it has only recently been demonstrated that the subthreshold membrane state also influences the effective spike threshold. The consequences for neural computation are not well understood yet. We address this question here using neural simulations and whole cell intracellular recordings in combination with information theoretic analysis. We show that an adaptive spike threshold leads to better stimulus discrimination for tight input correlations than would be achieved otherwise, independent from whether the stimulus is encoded in the rate or pattern of action potentials. The time scales of input selectivity are jointly governed by membrane and threshold dynamics. Encoding information using adaptive thresholds further ensures robust information transmission across cortical states i.e. decoding from different states is less state dependent in the adaptive threshold case, if the decoding is performed in reference to the timing of the population response. Results from in vitro neural recordings were consistent with simulations from adaptive threshold neurons. In summary, the adaptive spike threshold reduces information loss during intracellular information transfer, improves stimulus discriminability and ensures robust decoding across membrane states in a regime of highly correlated inputs, similar to those seen in sensory nuclei during the encoding of sensory information.

  1. Yeast prions form infectious amyloid inclusion bodies in bacteria

    Directory of Open Access Journals (Sweden)

    Espargaró Alba

    2012-06-01

    Full Text Available Abstract Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM and the Ure2 protein (Ure2p form inclusion bodies (IBs displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.

  2. A Salmonella small non-coding RNA facilitates bacterial invasion and intracellular replication by modulating the expression of virulence factors.

    Directory of Open Access Journals (Sweden)

    Hao Gong

    2011-09-01

    Full Text Available Small non-coding RNAs (sRNAs that act as regulators of gene expression have been identified in all kingdoms of life, including microRNA (miRNA and small interfering RNA (siRNA in eukaryotic cells. Numerous sRNAs identified in Salmonella are encoded by genes located at Salmonella pathogenicity islands (SPIs that are commonly found in pathogenic strains. Whether these sRNAs are important for Salmonella pathogenesis and virulence in animals has not been reported. In this study, we provide the first direct evidence that a pathogenicity island-encoded sRNA, IsrM, is important for Salmonella invasion of epithelial cells, intracellular replication inside macrophages, and virulence and colonization in mice. IsrM RNA is expressed in vitro under conditions resembling those during infection in the gastrointestinal tract. Furthermore, IsrM is found to be differentially expressed in vivo, with higher expression in the ileum than in the spleen. IsrM targets the mRNAs coding for SopA, a SPI-1 effector, and HilE, a global regulator of the expression of SPI-1 proteins, which are major virulence factors essential for bacterial invasion. Mutations in IsrM result in disregulation of expression of HilE and SopA, as well as other SPI-1 genes whose expression is regulated by HilE. Salmonella with deletion of isrM is defective in bacteria invasion of epithelial cells and intracellular replication/survival in macrophages. Moreover, Salmonella with mutations in isrM is attenuated in killing animals and defective in growth in the ileum and spleen in mice. Our study has shown that IsrM sRNA functions as a pathogenicity island-encoded sRNA directly involved in Salmonella pathogenesis in animals. Our results also suggest that sRNAs may represent a distinct class of virulence factors that are important for bacterial infection in vivo.

  3. Spectrally encoded confocal microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Tearney, G.J.; Webb, R.H.; Bouma, B.E. [Wellman Laboratories of Photomedicine, Massachusetts General Hospital, 50 Blossom Street, BAR 703, Boston, Massachusetts 02114 (United States)

    1998-08-01

    An endoscope-compatible, submicrometer-resolution scanning confocal microscopy imaging system is presented. This approach, spectrally encoded confocal microscopy (SECM), uses a quasi-monochromatic light source and a transmission diffraction grating to detect the reflectivity simultaneously at multiple points along a transverse line within the sample. Since this method does not require fast spatial scanning within the probe, the equipment can be miniaturized and incorporated into a catheter or endoscope. Confocal images of an electron microscope grid were acquired with SECM to demonstrate the feasibility of this technique. {copyright} {ital 1998} {ital Optical Society of America}

  4. Intracardiac intracellular angiotensin system in diabetes.

    NARCIS (Netherlands)

    Kumar, R.; Yong, Q.C.; Thomas, C.M.G.; Baker, K.M.

    2012-01-01

    The renin-angiotensin system (RAS) has mainly been categorized as a circulating and a local tissue RAS. A new component of the local system, known as the intracellular RAS, has recently been described. The intracellular RAS is defined as synthesis and action of ANG II intracellularly. This RAS appea

  5. Engineering a predatory bacterium as a proficient killer agent for intracellular bio-products recovery

    DEFF Research Database (Denmark)

    Martinez, Virginia; Herencias, Cristina; Jurkevitch, Edouard;

    2016-01-01

    This work examines the potential of the predatory bacterium Bdellovibrio bacteriovorus HD100, an obligate predator of other Gram-negative bacteria, as an external cell-lytic agent for recovering valuable intracellular bio-products produced by prey cultures. The bio-product targets to be recovered...

  6. The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages

    Science.gov (United States)

    Mraheil, Mobarak A.; Billion, André; Mohamed, Walid; Mukherjee, Krishnendu; Kuenne, Carsten; Pischimarov, Jordan; Krawitz, Christian; Retey, Julia; Hartsch, Thomas; Chakraborty, Trinad; Hain, Torsten

    2011-01-01

    Small non-coding RNAs (sRNAs) are widespread effectors of post-transcriptional gene regulation in bacteria. Currently extensive information exists on the sRNAs of Listeria monocytogenes expressed during growth in extracellular environments. We used deep sequencing of cDNAs obtained from fractioned RNA (<500 nt) isolated from extracellularly growing bacteria and from L. monocytogenes infected macrophages to catalog the sRNA repertoire during intracellular bacterial growth. Here, we report on the discovery of 150 putative regulatory RNAs of which 71 have not been previously described. A total of 29 regulatory RNAs, including small non-coding antisense RNAs, are specifically expressed intracellularly. We validated highly expressed sRNAs by northern blotting and demonstrated by the construction and characterization of isogenic mutants of rli31, rli33-1 and rli50* for intracellular expressed sRNA candidates, that their expression is required for efficient growth of bacteria in macrophages. All three mutants were attenuated when assessed for growth in mouse and insect models of infection. Comparative genomic analysis revealed the presence of lineage specific sRNA candidates and the absence of sRNA loci in genomes of naturally occurring infection-attenuated bacteria, with additional loss in non-pathogenic listerial genomes. Our analyses reveal extensive sRNA expression as an important feature of bacterial regulation during intracellular growth. PMID:21278422

  7. Eradication of intracellular Francisella tularensis in THP-1 human macrophages with a novel autophagy inducing agent

    Directory of Open Access Journals (Sweden)

    Gunn John S

    2009-12-01

    Full Text Available Abstract Background Autophagy has been shown recently to play an important role in the intracellular survival of several pathogenic bacteria. In this study, we investigated the effect of a novel small-molecule autophagy-inducing agent, AR-12, on the survival of Francisella tularensis, the causative bacterium of tularemia in humans and a potential bioterrorism agent, in macrophages. Methods and results Our results show that AR-12 induces autophagy in THP-1 macrophages, as indicated by increased autophagosome formation, and potently inhibits the intracellular survival of F. tularensis (type A strain, Schu S4 and F. novicida in macrophages in association with increased bacterial co-localization with autophagosomes. The effect of AR-12 on intracellular F. novicida was fully reversed in the presence of the autophagy inhibitor, 3-methyl adenine or the lysosome inhibitor, chloroquine. Intracellular F. novicida were not susceptible to the inhibitory activity of AR-12 added at 12 h post-infection in THP-1 macrophages, and this lack of susceptibility was independent of the intracellular location of bacteria. Conclusion Together, AR-12 represents a proof-of-principle that intracellular F. tularensis can be eradicated by small-molecule agents that target innate immunity.

  8. Economic Game Theory to Model the Attenuation of Virulence of an Obligate Intracellular Bacterium

    Science.gov (United States)

    Tago, Damian; Meyer, Damien F.

    2016-01-01

    Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria. PMID:27610355

  9. Determining the repertoire of immunodominant proteins via whole-genome amplification of intracellular pathogens.

    Directory of Open Access Journals (Sweden)

    Michael J Dark

    Full Text Available Culturing many obligate intracellular bacteria is difficult or impossible. However, these organisms have numerous adaptations allowing for infection persistence and immune system evasion, making them some of the most interesting to study. Recent advancements in genome sequencing, pyrosequencing and Phi29 amplification, have allowed for examination of whole-genome sequences of intracellular bacteria without culture. We have applied both techniques to the model obligate intracellular pathogen Anaplasma marginale and the human pathogen Anaplasma phagocytophilum, in order to examine the ability of phi29 amplification to determine the sequence of genes allowing for immune system evasion and long-term persistence in the host. When compared to traditional pyrosequencing, phi29-mediated genome amplification had similar genome coverage, with no additional gaps in coverage. Additionally, all msp2 functional pseudogenes from two strains of A. marginale were detected and extracted from the phi29-amplified genomes, highlighting its utility in determining the full complement of genes involved in immune evasion.

  10. Economic Game Theory to Model the Attenuation of Virulence of an Obligate Intracellular Bacterium.

    Science.gov (United States)

    Tago, Damian; Meyer, Damien F

    2016-01-01

    Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria.

  11. Antimicrobial properties of cultivable bacteria associated with seaweeds in the Gulf of Mannar on the southeast coast of India

    National Research Council Canada - National Science Library

    Chakraborty, K; Thilakan, B; Chakraborty, R.D

    2016-01-01

    .... Antimicrobial activity analysis combined with the results of amplifying genes encoding for polyketide synthetase and nonribosomal peptide synthetase showed that seaweed-associated bacteria had broad...

  12. Charcot–Marie–Tooth disease and intracellular traffic

    Science.gov (United States)

    Bucci, Cecilia; Bakke, Oddmund; Progida, Cinzia

    2012-01-01

    Mutations of genes whose primary function is the regulation of membrane traffic are increasingly being identified as the underlying causes of various important human disorders. Intriguingly, mutations in ubiquitously expressed membrane traffic genes often lead to cell type- or organ-specific disorders. This is particularly true for neuronal diseases, identifying the nervous system as the most sensitive tissue to alterations of membrane traffic. Charcot–Marie–Tooth (CMT) disease is one of the most common inherited peripheral neuropathies. It is also known as hereditary motor and sensory neuropathy (HMSN), which comprises a group of disorders specifically affecting peripheral nerves. This peripheral neuropathy, highly heterogeneous both clinically and genetically, is characterized by a slowly progressive degeneration of the muscle of the foot, lower leg, hand and forearm, accompanied by sensory loss in the toes, fingers and limbs. More than 30 genes have been identified as targets of mutations that cause CMT neuropathy. A number of these genes encode proteins directly or indirectly involved in the regulation of intracellular traffic. Indeed, the list of genes linked to CMT disease includes genes important for vesicle formation, phosphoinositide metabolism, lysosomal degradation, mitochondrial fission and fusion, and also genes encoding endosomal and cytoskeletal proteins. This review focuses on the link between intracellular transport and CMT disease, highlighting the molecular mechanisms that underlie the different forms of this peripheral neuropathy and discussing the pathophysiological impact of membrane transport genetic defects as well as possible future ways to counteract these defects. PMID:22465036

  13. Charcot-Marie-Tooth disease and intracellular traffic.

    Science.gov (United States)

    Bucci, Cecilia; Bakke, Oddmund; Progida, Cinzia

    2012-12-01

    Mutations of genes whose primary function is the regulation of membrane traffic are increasingly being identified as the underlying causes of various important human disorders. Intriguingly, mutations in ubiquitously expressed membrane traffic genes often lead to cell type- or organ-specific disorders. This is particularly true for neuronal diseases, identifying the nervous system as the most sensitive tissue to alterations of membrane traffic. Charcot-Marie-Tooth (CMT) disease is one of the most common inherited peripheral neuropathies. It is also known as hereditary motor and sensory neuropathy (HMSN), which comprises a group of disorders specifically affecting peripheral nerves. This peripheral neuropathy, highly heterogeneous both clinically and genetically, is characterized by a slowly progressive degeneration of the muscle of the foot, lower leg, hand and forearm, accompanied by sensory loss in the toes, fingers and limbs. More than 30 genes have been identified as targets of mutations that cause CMT neuropathy. A number of these genes encode proteins directly or indirectly involved in the regulation of intracellular traffic. Indeed, the list of genes linked to CMT disease includes genes important for vesicle formation, phosphoinositide metabolism, lysosomal degradation, mitochondrial fission and fusion, and also genes encoding endosomal and cytoskeletal proteins. This review focuses on the link between intracellular transport and CMT disease, highlighting the molecular mechanisms that underlie the different forms of this peripheral neuropathy and discussing the pathophysiological impact of membrane transport genetic defects as well as possible future ways to counteract these defects.

  14. DNA topology and adaptation of Salmonella typhimurium to an intracellular environment.

    Science.gov (United States)

    Marshall, D G; Bowe, F; Hale, C; Dougan, G; Dorman, C J

    2000-01-01

    The expression of genes coding for determinants of DNA topology in the facultative intracellular pathogen Salmonella typhimurium was studied during adaptation by the bacteria to the intracellular environment of J774A.1 macrophage-like cells. A reporter plasmid was used to monitor changes in DNA supercoiling during intracellular growth. Induction of the dps and spv genes, previously shown to be induced in the macrophage, was detected, as was expression of genes coding for DNA gyrase, integration host factor and the nucleoid-associated protein H-NS. The topA gene, coding for the DNA relaxing enzyme topoisomerase I, was not induced. Reporter plasmid data showed that bacterial DNA became relaxed following uptake of S. typhimurium cells by the macrophage. These data indicate that DNA topology in S. typhimurium undergoes significant changes during adaptation to the intracellular environment. A model describing how this process may operate is discussed. PMID:10874730

  15. Intra-ChIP: studying gene regulation in an intracellular pathogen.

    Science.gov (United States)

    Hanson, Brett R; Tan, Ming

    2016-08-01

    Intracellular bacteria that reside within a host cell use a variety of strategies to exploit this unique niche. While these organisms are technically challenging to study in the context of an infected host cell, recent advances have led to an improved understanding of how the intracellular environment impacts bacterial gene expression. We recently demonstrated that chromatin immunoprecipitation (ChIP) can be used to quantify transcription factor binding in the obligate intracellular pathogen Chlamydia trachomatis within infected cells. Furthermore, we showed it was possible to experimentally modulate transcription factor binding while simultaneously measuring changes in transcription. Here we discuss these findings as well as other recent work that has used ChIP to study intracellular pathogens within infected cells. We also discuss technical considerations associated with this approach and its possible future applications.

  16. Intracellular survival of Staphylococcus aureus during persistent infection in the insect Tenebrio molitor.

    Science.gov (United States)

    McGonigle, John E; Purves, Joanne; Rolff, Jens

    2016-06-01

    Survival of bacteria within host cells and tissues presents a challenge to the immune systems of higher organisms. Escape from phagocytic immune cells compounds this issue, as immune cells become potential vehicles for pathogen dissemination. However, the duration of persistence within phagocytes and its contribution to pathogen load has yet to be determined. We investigate the immunological significance of intracellular persistence within the insect model Tenebrio molitor, assessing the extent, duration and location of bacterial recovery during a persistent infection. Relative abundance of Staphylococcus aureus in both intracellular and extracellular fractions was determined over 21 days, and live S. aureus were successfully recovered from both the hemolymph and within phagocytic immune cells across the entire time course. The proportion of bacteria recovered from within phagocytes also increased over time. Our results show that to accurately estimate pathogen load it is vital to account for bacteria persisting within immune cells.

  17. Collective Resistance in Microbial Communities by Intracellular Antibiotic Deactivation

    Science.gov (United States)

    Sorg, Robin A.; Lin, Leo; van Doorn, G. Sander; Sorg, Moritz; Olson, Joshua; Nizet, Victor; Veening, Jan-Willem

    2016-01-01

    The structure and composition of bacterial communities can compromise antibiotic efficacy. For example, the secretion of β-lactamase by individual bacteria provides passive resistance for all residents within a polymicrobial environment. Here, we uncover that collective resistance can also develop via intracellular antibiotic deactivation. Real-time luminescence measurements and single-cell analysis demonstrate that the opportunistic human pathogen Streptococcus pneumoniae grows in medium supplemented with chloramphenicol (Cm) when resistant bacteria expressing Cm acetyltransferase (CAT) are present. We show that CAT processes Cm intracellularly but not extracellularly. In a mouse pneumonia model, more susceptible pneumococci survive Cm treatment when coinfected with a CAT-expressing strain. Mathematical modeling predicts that stable coexistence is only possible when antibiotic resistance comes at a fitness cost. Strikingly, CAT-expressing pneumococci in mouse lungs were outcompeted by susceptible cells even during Cm treatment. Our results highlight the importance of the microbial context during infectious disease as a potential complicating factor to antibiotic therapy. PMID:28027306

  18. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival.

    Science.gov (United States)

    Lamberti, Yanina; Gorgojo, Juan; Massillo, Cintia; Rodriguez, Maria E

    2013-12-01

    Bordetella pertussis is the causative agent of pertussis, aka whooping cough. Although generally considered an extracellular pathogen, this bacterium has been found inside respiratory epithelial cells, which might represent a survival strategy inside the host. Relatively little is known, however, about the mechanism of internalization and the fate of B. pertussis inside the epithelia. We show here that B. pertussis is able to enter those cells by a mechanism dependent on microtubule assembly, lipid raft integrity, and the activation of a tyrosine-kinase-mediated signaling. Once inside the cell, a significant proportion of the intracellular bacteria evade phagolysosomal fusion and remain viable in nonacidic lysosome-associated membrane-protein-1-negative compartments. In addition, intracellular B. pertussis was found able to repopulate the extracellular environment after complete elimination of the extracellular bacteria with polymyxin B. Taken together, these data suggest that B. pertussis is able to survive within respiratory epithelial cells and by this means potentially contribute to host immune system evasion.

  19. Two novel functions of hyaluronidase from Streptococcus agalactiae are enhanced intracellular survival and inhibition of proinflammatory cytokine expression.

    Science.gov (United States)

    Wang, Zhaofei; Guo, Changming; Xu, Yannan; Liu, Guangjin; Lu, Chengping; Liu, Yongjie

    2014-06-01

    Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl(+) isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl(+) strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.

  20. Palladium-mediated intracellular chemistry

    Science.gov (United States)

    Yusop, Rahimi M.; Unciti-Broceta, Asier; Johansson, Emma M. V.; Sánchez-Martín, Rosario M.; Bradley, Mark

    2011-03-01

    Many important intracellular biochemical reactions are modulated by transition metals, typically in the form of metalloproteins. The ability to carry out selective transformations inside a cell would allow researchers to manipulate or interrogate innumerable biological processes. Here, we show that palladium nanoparticles trapped within polystyrene microspheres can enter cells and mediate a variety of Pd0-catalysed reactions, such as allylcarbamate cleavage and Suzuki-Miyaura cross-coupling. The work provides the basis for the customization of heterogeneous unnatural catalysts as tools to carry out artificial chemistries within cells. Such in cellulo synthesis has potential for a plethora of applications ranging from cellular labelling to synthesis of modulators or inhibitors of cell function.

  1. Purifying selection in mitochondria, free-living and obligate intracellular proteobacteria

    Directory of Open Access Journals (Sweden)

    Popadin Konstantin

    2007-02-01

    Full Text Available Abstract Background The effectiveness of elimination of slightly deleterious mutations depends mainly on drift and recombination frequency. Here we analyze the influence of these two factors on the strength of the purifying selection in mitochondrial and proteobacterial orthologous genes taking into account the differences in the organism lifestyles. Results (I We found that the probability of fixation of nonsynonymous substitutions (Kn/Ks in mitochondria is significantly lower compared to obligate intracellular bacteria and even marginally significantly lower compared to free-living bacteria. The comparison of bacteria of different lifestyles demonstrates more effective elimination of slightly deleterious mutations in (II free-living bacteria as compared to obligate intracellular species and in (III obligate intracellular parasites as compared to obligate intracellular symbionts. (IV Finally, we observed that the level of the purifying selection (i.e. 1-Kn/Ks increases with the density of mobile elements in bacterial genomes. Conclusion This study shows that the comparison of patterns of molecular evolution of orthologous genes between ecologically different groups of organisms allow to elucidate the genetic consequences of their various lifestyles. Comparing the strength of the purifying selection among proteobacteria with different lifestyles we obtained results, which are in concordance with theoretical expectations: (II low effective population size and level of recombination in obligate intracellular proteobacteria lead to less effective elimination of mutations compared to free-living relatives; (III rare horizontal transmissions, i.e. effectively zero recombination level in symbiotic obligate intracellular bacteria leads to less effective purifying selection than in parasitic obligate intracellular bacteria; (IV the increased frequency of recombination in bacterial genomes with high mobile element density leads to a more effective

  2. Selecting Operations for Assembler Encoding

    Directory of Open Access Journals (Sweden)

    Tomasz Praczyk

    2010-04-01

    Full Text Available Assembler Encoding is a neuro-evolutionary method in which a neural network is represented in the form of a simple program called Assembler Encoding Program. The task of the program is to create the so-called Network Definition Matrix which maintains all the information necessary to construct the network. To generate Assembler Encoding Programs and the subsequent neural networks evolutionary techniques are used.
    The performance of Assembler Encoding strongly depends on operations used in Assembler Encoding Programs. To select the most effective operations, experiments in the optimization and the predator-prey problem were carried out. In the experiments, Assembler Encoding Programs equipped with different types of operations were tested. The results of the tests are presented at the end of the paper.

  3. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    Science.gov (United States)

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  4. Comparative genomics of the lactic acid bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Makarova, K.; Slesarev, A.; Wolf, Y.; Sorokin, A.; Mirkin, B.; Koonin, E.; Pavlov, A.; Pavlova, N.; Karamychev, V.; Polouchine, N.; Shakhova, V.; Grigoriev, I.; Lou, Y.; Rokhsar, D.; Lucas, S.; Huang, K.; Goodstein, D. M.; Hawkins, T.; Plengvidhya, V.; Welker, D.; Hughes, J.; Goh, Y.; Benson, A.; Baldwin, K.; Lee, J. -H.; Diaz-Muniz, I.; Dosti, B.; Smeianov, V; Wechter, W.; Barabote, R.; Lorca, G.; Altermann, E.; Barrangou, R.; Ganesan, B.; Xie, Y.; Rawsthorne, H.; Tamir, D.; Parker, C.; Breidt, F.; Broadbent, J.; Hutkins, R.; O' Sullivan, D.; Steele, J.; Unlu, G.; Saier, M.; Klaenhammer, T.; Richardson, P.; Kozyavkin, S.; Weimer, B.; Mills, D.

    2006-06-01

    Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

  5. Buoyant densities of phototrophic sulfur bacteria and cyanobacteria

    Science.gov (United States)

    Guerrero, R.

    1985-01-01

    The buoyant densities of bacterial cells are greatly influenced by the accumulation of intracellular reserve material. The buoyant density of phototrophic bacteria that are planktonic is of particular interest, since these organisms must remain in the photic zone of the water column for optimal growth. Separation of cells by their buoyant density may also be of use in separating and identifying organisms from a natural population. The bacteria used were obtained from pure cultures, enrichments, or samples taken directly from the environment.

  6. Potentized homeopathic drug Arsenicum Album 30C inhibits intracellular reactive oxygen species generation and up-regulates expression of arsenic resistance gene in arsenine-exposed bacteria Escherichia coli%顺势疗法药物白砷剂抑制暴露于三氧化二砷的大肠杆菌细胞内活性氧的产生并上调其抗三氧化二砷基因的表达

    Institute of Scientific and Technical Information of China (English)

    Arnab De; Durba Das; Suman Dutta; Debrup Chakraborty; Naoual Boujedaini; Anisur Rahman Khuda-Bukhsh

    2012-01-01

    目的:检验顺势疗法药物Arsenicum Album 30C(Ars Alb 30C)是否能够降低亚砷酸钠对大肠杆菌(Escherichia coli)的毒性.方法:将在标准培养基中培养至对数生长期的大肠杆菌暴露于低剂量砷剂下.1或2 mmol/L亚砷酸钠单独作用于大肠杆菌作为对照,在此基础上加入Ars Alb 30C作为治疗组,或加入按顺势疗法原则配置的乙醇作为安慰剂组.分别于45 min和90 min后检测大肠杆菌的葡萄糖摄取量,细胞内己糖激酶、脂质过氧化物酶、超氧化物歧化酶及过氧化氢酶活性,细胞内外亚砷酸钠含量,细胞生长情况,细胞膜电位,DNA损伤情况,细胞内活性氧、三磷酸腺苷及自由型谷胱甘肽含量,以及arsB和ptsG基因表达情况.实验按照随机分组原则及盲法原则进行.结果:暴露于亚砷酸钠的大肠杆菌的葡萄糖摄取量、细胞内活性氧、脂质过氧化反应及DNA损伤增加;己糖激酶、超氧化物歧化酶及过氧化氢酶活性降低;细胞内三磷酸腺苷及自由型谷胱甘肽含量降低;细胞膜电位降低且细胞生长缓慢;arsB和ptsG基因表达水平增高.Ars Alb 30C作用后降低了亚砷酸钠对大肠杆菌的毒性,表现为抑制细胞内活性氧的生成和对细胞生长的促进作用.结论:Ars Alb 30C能够降低亚砷酸钠对大肠杆菌的毒性,证实了这一顺势疗法原则下高度稀释的药物的效用.%OBJECTIVE: To examine if potentized homeopathic drug Arsenicum Album 30C (Ars AIb 30C) can reduce sodium arsenite-induced toxicity in Escherichia coli.METHODS: E.coli were exposed to low arsenite insult after they grew up to log phase in standard Luria-Bertani medium.E.coli were treated with 1 or 2 mmol/L sodium arsenite alone (control),or Ars AIb 30C was added to the medium of a subset of sodium arsenite-treated bacteria (drug-treated),or homeopathically agitated alcohol was added to the medium containing a subset of sodium arsenite-treated bacteria (placebo

  7. Intracellular peptides: From discovery to function

    Directory of Open Access Journals (Sweden)

    Emer S. Ferro

    2014-06-01

    Full Text Available Peptidomics techniques have identified hundreds of peptides that are derived from proteins present mainly in the cytosol, mitochondria, and/or nucleus; these are termed intracellular peptides to distinguish them from secretory pathway peptides that function primarily outside of the cell. The proteasome and thimet oligopeptidase participate in the production and metabolism of intracellular peptides. Many of the intracellular peptides are common among mouse tissues and human cell lines analyzed and likely to perform a variety of functions within cells. Demonstrated functions include the modulation of signal transduction, mitochondrial stress, and development; additional functions will likely be found for intracellular peptides.

  8. Elevated Cholesterol in the Coxiella burnetii Intracellular Niche Is Bacteriolytic

    Science.gov (United States)

    Mulye, Minal; Samanta, Dhritiman; Winfree, Seth; Heinzen, Robert A.

    2017-01-01

    ABSTRACT Coxiella burnetii is an intracellular bacterial pathogen and a significant cause of culture-negative endocarditis in the United States. Upon infection, the nascent Coxiella phagosome fuses with the host endocytic pathway to form a large lysosome-like vacuole called the parasitophorous vacuole (PV). The PV membrane is rich in sterols, and drugs perturbing host cell cholesterol homeostasis inhibit PV formation and bacterial growth. Using cholesterol supplementation of a cholesterol-free cell model system, we found smaller PVs and reduced Coxiella growth as cellular cholesterol concentration increased. Further, we observed in cells with cholesterol a significant number of nonfusogenic PVs that contained degraded bacteria, a phenotype not observed in cholesterol-free cells. Cholesterol had no effect on axenic Coxiella cultures, indicating that only intracellular bacteria are sensitive to cholesterol. Live-cell microscopy revealed that both plasma membrane-derived cholesterol and the exogenous cholesterol carrier protein low-density lipoprotein (LDL) traffic to the PV. To test the possibility that increasing PV cholesterol levels affects bacterial survival, infected cells were treated with U18666A, a drug that traps cholesterol in lysosomes and PVs. U18666A treatment led to PVs containing degraded bacteria and a significant loss in bacterial viability. The PV pH was significantly more acidic in cells with cholesterol or cells treated with U18666A, and the vacuolar ATPase inhibitor bafilomycin blocked cholesterol-induced PV acidification and bacterial death. Additionally, treatment of infected HeLa cells with several FDA-approved cholesterol-altering drugs led to a loss of bacterial viability, a phenotype also rescued by bafilomycin. Collectively, these data suggest that increasing PV cholesterol further acidifies the PV, leading to Coxiella death. PMID:28246364

  9. Francisella tularensis harvests nutrients derived via ATG5-independent autophagy to support intracellular growth.

    Directory of Open Access Journals (Sweden)

    Shaun Steele

    2013-08-01

    Full Text Available Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.

  10. A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system

    Directory of Open Access Journals (Sweden)

    Guy Lionel

    2004-12-01

    Full Text Available Abstract Background The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs. Results On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC, involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. Conclusions A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between

  11. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells.

    Science.gov (United States)

    Ortega, Alvaro D; Quereda, Juan J; Pucciarelli, M Graciela; García-del Portillo, Francisco

    2014-01-01

    Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of "intact" intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

  12. Non-coding RNA regulation in pathogenic bacteria located inside eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Álvaro D. Ortega

    2014-11-01

    Full Text Available Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of ‘intact’ intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.

  13. Hydrophilic fluorescent nanogel thermometer for intracellular thermometry.

    Science.gov (United States)

    Gota, Chie; Okabe, Kohki; Funatsu, Takashi; Harada, Yoshie; Uchiyama, Seiichi

    2009-03-01

    The first methodology to measure intracellular temperature is described. A highly hydrophilic fluorescent nanogel thermometer developed for this purpose stays in the cytoplasm and emits stronger fluorescence at a higher temperature. Thus, intracellular temperature variations associated with biological processes can be monitored by this novel thermometer with a temperature resolution of better than 0.5 degrees C.

  14. Quorum sensing-controlled gene expression in lactic acid bacteria

    NARCIS (Netherlands)

    Kuipers, Oscar P.; Ruyter, Pascalle G.G.A. de; Kleerebezem, Michiel; Vos, Willem M. de

    1998-01-01

    Quorum sensing in lactic acid bacteria (LAB) involves peptides that are directly sensed by membrane-located histidine kinases, after which the signal is transmitted to an intracellular response regulator. This regulator in turn activates transcription of target genes, that commonly include the struc

  15. Quorum sensing-controlled gene expression in lactic acid bacteria

    NARCIS (Netherlands)

    Kuipers, Oscar P.; Ruyter, Pascalle G.G.A. de; Kleerebezem, Michiel; Vos, Willem M. de

    1998-01-01

    Quorum sensing in lactic acid bacteria (LAB) involves peptides that are directly sensed by membrane-located histidine kinases, after which the signal is transmitted to an intracellular response regulator. This regulator in turn activates transcription of target genes, that commonly include the

  16. Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution.

    Science.gov (United States)

    Liu, Xueliang; Lopez, Paola A; Giessen, Tobias W; Giles, Michael; Way, Jeffrey C; Silver, Pamela A

    2016-11-29

    Genetically encoding the synthesis of functional nanomaterials such as magnetic nanoparticles enables sensitive and non-invasive biological sensing and control. Via directed evolution of the natural iron-sequestering ferritin protein, we discovered key mutations that lead to significantly enhanced cellular magnetism, resulting in increased physical attraction of ferritin-expressing cells to magnets and increased contrast for cellular magnetic resonance imaging (MRI). The magnetic mutants further demonstrate increased iron biomineralization measured by a novel fluorescent genetic sensor for intracellular free iron. In addition, we engineered Escherichia coli cells with multiple genomic knockouts to increase cellular accumulation of various metals. Lastly to explore further protein candidates for biomagnetism, we characterized members of the DUF892 family using the iron sensor and magnetic columns, confirming their intracellular iron sequestration that results in increased cellular magnetization.

  17. Intracellular structure and nucleocytoplasmic transport.

    Science.gov (United States)

    Agutter, P S

    1995-01-01

    Intracellular movement of any solute or particle accords with one of two general schemes: either it takes place predominantly in the solution phase or it occurs by dynamic interactions with solid-state structures. If nucleocytoplasmic exchanges of macromolecules and complexes are predominantly solution-phase processes, i.e., if the former ("diffusionist") perspective applies, then the only significant structures in nucleocytoplasmic transport are the pore complexes. However, if such exchanges accord with the latter ("solid-state") perspective, then the roles of the nucleoskeleton and cytoskeleton in nucleocytoplasmic transport are potentially, at least, as important as that of the pore complexes. The role of the nucleoskeleton in mRNA transport is more difficult to evaluate than that of the cytoskeleton because it is less well characterized, and current evidence does not exclude either perspective. However, the balance of evidence favors a solid-state scheme. It is argued that ribosomal subunits are also more likely to migrate by a solid-state rather than a diffusionist mechanism, though the opposite is true of proteins and tRNAs. Moreover, recent data on the effects of viral proteins on intranuclear RNA processing and migration accord with the solid-state perspective. In view of this balance of evidence, three possible solid-state mechanisms for nucleocytoplasmic mRNA transport are described and evaluated. The explanatory advantage of solid-state models is contrasted with the heuristic advantage of diffusion theory, but it is argued that diffusion theory itself, even aided by modern computational techniques and numerical and graphical approaches, cannot account for data describing the movements of materials within the cell. Therefore, the mechanisms envisaged in a diffusionist perspective cannot be confined to diffusion alone, but must include other processes such as bulk fluid flow.

  18. PNA-encoded chemical libraries.

    Science.gov (United States)

    Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

    2015-06-01

    Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

  19. Bacteria isolated from amoebae/bacteria consortium

    Science.gov (United States)

    Tyndall, Richard L.

    1995-01-01

    New protozoan derived microbial consortia and method for their isolation are provided. Consortia and bacteria isolated therefrom are useful for treating wastes such as trichloroethylene and trinitrotoluene. Consortia, bacteria isolated therefrom, and dispersants isolated therefrom are useful for dispersing hydrocarbons such as oil, creosote, wax, and grease.

  20. From microbiology to cell biology: when an intracellular bacterium becomes part of its host cell.

    Science.gov (United States)

    McCutcheon, John P

    2016-08-01

    Mitochondria and chloroplasts are now called organelles, but they used to be bacteria. As they transitioned from endosymbionts to organelles, they became more and more integrated into the biochemistry and cell biology of their hosts. Work over the last 15 years has shown that other symbioses show striking similarities to mitochondria and chloroplasts. In particular, many sap-feeding insects house intracellular bacteria that have genomes that overlap mitochondria and chloroplasts in terms of size and coding capacity. The massive levels of gene loss in some of these bacteria suggest that they, too, are becoming highly integrated with their host cells. Understanding these bacteria will require inspiration from eukaryotic cell biology, because a traditional microbiological framework is insufficient for understanding how they work.

  1. Dynamical quorum sensing: Population density encoded in cellular dynamics

    Science.gov (United States)

    De Monte, Silvia; d'Ovidio, Francesco; Danø, Sune; Sørensen, Preben Graae

    2007-01-01

    Mutual synchronization by exchange of chemicals is a mechanism for the emergence of collective dynamics in cellular populations. General theories exist on the transition to coherence, but no quantitative, experimental demonstration has been given. Here, we present a modeling and experimental analysis of cell-density-dependent glycolytic oscillations in yeast. We study the disappearance of oscillations at low cell density and show that this phenomenon occurs synchronously in all cells and not by desynchronization, as previously expected. This study identifies a general scenario for the emergence of collective cellular oscillations and suggests a quorum-sensing mechanism by which the cell density information is encoded in the intracellular dynamical state. PMID:18003917

  2. DMTB: the magnetotactic bacteria database

    Science.gov (United States)

    Pan, Y.; Lin, W.

    2012-12-01

    Magnetotactic bacteria (MTB) are of interest in biogeomagnetism, rock magnetism, microbiology, biomineralization, and advanced magnetic materials because of their ability to synthesize highly ordered intracellular nano-sized magnetic minerals, magnetite or greigite. Great strides for MTB studies have been made in the past few decades. More than 600 articles concerning MTB have been published. These rapidly growing data are stimulating cross disciplinary studies in such field as biogeomagnetism. We have compiled the first online database for MTB, i.e., Database of Magnestotactic Bacteria (DMTB, http://database.biomnsl.com). It contains useful information of 16S rRNA gene sequences, oligonucleotides, and magnetic properties of MTB, and corresponding ecological metadata of sampling sites. The 16S rRNA gene sequences are collected from the GenBank database, while all other data are collected from the scientific literature. Rock magnetic properties for both uncultivated and cultivated MTB species are also included. In the DMTB database, data are accessible through four main interfaces: Site Sort, Phylo Sort, Oligonucleotides, and Magnetic Properties. References in each entry serve as links to specific pages within public databases. The online comprehensive DMTB will provide a very useful data resource for researchers from various disciplines, e.g., microbiology, rock magnetism and paleomagnetism, biogeomagnetism, magnetic material sciences and others.

  3. PlyC, a bacteriophage endolysin that is internalized by epithelial cells and retains bacteriolytic activity against intracellular streptococci

    Science.gov (United States)

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial...

  4. Compressed Encoding for Rank Modulation

    CERN Document Server

    Gad, Eyal En; Jiang,; Bruck, Jehoshua

    2011-01-01

    Rank modulation has been recently proposed as a scheme for storing information in flash memories. While rank modulation has advantages in improving write speed and endurance, the current encoding approach is based on the "push to the top" operation that is not efficient in the general case. We propose a new encoding procedure where a cell level is raised to be higher than the minimal necessary subset - instead of all - of the other cell levels. This new procedure leads to a significantly more compressed (lower charge levels) encoding. We derive an upper bound for a family of codes that utilize the proposed encoding procedure, and consider code constructions that achieve that bound for several special cases.

  5. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  6. Self-Organising Stochastic Encoders

    CERN Document Server

    Luttrell, Stephen

    2010-01-01

    The processing of mega-dimensional data, such as images, scales linearly with image size only if fixed size processing windows are used. It would be very useful to be able to automate the process of sizing and interconnecting the processing windows. A stochastic encoder that is an extension of the standard Linde-Buzo-Gray vector quantiser, called a stochastic vector quantiser (SVQ), includes this required behaviour amongst its emergent properties, because it automatically splits the input space into statistically independent subspaces, which it then separately encodes. Various optimal SVQs have been obtained, both analytically and numerically. Analytic solutions which demonstrate how the input space is split into independent subspaces may be obtained when an SVQ is used to encode data that lives on a 2-torus (e.g. the superposition of a pair of uncorrelated sinusoids). Many numerical solutions have also been obtained, using both SVQs and chains of linked SVQs: (1) images of multiple independent targets (encod...

  7. Bleach vs. Bacteria

    Science.gov (United States)

    ... Articles | Inside Life Science Home Page Bleach vs. Bacteria By Sharon Reynolds Posted April 2, 2014 Your ... hypochlorous acid to help kill invading microbes, including bacteria. Researchers funded by the National Institutes of Health ...

  8. Stochastic resonance in an intracellular genetic perceptron

    Science.gov (United States)

    Bates, Russell; Blyuss, Oleg; Zaikin, Alexey

    2014-03-01

    Intracellular genetic networks are more intelligent than was first assumed due to their ability to learn. One of the manifestations of this intelligence is the ability to learn associations of two stimuli within gene-regulating circuitry: Hebbian-type learning within the cellular life. However, gene expression is an intrinsically noisy process; hence, we investigate the effect of intrinsic and extrinsic noise on this kind of intracellular intelligence. We report a stochastic resonance in an intracellular associative genetic perceptron, a noise-induced phenomenon, which manifests itself in noise-induced increase of response in efficiency after the learning event under the conditions of optimal stochasticity.

  9. Tyramine and phenylethylamine biosynthesis by food bacteria.

    Science.gov (United States)

    Marcobal, Angela; De las Rivas, Blanca; Landete, José María; Tabera, Laura; Muñoz, Rosario

    2012-01-01

    Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products.

  10. Probing the metabolic water contribution to intracellular water using oxygen isotope ratios of PO4

    Science.gov (United States)

    Li, Hui; Yu, Chan; Wang, Fei; Chang, Sae Jung; Yao, Jun; Blake, Ruth E.

    2016-05-01

    Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition—including metabolic water produced as a byproduct of metabolism—based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the 18O/16O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ˜30-40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.

  11. Urothelial cultures support intracellular bacterial community formation by uropathogenic Escherichia coli.

    Science.gov (United States)

    Berry, Ruth E; Klumpp, David J; Schaeffer, Anthony J

    2009-07-01

    Uropathogenic Escherichia coli (UPEC) causes most community-acquired and nosocomial urinary tract infections (UTI). In a mouse model of UTI, UPEC invades superficial bladder cells and proliferates rapidly, forming biofilm-like structures called intracellular bacterial communities (IBCs). Using a gentamicin protection assay and fluorescence microscopy, we developed an in vitro model for studying UPEC proliferation within immortalized human urothelial cells. By pharmacologic manipulation of urothelial cells with the cholesterol-sequestering drug filipin, numbers of intracellular UPEC CFU increased 8 h and 24 h postinfection relative to untreated cultures. Enhanced UPEC intracellular proliferation required that the urothelial cells, but not the bacteria, be filipin treated prior to infection. However, neither UPEC frequency of invasion nor early intracellular trafficking events to a Lamp1-positive compartment were modulated by filipin. Upon inspection by fluorescence microscopy, cultures with enhanced UPEC intracellular proliferation exhibited large, dense bacterial aggregates within cells that resembled IBCs but were contained with Lamp1-positive vacuoles. While an isogenic fimH mutant was capable of forming these IBC-like structures, the mutant formed significantly fewer than wild-type UPEC. Similar to IBCs, expression of E. coli iron acquisition systems was upregulated by intracellular UPEC. Expression of other putative virulence factors, including hlyA, cnf1, fliC, kpsD, and the biofilm adhesin yfaL also increased, while expression of fimA decreased and that of flu did not change. These results indicate that UPEC differentially regulates virulence factors in the intracellular environment. Thus, immortalized urothelial cultures that recapitulate IBC formation in vitro represent a novel system for the molecular and biochemical characterization of the UPEC intracellular life cycle.

  12. Bacteria and lignin degradation

    Institute of Scientific and Technical Information of China (English)

    Jing LI; Hongli YUAN; Jinshui YANG

    2009-01-01

    Lignin is both the most abundant aromatic (phenolic) polymer and the second most abundant raw material.It is degraded and modified by bacteria in the natural world,and bacteria seem to play a leading role in decomposing lignin in aquatic ecosystems.Lignin-degrading bacteria approach the polymer by mechanisms such as tunneling,erosion,and cavitation.With the advantages of immense environmental adaptability and biochemical versatility,bacteria deserve to be studied for their ligninolytic potential.

  13. Multistability and dynamic transitions of intracellular Min protein patterns.

    Science.gov (United States)

    Wu, Fabai; Halatek, Jacob; Reiter, Matthias; Kingma, Enzo; Frey, Erwin; Dekker, Cees

    2016-06-08

    Cells owe their internal organization to self-organized protein patterns, which originate and adapt to growth and external stimuli via a process that is as complex as it is little understood. Here, we study the emergence, stability, and state transitions of multistable Min protein oscillation patterns in live Escherichia coli bacteria during growth up to defined large dimensions. De novo formation of patterns from homogenous starting conditions is observed and studied both experimentally and in simulations. A new theoretical approach is developed for probing pattern stability under perturbations. Quantitative experiments and simulations show that, once established, Min oscillations tolerate a large degree of intracellular heterogeneity, allowing distinctly different patterns to persist in different cells with the same geometry. Min patterns maintain their axes for hours in experiments, despite imperfections, expansion, and changes in cell shape during continuous cell growth. Transitions between multistable Min patterns are found to be rare events induced by strong intracellular perturbations. The instances of multistability studied here are the combined outcome of boundary growth and strongly nonlinear kinetics, which are characteristic of the reaction-diffusion patterns that pervade biology at many scales.

  14. Brucella canis Is an Intracellular Pathogen That Induces a Lower Proinflammatory Response than Smooth Zoonotic Counterparts

    Science.gov (United States)

    Chacón-Díaz, Carlos; Altamirano-Silva, Pamela; González-Espinoza, Gabriela; Medina, María-Concepción; Alfaro-Alarcón, Alejandro; Bouza-Mora, Laura; Jiménez-Rojas, César; Wong, Melissa; Barquero-Calvo, Elías; Rojas, Norman; Guzmán-Verri, Caterina

    2015-01-01

    Canine brucellosis caused by Brucella canis is a disease of dogs and a zoonotic risk. B. canis harbors most of the virulence determinants defined for the genus, but its pathogenic strategy remains unclear since it has not been demonstrated that this natural rough bacterium is an intracellular pathogen. Studies of B. canis outbreaks in kennel facilities indicated that infected dogs displaying clinical signs did not present hematological alterations. A virulent B. canis strain isolated from those outbreaks readily replicated in different organs of mice for a protracted period. However, the levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-12 in serum were close to background levels. Furthermore, B. canis induced lower levels of gamma interferon, less inflammation of the spleen, and a reduced number of granulomas in the liver in mice than did B. abortus. When the interaction of B. canis with cells was studied ex vivo, two patterns were observed, a predominant scattered cell-associated pattern of nonviable bacteria and an infrequent intracellular replicative pattern of viable bacteria in a perinuclear location. The second pattern, responsible for the increase in intracellular multiplication, was dependent on the type IV secretion system VirB and was seen only if the inoculum used for cell infections was in early exponential phase. Intracellular replicative B. canis followed an intracellular trafficking route undistinguishable from that of B. abortus. Although B. canis induces a lower proinflammatory response and has a stealthier replication cycle, it still displays the pathogenic properties of the genus and the ability to persist in infected organs based on the ability to multiply intracellularly. PMID:26438796

  15. Differentiating intracellular from extracellular alkaline phosphatase activity in soil by sonication.

    Directory of Open Access Journals (Sweden)

    Shuping Qin

    Full Text Available Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v and power density  =  15 watt ml(-1], the activity of alkaline phosphomonoesterase (phosphatase in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80% of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.

  16. Differentiating intracellular from extracellular alkaline phosphatase activity in soil by sonication.

    Science.gov (United States)

    Qin, Shuping; Hu, Chunsheng; Oenema, Oene

    2013-01-01

    Differentiating intracellular from extracellular enzyme activity is important in soil enzymology, but not easy. Here, we report on an adjusted sonication method for the separation of intracellular from extracellular phosphatase activity in soil. Under optimal sonication conditions [soil:water ratio  =  1/8 (w/v) and power density  =  15 watt ml(-1)], the activity of alkaline phosphomonoesterase (phosphatase) in a Haplic Cambisol soil increased with sonication time in two distinct steps. A first plateau of enzyme activity was reached between 60 and 100 s, and a second higher plateau after 300 s. We also found that sonication for 100 s under optimal conditions activated most (about 80%) of the alkaline phosphatase that was added to an autoclaved soil, while total bacteria number was not affected. Sonication for 300 s reduced the total bacteria number by three orders of magnitude but had no further effects on enzyme activity. Our results indicate that the first plateau of alkaline phosphatase activity was derived from extracellular enzymes attached to soil particles, and the second plateau to the combination of extracellular and intracellular enzymes after cell lysis. We conclude that our adjusted sonication method may be an alternative to the currently used physiological and chloroform-fumigation methods for differentiating intracellular from extracellular phosphatase activity in soil. Further testing is needed to find out whether this holds for other soil types.

  17. 慢病毒介导的 TACO-shRNA 通过促进吞噬体与溶酶体融合提高巨噬细胞清除结核分枝杆菌的能力%Lentivirus-mediated RNA interference targeting TACO gene increases the intracellular killing of Mycobacterium tuberculosis by promoting the fusion of bacteria-containing phagosomes and lysosomes

    Institute of Scientific and Technical Information of China (English)

    陈洁; 呙阳; 邓亚婷; 江红; 黄自坤; 罗清; 叶建青; 李俊明

    2015-01-01

    Objective To construct a lentiviral vector-based short hairpin RNA (shRNA) targeting the gene encoding tryptophan-aspartate containing coat protein ( TACO) and to evaluate its inhibitory effect on the expression of TACO , and to further elucidate its effects on the phagocytosing and intracellular killing of My-cobacterium tuberculosis (M.tb) by macrophages and the possible mechanisms.Methods Three shRNA frag-ments targeting TACO gene and a scrambling control shRNA fragments were designed and cloned into the lentivi -ral vector pSicoR .The recombinant lentiviral vectors were identified by sequencing analysis and then packed in 293T cells.Real-time RT-PCR and Western blot assay were performed to evaluate the gene-silencing efficiency of the recombinant lentiviral vectors among RAW 264.7 cells transfected with the concentrated lentivirus .The most effective lentivirus was screened out to transfect the RAW 264.7 cells for 48 hours, followed by infection those cells with M.tb strains.The entry and intracellular survival of M .tb strains in RAW264.7 cells were de-termined by bacterial culture at indicated time points .The colocalization of M .tb and lysosomes was detected by immunofluorescence staining .The cyto-ID autophagy kit was used to detect the cellular autophagy and the auto-phagy-associated protein LC 3 was determined by Western blot assay .Results The recombinant lentiviral vec-tors were successfully constructed and confirmed by sequencing analysis .Decreased expression of TACO in RAW264 .7 cells was detected after transfecting the cells with the lentiviral vector-based shRNA vectors targeting TACO gene for 48 hours.The most effective lentivirus , LV-pSRT1, decreased the expression of TACO by 85.24%and 69.00%at the mRNA and protein levels, respectively.The bacterial loads in LV-pSRT1 trans-fected RAW264.7 cells were significantly decreased at the time point of 0 h after M.tb infection as compared with those in the control lentivirus treated cells (5.50×104 vs

  18. Intracellular Biopotentials During Static Extracellular Stimulation

    Science.gov (United States)

    Klee, Maurice

    1973-01-01

    Two properties of the intracellular potentials and electric fields resulting from static extracellular stimulation are obtained for arbitrarily shaped cells. First, the values of intracellular potential are shown to be bounded by the maximum and minimum values of extracellular potential on the surface of the cell. Second, the volume average of the magnitude of intracellular electric field is shown to have an upper bound given by the ratio of the magnitude of the largest extracellular potential difference on the surface of the cell to a generalized length constant λ = [σintraVcell/(σmemb Acell)]1/2, where Vcell and Acell are the volume and surface area of the cell, σintra is the intracellular conductivity (reciprocal ohms per centimeter), and σmemb is the membrane conductivity (reciprocal ohms per square centimeter). The use of the upper bound on the volume average of the magnitude of intracellular electric field as an estimate for intracellular isopotentiality is discussed and the use of the generalized length constant for electrically describing arbitrary cells is illustrated for cylindrical- and spheroidal-shaped cells. PMID:4726882

  19. Multidimensionally encoded magnetic resonance imaging.

    Science.gov (United States)

    Lin, Fa-Hsuan

    2013-07-01

    Magnetic resonance imaging (MRI) typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here, we propose the multidimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel radiofrequency coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled.

  20. A method for functional trans-complementation of intracellular Francisella tularensis.

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    Shaun Steele

    Full Text Available Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional

  1. The Francisella intracellular life cycle: towards molecular mechanisms of intracellular survival and proliferation

    Directory of Open Access Journals (Sweden)

    Audrey eChong

    2010-12-01

    Full Text Available The tularemia-causing bacterium Francisella tularensis is a facultative intracellular organism with a complex intracellular lifecycle that ensures its survival and proliferation in a variety of mammalian cell types, including professional phagocytes. Because this cycle is essential to Francisella pathogenesis and virulence, much research has focused on deciphering the mechanisms of its intracellular survival and replication and characterizing both bacterial and host determinants of the bacterium’s intracellular cycle. Studies of various strains and host cell models have led to the consensual paradigm of Francisella as a cytosolic pathogen, but also to some controversy about its intracellular cycle. In this review, we will detail major findings that have advanced our knowledge of Francisella intracellular survival strategies and also attempt to reconcile discrepancies that exist in our molecular understanding of the Francisella-phagocyte interaction.

  2. Learning from Bacteriophages - Advantages and Limitations of Phage and Phage-Encoded Protein Applications

    Science.gov (United States)

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grażyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

    2012-01-01

    The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application. PMID:23305359

  3. Learning from bacteriophages - advantages and limitations of phage and phage-encoded protein applications.

    Science.gov (United States)

    Drulis-Kawa, Zuzanna; Majkowska-Skrobek, Grazyna; Maciejewska, Barbara; Delattre, Anne-Sophie; Lavigne, Rob

    2012-12-01

    The emergence of bacteria resistance to most of the currently available antibiotics has become a critical therapeutic problem. The bacteria causing both hospital and community-acquired infections are most often multidrug resistant. In view of the alarming level of antibiotic resistance between bacterial species and difficulties with treatment, alternative or supportive antibacterial cure has to be developed. The presented review focuses on the major characteristics of bacteriophages and phage-encoded proteins affecting their usefulness as antimicrobial agents. We discuss several issues such as mode of action, pharmacodynamics, pharmacokinetics, resistance and manufacturing aspects of bacteriophages and phage-encoded proteins application.

  4. Virally encoded 7TM receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Waldhoer, M; Lüttichau, H R

    2001-01-01

    A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion...... in various parts of the viral life cyclus. Most of the receptors encoded by human pathogenic virus are still orphan receptors, i.e. the endogenous ligand is unknown. In the few cases where it has been possible to characterize these receptors pharmacologically, they have been found to bind a broad spectrum...... expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets....

  5. Defining a role for Hfq in Gram-positive bacteria

    DEFF Research Database (Denmark)

    Nielsen, Jesper Sejrup; Lei, Lisbeth Kristensen; Ebersbach, Tine

    2010-01-01

    Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species...... mechanism, and that Hfq facilitates the binding of LhrA to its target. The work presented here provides the first experimental evidence for Hfq-dependent riboregulation in a Gram-positive bacterium. Our findings indicate that modulation of translation by trans-encoded sRNAs may occur by both Hfq...

  6. Synaptic encoding of temporal contiguity

    Directory of Open Access Journals (Sweden)

    Srdjan eOstojic

    2013-04-01

    Full Text Available Often we need to perform tasks in an environment that changes stochastically. In these situations it is important to learn the statistics of sequences of events in order to predict the future and the outcome of our actions. The statistical description of many of these sequences can be reduced to the set of probabilities that a particular event follows another event (temporal contiguity. Under these conditions, it is important to encode and store in our memory these transition probabilities. Here we show that for a large class of synaptic plasticity models, the distribution of synaptic strengths encodes transitions probabilities. Specifically, when the synaptic dynamics depend on pairs of contiguous events and the synapses can remember multiple instances of the transitions, then the average synaptic weights are a monotonic function of the transition probabilities. The synaptic weights converge to the distribution encoding the probabilities also when the correlations between consecutive synaptic modifications are considered. We studied how this distribution depends on the number of synaptic states for a specific model of a multi-state synapse with hard bounds. In the case of bistable synapses, the average synaptic weights are a smooth function of the transition probabilities and the accuracy of the encoding depends on the learning rate. As the number of synaptic states increases, the average synaptic weights become a step function of the transition probabilities. We finally show that the information stored in the synaptic weights can be read out by a simple rate-based neural network. Our study shows that synapses encode transition probabilities under general assumptions and this indicates that temporal contiguity is likely to be encoded and harnessed in almost every neural circuit in the brain.

  7. Bacteriophage-encoded shiga toxin gene in atypical bacterial host

    Directory of Open Access Journals (Sweden)

    Casas Veronica

    2011-07-01

    Full Text Available Abstract Background Contamination from fecal bacteria in recreational waters is a major health concern since bacteria capable of causing human disease can be found in animal feces. The Dog Beach area of Ocean Beach in San Diego, California is a beach prone to closures due to high levels of fecal indicator bacteria (FIB. A potential source of these FIB could be the canine feces left behind by owners who do not clean up after their pets. We tested this hypothesis by screening the DNA isolated from canine feces for the bacteriophage-encoded stx gene normally found in the virulent strains of the fecal bacterium Escherichia coli. Results Twenty canine fecal samples were collected, processed for total and bacterial fraction DNA, and screened by PCR for the stx gene. The stx gene was detected in the total and bacterial fraction DNA of one fecal sample. Bacterial isolates were then cultivated from the stx-positive fecal sample. Eighty nine of these canine fecal bacterial isolates were screened by PCR for the stx gene. The stx gene was detected in five of these isolates. Sequencing and phylogenetic analyses of 16S rRNA gene PCR products from the canine fecal bacterial isolates indicated that they were Enterococcus and not E. coli. Conclusions The bacteriophage-encoded stx gene was found in multiple species of bacteria cultivated from canine fecal samples gathered at the shoreline of the Dog Beach area of Ocean Beach in San Diego, California. The canine fecal bacteria carrying the stx gene were not the typical E. coli host and were instead identified through phylogenetic analyses as Enterococcus. This suggests a large degree of horizontal gene transfer of exotoxin genes in recreational waters.

  8. Manganese (Mn) Oxidation Increases Intracellular Mn in Pseudomonas putida GB-1

    Science.gov (United States)

    Banh, Andy; Chavez, Valarie; Doi, Julia; Nguyen, Allison; Hernandez, Sophia; Ha, Vu; Jimenez, Peter; Espinoza, Fernanda; Johnson, Hope A.

    2013-01-01

    Bacterial manganese (Mn) oxidation plays an important role in the global biogeochemical cycling of Mn and other compounds, and the diversity and prevalence of Mn oxidizers have been well established. Despite many hypotheses of why these bacteria may oxidize Mn, the physiological reasons remain elusive. Intracellular Mn levels were determined for Pseudomonas putida GB-1 grown in the presence or absence of Mn by inductively coupled plasma mass spectrometry (ICP-MS). Mn oxidizing wild type P. putida GB-1 had higher intracellular Mn than non Mn oxidizing mutants grown under the same conditions. P. putida GB-1 had a 5 fold increase in intracellular Mn compared to the non Mn oxidizing mutant P. putida GB-1-007 and a 59 fold increase in intracellular Mn compared to P. putida GB-1 ∆2665 ∆2447. The intracellular Mn is primarily associated with the less than 3 kDa fraction, suggesting it is not bound to protein. Protein oxidation levels in Mn oxidizing and non oxidizing cultures were relatively similar, yet Mn oxidation did increase survival of P. putida GB-1 when oxidatively stressed. This study is the first to link Mn oxidation to Mn homeostasis and oxidative stress protection. PMID:24147089

  9. The role of TREM-2 in internalization and intracellular survival of Brucella abortus in murine macrophages.

    Science.gov (United States)

    Wei, Pan; Lu, Qiang; Cui, Guimei; Guan, Zhenhong; Yang, Li; Sun, Changjiang; Sun, Wanchun; Peng, Qisheng

    2015-02-15

    Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor primarily expressed on macrophages and dendritic cells. TREM-2 functions as a phagocytic receptor for bacteria as well as an inhibitor of Toll like receptors (TLR) induced inflammatory cytokines. However, the role of TREM-2 in Brucella intracellular growth remains unknown. To investigate whether TREM-2 is involved in Brucella intracellular survival, we chose bone marrow derived macrophages (BMDMs), in which TREM-2 is stably expressed, as cell model. Colony formation Units (CFUs) assay suggests that TREM-2 is involved in the internalization of Brucella abortus (B. abortus) by macrophages, while silencing of TREM-2 decreases intracellular survival of B. abortus. To further study the underlying mechanisms of TREM-2-mediated bacterial intracellular survival, we examined the activation of B. abortus-infected macrophages through determining the kinetics of activation of the three MAPKs, including ERK, JNK and p38, and measuring TNFα production in response to lipopolysaccharide (LPS) of Brucella (BrLPS) or B. abortus stimulation. Our data show that TREM-2 deficiency promotes activation of Brucella-infected macrophages. Moreover, our data also demonstrate that macrophage activation promotes killing of Brucella by enhancing nitric oxygen (NO), but not reactive oxygen species (ROS) production, macrophage apoptosis or cellular death. Taken together, these findings provide a novel interpretation of Brucella intracellular growth through inhibition of NO production produced by TREM-2-mediated activated macrophages.

  10. IcgA is a virulence factor of Rhodococcus equi that modulates intracellular growth.

    Science.gov (United States)

    Wang, Xiaoguang; Coulson, Garry B; Miranda-Casoluengo, Aleksandra A; Miranda-Casoluengo, Raúl; Hondalus, Mary K; Meijer, Wim G

    2014-05-01

    Virulence of the intracellular pathogen Rhodococcus equi depends on a 21.3-kb pathogenicity island located on a conjugative plasmid. To date, the only nonregulatory pathogenicity island-encoded virulence factor identified is the cell envelope-associated VapA protein. Although the pathogenicity islands from porcine and equine R. equi isolates have undergone major rearrangements, the virR operon (virR-icgA-vapH-orf7-virS) is highly conserved in both, suggesting these genes play an important role in pathogenicity. VirR and VirS are transcriptional regulators controlling expression of pathogenicity island genes, including vapA. Here, we show that while vapH and orf7 are dispensable for intracellular growth of R. equi, deletion of icgA, formerly known as orf5, encoding a major facilitator superfamily transport protein, elicited an enhanced growth phenotype in macrophages and a significant reduction in macrophage viability, while extracellular growth in broth remained unaffected. Transcription of virS, located downstream of icgA, and vapA was not affected by the icgA deletion during growth in broth or in macrophages, showing that the enhanced growth phenotype caused by deletion of icgA was not mediated through abnormal transcription of these genes. Transcription of icgA increased 6-fold within 2 h following infection of macrophages and remained significantly higher 48 h postinfection compared to levels at the start of the infection. The major facilitator superfamily transport protein IcgA is the first factor identified in R. equi that negatively affects intracellular replication. Aside from VapA, it is only the second pathogenicity island-encoded structural protein shown to play a direct role in intracellular growth of this pathogenic actinomycete.

  11. New combined assay of phagocytosis and intracellular killing of Escherichia coli by polymorphonuclear leukocytes

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, P.J.; Ford, J.M. (Saint Bartholomew' s Hospital, London (UK))

    1982-03-12

    A new combined radiometric assay is described in which adherence, and phagocytosis and killing of Escherichia coli by human polymorphonuclear leucocytes (PMN) are simultaneously measured in the same sample. Pure monolayers of PMN in Petri dishes are allowed to ingest (/sup 14/C)phenylalanine labelled E. coli and excess bacteria are removed by washing. A period of incubation allows intracellular killing to occur while polymyxin-B is added to half the dishes to kill extracellular bacteria. The remaining viable bacteria in all dishes are labelled with (/sup 3/H)thymidine. The number of ingested bacteria and the percentage of intracellular organisms killed is determined from the /sup 14/C and /sup 3/H counts by a simple subtraction technique. By performing protein assays on representative monolayers, the number of PMN adhered in the monolayers and hence the mean bacterial uptake per PMN is estimated. The assay detected killing efficiencies reduced below the normal range, in monolayers treated with sodium azide, phenylbutazone, in polymorphonuclear leukocytes from patients with chronic granulomatous disease, and in immature neutrophils from the promyelocytic leukaemic cell line, HL60. The assay was adapted to measure phagocytosis and killing by cells in suspension.

  12. Exploration of the core metabolism of symbiotic bacteria.

    Science.gov (United States)

    Klein, Cecilia Coimbra; Cottret, Ludovic; Kielbassa, Janice; Charles, Hubert; Gautier, Christian; Ribeiro de Vasconcelos, Ana Tereza; Lacroix, Vincent; Sagot, Marie-France

    2012-08-31

    A large number of genome-scale metabolic networks is now available for many organisms, mostly bacteria. Previous works on minimal gene sets, when analysing host-dependent bacteria, found small common sets of metabolic genes. When such analyses are restricted to bacteria with similar lifestyles, larger portions of metabolism are expected to be shared and their composition is worth investigating. Here we report a comparative analysis of the small molecule metabolism of symbiotic bacteria, exploring common and variable portions as well as the contribution of different lifestyle groups to the reduction of a common set of metabolic capabilities. We found no reaction shared by all the bacteria analysed. Disregarding those with the smallest genomes, we still do not find a reaction core, however we did find a core of biochemical capabilities. While obligate intracellular symbionts have no core of reactions within their group, extracellular and cell-associated symbionts do have a small core composed of disconnected fragments. In agreement with previous findings in Escherichia coli, their cores are enriched in biosynthetic processes whereas the variable metabolisms have similar ratios of biosynthetic and degradation reactions. Conversely, the variable metabolism of obligate intracellular symbionts is enriched in anabolism. Even when removing the symbionts with the most reduced genomes, there is no core of reactions common to the analysed symbiotic bacteria. The main reason is the very high specialisation of obligate intracellular symbionts, however, host-dependence alone is not an explanation for such absence. The composition of the metabolism of cell-associated and extracellular bacteria shows that while they have similar needs in terms of the building blocks of their cells, they have to adapt to very distinct environments. On the other hand, in obligate intracellular bacteria, catabolism has largely disappeared, whereas synthetic routes appear to have been selected for

  13. Genomics of Probiotic Bacteria

    Science.gov (United States)

    O'Flaherty, Sarah; Goh, Yong Jun; Klaenhammer, Todd R.

    Probiotic bacteria from the Lactobacillus and Bifidobacterium species belong to the Firmicutes and the Actinobacteria phylum, respectively. Lactobacilli are members of the lactic acid bacteria (LAB) group, a broadly defined family of microorganisms that ferment various hexoses into primarily lactic acid. Lactobacilli are typically low G + C gram-positive species which are phylogenetically diverse, with over 100 species documented to date. Bifidobacteria are heterofermentative, high G + C content bacteria with about 30 species of bifidobacteria described to date.

  14. Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

    Science.gov (United States)

    Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

    2015-06-21

    High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics.

  15. Functional characterization of the incomplete phosphotransferase system (PTS of the intracellular pathogen Brucella melitensis.

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    Marie Dozot

    Full Text Available BACKGROUND: In many bacteria, the phosphotransferase system (PTS is a key player in the regulation of the assimilation of alternative carbon sources notably through catabolic repression. The intracellular pathogens Brucella spp. possess four PTS proteins (EINtr, NPr, EIIANtr and an EIIA of the mannose family but no PTS permease suggesting that this PTS might serve only regulatory functions. METHODOLOGY/PRINCIPAL FINDINGS: In vitro biochemical analyses and in vivo detection of two forms of EIIANtr (phosphorylated or not established that the four PTS proteins of Brucella melitensis form a functional phosphorelay. Moreover, in vitro the protein kinase HprK/P phosphorylates NPr on a conserved serine residue, providing an additional level of regulation to the B. melitensis PTS. This kinase activity was inhibited by inorganic phosphate and stimulated by fructose-1,6 bisphosphate. The genes encoding HprK/P, an EIIAMan-like protein and NPr are clustered in a locus conserved among α-proteobacteria and also contain the genes for the crucial two-component system BvrR-BvrS. RT-PCR revealed a transcriptional link between these genes suggesting an interaction between PTS and BvrR-BvrS. Mutations leading to the inactivation of EINtr or NPr significantly lowered the synthesis of VirB proteins, which form a type IV secretion system. These two mutants also exhibit a small colony phenotype on solid media. Finally, interaction partners of PTS proteins were identified using a yeast two hybrid screen against the whole B. melitensis ORFeome. Both NPr and HprK/P were shown to interact with an inorganic pyrophosphatase and the EIIAMan-like protein with the E1 component (SucA of 2-oxoglutarate dehydrogenase. CONCLUSIONS/SIGNIFICANCE: The B. melitensis can transfer the phosphoryl group from PEP to the EIIAs and a link between the PTS and the virulence of this organism could be established. Based on the protein interaction data a preliminary model is proposed in which

  16. GTPases in intracellular trafficking: an overview.

    Science.gov (United States)

    Segev, Nava

    2011-02-01

    Small GTPases that belong to the ras sub-families of Rab, Arf, and Rho, and the large GTPase dynamin, regulate intracellular trafficking. This issue of Seminars of Cell and Developmental Biology highlights topics regarding mechanisms by which these GTPases regulate the different steps of vesicular transport: vesicle formation, scission, targeting and fusion. In addition, the emerging roles of GTPases in coordination of individual transport steps as well as coordination of intracellular trafficking with other cellular processes are reviewed. Finally, common structures and mechanisms underlying the function of the ras-like GTPases and the importance of their function to human health and disease are discussed.

  17. Nitric oxide from IFNγ-primed macrophages modulates the antimicrobial activity of β-lactams against the intracellular pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella.

    Directory of Open Access Journals (Sweden)

    Jessica Jones-Carson

    2014-08-01

    Full Text Available Our investigations show that nonlethal concentrations of nitric oxide (NO abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2, while stimulating hydrogen peroxide (H2O2 synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF, and thus the dissipation of ΔH+ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics.

  18. Sulfur cycling and metabolism of phototrophic and filamentous sulfur bacteria

    Science.gov (United States)

    Guerrero, R.; Brune, D.; Poplawski, R.; Schmidt, T. M.

    1985-01-01

    Phototrophic sulfur bacteria taken from different habitate (Alum Rock State Park, Palo Alto salt marsh, and Big Soda Lake) were grown on selective media, characterized by morphological and pigment analysis, and compared with bacteria maintained in pure culture. A study was made of the anaerobic reduction of intracellular sulfur globules by a phototrophic sulfur bacterium (Chromatium vinosum) and a filamentous aerobic sulfur bacterium (Beggiatoa alba). Buoyant densities of different bacteria were measured in Percoll gradients. This method was also used to separate different chlorobia in mixed cultures and to assess the relative homogeneity of cultures taken directly or enriched from natural samples (including the purple bacterial layer found at a depth of 20 meters at Big Soda Lake.) Interactions between sulfide oxidizing bacteria were studied.

  19. Intracellular protozoan parasites of humans: the role of molecular chaperones in development and pathogenesis.

    Science.gov (United States)

    Shonhai, Addmore; Maier, Alexander G; Przyborski, Jude M; Blatch, Gregory L

    2011-02-01

    Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.

  20. A Rebeccamycin Analog Provides Plasmid-Encoded Niche Defense.

    Science.gov (United States)

    Van Arnam, Ethan B; Ruzzini, Antonio C; Sit, Clarissa S; Currie, Cameron R; Clardy, Jon

    2015-11-18

    Bacterial symbionts of fungus-growing ants occupy a highly specialized ecological niche and face the constant existential threat of displacement by another strain of ant-adapted bacteria. As part of a systematic study of the small molecules underlying this fraternal competition, we discovered an analog of the antitumor agent rebeccamycin, a member of the increasingly important indolocarbazole family. While several gene clusters consistent with this molecule's newly reported modification had previously been identified in metagenomic studies, the metabolite itself has been cryptic. The biosynthetic gene cluster for 9-methoxyrebeccamycin is encoded on a plasmid in a manner reminiscent of plasmid-derived peptide antimicrobials that commonly mediate antagonism among closely related Gram-negative bacteria.

  1. How honey kills bacteria

    NARCIS (Netherlands)

    P.H.S. Kwakman; A.A. te Velde; L. de Boer; D. Speijer; C.M.J.E. Vandenbroucke-Grauls; S.A.J. Zaat

    2010-01-01

    With the rise in prevalence of antibiotic-resistant bacteria, honey is increasingly valued for its antibacterial activity. To characterize all bactericidal factors in a medical-grade honey, we used a novel approach of successive neutralization of individual honey bactericidal factors. All bacteria t

  2. Encoding information into precipitation structures

    Science.gov (United States)

    Martens, Kirsten; Bena, Ioana; Droz, Michel; Rácz, Zoltan

    2008-12-01

    Material design at submicron scales would be profoundly affected if the formation of precipitation patterns could be easily controlled. It would allow the direct building of bulk structures, in contrast to traditional techniques which consist of removing material in order to create patterns. Here, we discuss an extension of our recent proposal of using electrical currents to control precipitation bands which emerge in the wake of reaction fronts in A+ + B- → C reaction-diffusion processes. Our main result, based on simulating the reaction-diffusion-precipitation equations, is that the dynamics of the charged agents can be guided by an appropriately designed time-dependent electric current so that, in addition to the control of the band spacing, the width of the precipitation bands can also be tuned. This makes straightforward the encoding of information into precipitation patterns and, as an amusing example, we demonstrate the feasibility by showing how to encode a musical rhythm.

  3. The Vacuolar ATPase from Entamoeba histolytica: Molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein

    Directory of Open Access Journals (Sweden)

    Luna-Arias Juan

    2008-12-01

    Full Text Available Abstract Background Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. Results We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. Conclusion We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits

  4. Endosymbiotic calcifying bacteria across sponge species and oceans

    Science.gov (United States)

    Garate, Leire; Sureda, Jan; Agell, Gemma; Uriz, Maria J.

    2017-01-01

    From an evolutionary point of view, sponges are ideal targets to study marine symbioses as they are the most ancient living metazoans and harbour highly diverse microbial communities. A recently discovered association between the sponge Hemimycale columella and an intracellular bacterium that generates large amounts of calcite spherules has prompted speculation on the possible role of intracellular bacteria in the evolution of the skeleton in early animals. To gain insight into this purportedly ancestral symbiosis, we investigated the presence of symbiotic bacteria in Mediterranean and Caribbean sponges. We found four new calcibacteria OTUs belonging to the SAR116 in two orders (Poecilosclerida and Clionaida) and three families of Demospongiae, two additional OTUs in cnidarians and one more in seawater (at 98.5% similarity). Using a calcibacteria targeted probe and CARD-FISH, we also found calcibacteria in Spirophorida and Suberitida and proved that the calcifying bacteria accumulated at the sponge periphery, forming a skeletal cortex, analogous to that of siliceous microscleres in other demosponges. Bacteria-mediated skeletonization is spread in a range of phylogenetically distant species and thus the purported implication of bacteria in skeleton formation and evolution of early animals gains relevance. PMID:28262822

  5. Endosymbiotic calcifying bacteria across sponge species and oceans

    Science.gov (United States)

    Garate, Leire; Sureda, Jan; Agell, Gemma; Uriz, Maria J.

    2017-03-01

    From an evolutionary point of view, sponges are ideal targets to study marine symbioses as they are the most ancient living metazoans and harbour highly diverse microbial communities. A recently discovered association between the sponge Hemimycale columella and an intracellular bacterium that generates large amounts of calcite spherules has prompted speculation on the possible role of intracellular bacteria in the evolution of the skeleton in early animals. To gain insight into this purportedly ancestral symbiosis, we investigated the presence of symbiotic bacteria in Mediterranean and Caribbean sponges. We found four new calcibacteria OTUs belonging to the SAR116 in two orders (Poecilosclerida and Clionaida) and three families of Demospongiae, two additional OTUs in cnidarians and one more in seawater (at 98.5% similarity). Using a calcibacteria targeted probe and CARD-FISH, we also found calcibacteria in Spirophorida and Suberitida and proved that the calcifying bacteria accumulated at the sponge periphery, forming a skeletal cortex, analogous to that of siliceous microscleres in other demosponges. Bacteria-mediated skeletonization is spread in a range of phylogenetically distant species and thus the purported implication of bacteria in skeleton formation and evolution of early animals gains relevance.

  6. Metallization of bacteria cells

    Institute of Scientific and Technical Information of China (English)

    黎向锋; 李雅芹; 蔡军; 张德远

    2003-01-01

    Bacteria cells with different standard shapes are well suited for use as templates for the fabrication of magnetic and electrically conductive microstructures. In this paper, metallization of bacteria cells is demonstrated by an electroless deposition technique of nickel-phosphorus initiated by colloid palladium-tin catalyst on the surfaces of Citeromyces matritensis and Bacillus cereus. The activated and metallized bacteria cells have been characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction analysis (XRD). Results showed that both Citeromyces matritensis and Bacillus cereus had no deformation in shape after metallization; the metallized films deposited on the surfaces of bacteria cells are homogeneous in thickness and noncrystalline in phase structure. The kinetics of colloid palladium-tin solution and electroless plating on bacteria cells is discussed.

  7. Geometric Hyperplanes: Desargues Encodes Doily

    CERN Document Server

    Saniga, Metod

    2011-01-01

    It is shown that the structure of the generalized quadrangle of order two is fully encoded in the properties of the Desargues configuration. A point of the quadrangle is represented by a geometric hyperplane of the Desargues configuration and its line by a set of three hyperplanes such that one of them is the complement of the symmetric difference of the remaining two and they all share a pair of non-collinear points.

  8. Transcriptomic Analysis of Yersinia enterocolitica Biovar 1B Infecting Murine Macrophages Reveals New Mechanisms of Extracellular and Intracellular Survival.

    Science.gov (United States)

    Bent, Zachary W; Poorey, Kunal; Brazel, David M; LaBauve, Annette E; Sinha, Anupama; Curtis, Deanna J; House, Samantha E; Tew, Karen E; Hamblin, Rachelle Y; Williams, Kelly P; Branda, Steven S; Young, Glenn M; Meagher, Robert J

    2015-07-01

    Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocolitica biovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26 °C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37 °C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

  9. Vector Encoding in Biochemical Networks

    Science.gov (United States)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  10. [Magnetic nanoparticles and intracellular delivery of biopolymers].

    Science.gov (United States)

    Kornev, A A; Dubina, M V

    2014-03-01

    The basic methods of intracellular delivery of biopolymers are present in this review. The structure and synthesis of magnetic nanoparticles, their stabilizing surfactants are described. The examples of the interaction of nanoparticles with biopolymers such as nucleic acids and proteins are considered. The final part of the review is devoted to problems physiology and biocompatibility of magnetic nanoparticles.

  11. Real-time detection of viable microorganisms by intracellular phototautomerism

    Directory of Open Access Journals (Sweden)

    Schuren Frank

    2010-06-01

    Full Text Available Abstract Background To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner. Results The developed method includes exposure of cells to a weak acid probe at low pH. The neutral probe rapidly permeates the membrane and enters the cytosol. In dead cells no signal is obtained, as the cytosolic pH reflects that of the acidic extracellular environment. In live cells with a neutral internal pH, the probe dissociates into a fluorescent phototautomeric anion. After reaching peak fluorescence, the population of live cells decays. This decay can be followed real-time as cell death coincides with intracellular acidification and return of the probe to its uncharged non-fluorescent state. The rise and decay of the fluorescence signal depends on the probe structure and appears discriminative for bacteria, fungi, and spores. We identified 13 unique probes, which can be applied in the real-time viability method described here. Under the experimental conditions used in a microplate reader, the reported method shows a detection limit of 106 bacteria ml-1, while the frequently used LIVE/DEAD BacLight™ Syto9 and propidium iodide stains show detection down to 106 and 107 bacteria ml-1, respectively. Conclusions We present a novel fluorescence-based method for viability assessment, which is applicable to all bacteria and eukaryotic cell types tested so far. The RTV method will have a significant impact in many areas of applied microbiology including research on biocidal activity, improvement of

  12. Subversion of inflammasome activation and pyroptosis by pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Larissa D Cunha

    2013-11-01

    Full Text Available Activation of the inflammasome occurs in response to a notably high number of pathogenic microbes and is a broad innate immune response that effectively contributes to restriction of pathogen replication and generation of adaptive immunity. Activation of these platforms leads to caspase-1- and/or caspase-11-dependent secretion of proteins, including cytokines, and induction of a specific form of cell death called pyroptosis, which directly or indirectly contribute for restriction of pathogen replication. Not surprisingly, bona fide intracellular pathogens developed strategies for manipulation of cell death to guarantee intracellular replication. In this sense, the remarkable advances in the knowledge of the inflammasome field have been accompanied by several reports characterizing the inhibition of this platform by several pathogenic bacteria. Herein, we review some processes used by pathogenic bacteria, including Yersinia spp., Pseudomonas aeruginosa, Vibrio parahaemolyticus, Chlamydia trachomatis, Francisella tularensis, Shigella flexneri, Legionella pneumophila and Coxiella burnetii to evade the activation of the inflammasome and the induction of pyroptosis.

  13. Regulation of persistent sodium currents by glycogen synthase kinase 3 encodes daily rhythms of neuronal excitability

    Science.gov (United States)

    Paul, Jodi R.; Dewoskin, Daniel; McMeekin, Laura J.; Cowell, Rita M.; Forger, Daniel B.; Gamble, Karen L.

    2016-11-01

    How neurons encode intracellular biochemical signalling cascades into electrical signals is not fully understood. Neurons in the central circadian clock in mammals provide a model system to investigate electrical encoding of biochemical timing signals. Here, using experimental and modelling approaches, we show how the activation of glycogen synthase kinase 3 (GSK3) contributes to neuronal excitability through regulation of the persistent sodium current (INaP). INaP exhibits a day/night difference in peak magnitude and is regulated by GSK3. Using mathematical modelling, we predict and confirm that GSK3 activation of INaP affects the action potential afterhyperpolarization, which increases the spontaneous firing rate without affecting the resting membrane potential. Together, these results demonstrate a crucial link between the molecular circadian clock and electrical activity, providing examples of kinase regulation of electrical activity and the propagation of intracellular signals in neuronal networks.

  14. Hijacking host cell highways: manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens

    Directory of Open Access Journals (Sweden)

    Punsiri M Colonne

    2016-09-01

    Full Text Available Intracellular bacterial pathogens replicate within eukaryotic cells and display unique adaptations that support key infection events including invasion, replication, immune evasion, and dissemination. From invasion to dissemination, all stages of the intracellular bacterial life cycle share the same three-dimensional cytosolic space containing the host cytoskeleton. For successful infection and replication, many pathogens hijack the cytoskeleton using effector proteins introduced into the host cytosol by specialized secretion systems. A subset of effectors contains eukaryotic-like motifs that mimic host proteins to exploit signaling and modify specific cytoskeletal components such as actin and microtubules. Cytoskeletal rearrangement promotes numerous events that are beneficial to the pathogen, including internalization of bacteria, subversion of cell intrinsic immunity, structural support for bacteria-containing vacuoles, altered vesicular trafficking, actin-dependent bacterial movement, and pathogen dissemination. This review highlights a diverse group of obligate intracellular bacterial pathogens that manipulate the host cytoskeleton to thrive within eukaryotic cells and discusses underlying molecular mechanisms that promote these dynamic host-pathogen interactions.

  15. Economic game theory to model the attenuation of virulence of an obligate intracellular bacterium

    Directory of Open Access Journals (Sweden)

    Damian Tago

    2016-08-01

    Full Text Available Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host’s defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g. with Ehrlichia ruminantium, there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria.

  16. Anhydrobiotic engineering of bacterial and mammalian cells: is intracellular trehalose sufficient?

    Science.gov (United States)

    Tunnacliffe, A; García de Castro, A; Manzanera, M

    2001-09-01

    Anhydrobiotic engineering aims to confer a high degree of desiccation tolerance on otherwise sensitive living organisms and cells by adopting the strategies of anhydrobiosis. Nonreducing disaccharides such as trehalose and sucrose are thought to play a pivotal role in resistance to desiccation stress in many microorganisms, invertebrates, and plants, and in vitro trehalose is known to confer stability on dried biomolecules and biomembranes. We have therefore tested the hypothesis that intracellular trehalose (or a similar molecule) may be not only necessary for anhydrobiosis but also sufficient. High concentrations of trehalose were produced in bacteria by osmotic preconditioning, and in mammalian cells by genetic engineering, but in neither system was desiccation tolerance similar to that seen in anhydrobiotic organisms, suggesting that trehalose alone is not sufficient for anhydrobiosis. In Escherichia coli such desiccation tolerance was achievable, but only when bacteria were dried in the presence of both extracellular trehalose and intracellular trehalose. In mouse L cells, improved osmotolerance was observed with up to 100 mM intracellular trehalose, but desiccation was invariably lethal even with extracellular trehalose present. We conclude that anhydrobiotic engineering of at least some microorganisms is achievable with present technology, but that further advances are needed for similar desiccation tolerance of mammalian cells. Copyright 2001 Elsevier Science (USA).

  17. Autophagy Induced by Intracellular Infection of Propionibacterium acnes

    Science.gov (United States)

    Nakamura, Teruko; Furukawa, Asuka; Uchida, Keisuke; Ogawa, Tomohisa; Tamura, Tomoki; Sakonishi, Daisuke; Wada, Yuriko; Suzuki, Yoshimi; Ishige, Yuki; Minami, Junko; Akashi, Takumi

    2016-01-01

    Background Sarcoidosis is caused by Th1-type immune responses to unknown agents, and is linked to the infectious agent Propionibacterium acnes. Many strains of P. acnes isolated from sarcoid lesions cause intracellular infection and autophagy may contribute to the pathogenesis of sarcoidosis. We examined whether P. acnes induces autophagy. Methods Three cell lines from macrophages (Raw264.7), mesenchymal cells (MEF), and epithelial cells (HeLa) were infected by viable or heat-killed P. acnes (clinical isolate from sarcoid lymph node) at a multiplicity of infection (MOI) of 100 or 1000 for 1 h. Extracellular bacteria were killed by washing and culturing infected cells with antibiotics. Samples were examined by colony assay, electron-microscopy, and fluorescence-microscopy with anti-LC3 and anti-LAMP1 antibodies. Autophagy-deficient (Atg5-/-) MEF cells were also used. Results Small and large (≥5 μm in diameter) LC3-positive vacuoles containing few or many P. acnes cells (LC3-positive P. acnes) were frequently found in the three cell lines when infected by viable P. acnes at MOI 1000. LC3-positive large vacuoles were mostly LAMP1-positive. A few small LC3-positive/LAMP1-negative vacuoles were consistently observed in some infected cells for 24 h postinfection. The number of LC3-positive P. acnes was decreased at MOI 100 and completely abolished when heat-killed P. acnes was used. LC3-positive P. acnes was not found in autophagy-deficient Atg5-/- cells where the rate of infection was 25.3 and 17.6 times greater than that in wild-type Atg5+/+ cells at 48 h postinfection at MOI 100 and 1000, respectively. Electron-microscopic examination revealed bacterial cells surrounded mostly by a single-membrane including the large vacuoles and sometimes a double or multi-layered membrane, with occasional undigested bacterial cells in ruptured late endosomes or in the cytoplasm. Conclusion Autophagy was induced by intracellular P. acnes infection and contributed to intracellular

  18. Genome evolution in an ancient bacteria-ant symbiosis: parallel gene loss among Blochmannia spanning the origin of the ant tribe Camponotini

    Directory of Open Access Journals (Sweden)

    Laura E. Williams

    2015-04-01

    coresident symbionts other than Wolbachia. Although gene order is strictly conserved in four Blochmannia of Camponotus sensu stricto, comparisons with deeply divergent lineages revealed inversions in eight genomic regions, indicating ongoing recombination despite ancestral loss of recA. In sum, the addition of two Blochmannia genomes of divergent host lineages enables reconstruction of early events in evolution of this symbiosis and suggests that Blochmannia lineages may experience distinct, host-associated selective pressures. Understanding how evolutionary forces shape genome reduction in this system may help to clarify forces driving gene loss in other bacteria, including intracellular pathogens.

  19. Metabolic engineering of bacteria for ethanol production

    Energy Technology Data Exchange (ETDEWEB)

    Ingram, L.O.; Gomez, P.F.; Lai, X.; Moniruzzaman, M.; Wood, B.E.; Yomano, L.P.; York, S.W. [Univ. of Florida, Gainesville, FL (United States). Dept. of Microbiology and Cell Science

    1998-04-20

    Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. The authors` work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes.

  20. Antibiotics from predatory bacteria

    Directory of Open Access Journals (Sweden)

    Juliane Korp

    2016-03-01

    Full Text Available Bacteria, which prey on other microorganisms, are commonly found in the environment. While some of these organisms act as solitary hunters, others band together in large consortia before they attack their prey. Anecdotal reports suggest that bacteria practicing such a wolfpack strategy utilize antibiotics as predatory weapons. Consistent with this hypothesis, genome sequencing revealed that these micropredators possess impressive capacities for natural product biosynthesis. Here, we will present the results from recent chemical investigations of this bacterial group, compare the biosynthetic potential with that of non-predatory bacteria and discuss the link between predation and secondary metabolism.

  1. Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines

    Science.gov (United States)

    Hoshina, Shiho; Sekizuka, Tsuyoshi; Kataoka, Michiyo; Hasegawa, Hideki; Hamada, Hiromichi; Kuroda, Makoto; Katano, Harutaka

    2016-01-01

    Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein—Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. PMID:27611973

  2. Sterol Synthesis in Diverse Bacteria.

    Science.gov (United States)

    Wei, Jeremy H; Yin, Xinchi; Welander, Paula V

    2016-01-01

    Sterols are essential components of eukaryotic cells whose biosynthesis and function has been studied extensively. Sterols are also recognized as the diagenetic precursors of steranes preserved in sedimentary rocks where they can function as geological proxies for eukaryotic organisms and/or aerobic metabolisms and environments. However, production of these lipids is not restricted to the eukaryotic domain as a few bacterial species also synthesize sterols. Phylogenomic studies have identified genes encoding homologs of sterol biosynthesis proteins in the genomes of several additional species, indicating that sterol production may be more widespread in the bacterial domain than previously thought. Although the occurrence of sterol synthesis genes in a genome indicates the potential for sterol production, it provides neither conclusive evidence of sterol synthesis nor information about the composition and abundance of basic and modified sterols that are actually being produced. Here, we coupled bioinformatics with lipid analyses to investigate the scope of bacterial sterol production. We identified oxidosqualene cyclase (Osc), which catalyzes the initial cyclization of oxidosqualene to the basic sterol structure, in 34 bacterial genomes from five phyla (Bacteroidetes, Cyanobacteria, Planctomycetes, Proteobacteria, and Verrucomicrobia) and in 176 metagenomes. Our data indicate that bacterial sterol synthesis likely occurs in diverse organisms and environments and also provides evidence that there are as yet uncultured groups of bacterial sterol producers. Phylogenetic analysis of bacterial and eukaryotic Osc sequences confirmed a complex evolutionary history of sterol synthesis in this domain. Finally, we characterized the lipids produced by Osc-containing bacteria and found that we could generally predict the ability to synthesize sterols. However, predicting the final modified sterol based on our current knowledge of sterol synthesis was difficult. Some bacteria

  3. Trade-Offs of Escherichia coli Adaptation to an Intracellular Lifestyle in Macrophages.

    Directory of Open Access Journals (Sweden)

    M Azevedo

    Full Text Available The bacterium Escherichia coli exhibits remarkable genomic and phenotypic variation, with some pathogenic strains having evolved to survive and even replicate in the harsh intra-macrophage environment. The rate and effects of mutations that can cause pathoadaptation are key determinants of the pace at which E. coli can colonize such niches and become pathogenic. We used experimental evolution to determine the speed and evolutionary paths undertaken by a commensal strain of E. coli when adapting to intracellular life. We estimated the acquisition of pathoadaptive mutations at a rate of 10-6 per genome per generation, resulting in the fixation of more virulent strains in less than a hundred generations. Whole genome sequencing of independently evolved clones showed that the main targets of intracellular adaptation involved loss of function mutations in genes implicated in the assembly of the lipopolysaccharide core, iron metabolism and di- and tri-peptide transport, namely rfaI, fhuA and tppB, respectively. We found a substantial amount of antagonistic pleiotropy in evolved populations, as well as metabolic trade-offs, commonly found in intracellular bacteria with reduced genome sizes. Overall, the low levels of clonal interference detected indicate that the first steps of the transition of a commensal E. coli into intracellular pathogens are dominated by a few pathoadaptive mutations with very strong effects.

  4. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii

    Directory of Open Access Journals (Sweden)

    Salah Shanan

    2016-04-01

    Full Text Available Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria.

  5. Swedish isolates of Vibrio cholerae enhance their survival when interacted intracellularly with Acanthamoeba castellanii.

    Science.gov (United States)

    Shanan, Salah; Bayoumi, Magdi; Saeed, Amir; Sandström, Gunnar; Abd, Hadi

    2016-01-01

    Vibrio cholerae is a Gram-negative bacterium that occurs naturally in aquatic environment. Only V. cholerae O1 and V. cholerae O139 produce cholera toxin and cause cholera, other serogroups can cause gastroenteritis, open wounds infection, and septicaemia. V. cholerae O1 and V. cholerae O139 grow and survive inside Acanthamoeba castellanii. The aim of this study is to investigate the interactions of the Swedish clinical isolates V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 with A. castellanii. The interaction between A. castellanii and V. cholerae strains was studied by means of amoeba cell counts, viable counts of the bacteria in the absence or presence of amoebae, and of the intracellularly growing bacteria, visualised by electron microscopy. These results show that all V. cholerae can grow and survive outside and inside the amoebae, disclosing that V. cholerae O3, V. cholerae O4, V. cholerae O5, V. cholerae O11, and V. cholerae O160 all can be considered as facultative intracellular bacteria.

  6. Hall effect encoding of brushless dc motors

    Science.gov (United States)

    Berard, C. A.; Furia, T. J.; Goldberg, E. A.; Greene, R. C.

    1970-01-01

    Encoding mechanism integral to the motor and using the permanent magnets embedded in the rotor eliminates the need for external devices to encode information relating the position and velocity of the rotating member.

  7. [Darwin and bacteria].

    Science.gov (United States)

    Ledermann D, Walter

    2009-02-01

    As in 2009 the scientific world celebrates two hundreds years from the birthday of Charles Darwin and one hundred and fifty from the publication of The Origin of Species, an analysis of his complete work is performed, looking for any mention of bacteria. But it seems that the great naturahst never took knowledge about its existence, something rather improbable in a time when the discovery of bacteria shook the medical world, or he deliberately ignored them, not finding a place for such microscopic beings into his theory of evolution. But the bacteria badly affected his familiar life, killing scarlet fever one of his children and worsening to death the evolution of tuberculosis of his favourite Annie. Darwin himself could suffer the sickness of Chagas, whose etiological agent has a similar level to bacteria in the scale of evolution.

  8. Extracellular communication in bacteria

    DEFF Research Database (Denmark)

    Chhabra, S.R.; Philipp, B.; Eberl, L.

    2005-01-01

    molecules, in different Gram-positive and Gram-negative bacteria they control pathogenicity, secondary metabolite production, biofilm differentiation, DNA transfer and bioluminescence. The development of biosensors for the detection of these signal molecules has greatly facilitated their subsequent chemical...

  9. The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Charles Godbout

    Full Text Available Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.

  10. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    Science.gov (United States)

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

  11. Characterization of the mutualistic endosymbiosis between intracellular bacteria and mealybugs (Hemiptera: Pseudococcidae)

    OpenAIRE

    López Madrigal, Sergio

    2015-01-01

    Simbiosis, del griego sym “con” y biosis “vivir”, hace referencia a la asociación estable entre individuos de dos o más especies (simbiontes) que muestran interdependencia a cualquier nivel biológico. La ubicuidad de las asociaciones simbióticas en las ramas principales del árbol de la vida evidencia la relevancia global de éstas en la evolución de la vida. Las asociaciones más estudiadas son aquellas que se establecen entre procariotas y eucariotas que, habitualmente, suponen la ampliación d...

  12. Intracellular mechanisms of solar water disinfection

    Science.gov (United States)

    Castro-Alférez, María; Polo-López, María Inmaculada; Fernández-Ibáñez, Pilar

    2016-12-01

    Solar water disinfection (SODIS) is a zero-cost intervention measure to disinfect drinking water in areas of poor access to improved water sources, used by more than 6 million people in the world. The bactericidal action of solar radiation in water has been widely proven, nevertheless the causes for this remain still unclear. Scientific literature points out that generation of reactive oxygen species (ROS) inside microorganisms promoted by solar light absorption is the main reason. For the first time, this work reports on the experimental measurement of accumulated intracellular ROS in E. coli during solar irradiation. For this experimental achievement, a modified protocol based on the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA), widely used for oxidative stress in eukaryotic cells, has been tested and validated for E. coli. Our results demonstrate that ROS and their accumulated oxidative damages at intracellular level are key in solar water disinfection.

  13. Dynamics of gradient formation by intracellular shuttling

    Energy Technology Data Exchange (ETDEWEB)

    Berezhkovskii, Alexander M. [Mathematical and Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892 (United States); Shvartsman, Stanislav Y. [Department of Chemical and Biological Engineering and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544 (United States)

    2015-08-21

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  14. Dynamics of gradient formation by intracellular shuttling

    Science.gov (United States)

    Berezhkovskii, Alexander M.; Shvartsman, Stanislav Y.

    2015-08-01

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  15. An intracellular replication niche for Vibrio cholerae in the amoeba Acanthamoeba castellanii.

    Science.gov (United States)

    Van der Henst, Charles; Scrignari, Tiziana; Maclachlan, Catherine; Blokesch, Melanie

    2016-04-01

    Vibrio cholerae is a human pathogen and the causative agent of cholera. The persistence of this bacterium in aquatic environments is a key epidemiological concern, as cholera is transmitted through contaminated water. Predatory protists, such as amoebae, are major regulators of bacterial populations in such environments. Therefore, we investigated the interaction between V. cholerae and the amoeba Acanthamoeba castellanii at the single-cell level. We observed that V. cholerae can resist intracellular killing. The non-digested bacteria were either released or, alternatively, established a replication niche within the contractile vacuole of A. castellanii. V. cholerae was maintained within this compartment even upon encystment. The pathogen ultimately returned to its aquatic habitat through lysis of A. castellanii, a process that was dependent on the production of extracellular polysaccharide by the pathogen. This study reinforces the concept that V. cholerae is a facultative intracellular bacterium and describes a new host-pathogen interaction.

  16. Lipopolysaccharides in diazotrophic bacteria

    Directory of Open Access Journals (Sweden)

    Rodrigo Vassoler Serrato

    2014-09-01

    Full Text Available Biological nitrogen fixation is a process in which the atmospheric nitrogen (N2 is transformed into ammonia (NH3 by a select group of nitrogen-fixing organisms, or diazotrophic bacteria. In order to furnish the biologically useful nitrogen to plants, these bacteria must be in constant molecular communication with their host plants. Some of these molecular plant-microbe interactions are very specific, resulting in a symbiotic relationship between the diazotroph and the host. Others are found between associative diazotrophs and plants, resulting in plant infection and colonization of internal tissues. Independent of the type of ecological interaction, glycans and glycoconjugates produced by these bacteria play an important role in the molecular communication prior and during colonization. Even though exopolysaccharides (EPS and lipochitooligosaccharides (LCO produced by diazotrophic bacteria and released onto the environment have their importance in the microbe-plant interaction, it is the lipopolysaccharides (LPS, anchored on the external membrane of these bacteria, that mediates the direct contact of the diazotroph with the host cells. These molecules are extremely variable among the several species of nitrogen fixing-bacteria, and there are evidences of the mechanisms of infection being closely related to their structure.

  17. Lipopolysaccharides in diazotrophic bacteria.

    Science.gov (United States)

    Serrato, Rodrigo V

    2014-01-01

    Biological nitrogen fixation (BNF) is a process in which the atmospheric nitrogen (N2) is transformed into ammonia (NH3) by a select group of nitrogen-fixing organisms, or diazotrophic bacteria. In order to furnish the biologically useful nitrogen to plants, these bacteria must be in constant molecular communication with their host plants. Some of these molecular plant-microbe interactions are very specific, resulting in a symbiotic relationship between the diazotroph and the host. Others are found between associative diazotrophs and plants, resulting in plant infection and colonization of internal tissues. Independent of the type of ecological interaction, glycans, and glycoconjugates produced by these bacteria play an important role in the molecular communication prior and during colonization. Even though exopolysaccharides (EPS) and lipochitooligosaccharides (LCO) produced by diazotrophic bacteria and released onto the environment have their importance in the microbe-plant interaction, it is the lipopolysaccharides (LPS), anchored on the external membrane of these bacteria, that mediates the direct contact of the diazotroph with the host cells. These molecules are extremely variable among the several species of nitrogen fixing-bacteria, and there are evidences of the mechanisms of infection being closely related to their structure.

  18. [Neurons that encode sound direction].

    Science.gov (United States)

    Peña, J L

    In the auditory system, the inner ear breaks down complex signals into their spectral components, and encodes the amplitude and phase of each. In order to infer sound direction in space, a computation on each frequency component of the sound must be performed. Space specific neurons in the owl s inferior colliculus respond only to sounds coming from a particular direction and represent the results of this computation. The interaural time difference (ITD) and interaural level difference (ILD define the auditory space for the owl and are processed in separate neural pathways. The parallel pathways that process these cues merge in the external nucleus of the inferior colliculus where the space specific neurons are selective to combinations of ITD and ILD. How do inputs from the two sources interact to produce combination selectivity to ITD ILD pairs? A multiplication of postsynaptic potentials tuned to ITD and ILD can account for the subthreshold responses of these neurons to ITD ILD pairs. Examples of multiplication by neurons or neural circuits are scarce, but many computational models assume the existence of this basic operation. The owl s auditory system uses such operation to create a 2 dimensional map of auditory space. The map of space in the owl s auditory system shows important similarities with representations of space in the cerebral cortex and other sensory systems. In encoding space or other stimulus features, individual neurons appear to possess analogous functional properties related to the synthesis of high order receptive fields.

  19. Fluorescent nanoparticles for intracellular sensing: A review

    Energy Technology Data Exchange (ETDEWEB)

    Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  20. Invasion and intracellular survival by protozoan parasites.

    Science.gov (United States)

    Sibley, L David

    2011-03-01

    Intracellular parasitism has arisen only a few times during the long ancestry of protozoan parasites including in diverse groups such as microsporidians, kinetoplastids, and apicomplexans. Strategies used to gain entry differ widely from injection (e.g. microsporidians), active penetration of the host cell (e.g. Toxoplasma), recruitment of lysosomes to a plasma membrane wound (e.g. Trypanosoma cruzi), to host cell-mediated phagocytosis (e.g. Leishmania). The resulting range of intracellular niches is equally diverse ranging from cytosolic (e.g. T. cruzi) to residing within a non-fusigenic vacuole (e.g. Toxoplasma, Encephalitozoon) or a modified phagolysosome (e.g. Leishmania). These lifestyle choices influence access to nutrients, interaction with host cell signaling pathways, and detection by pathogen recognition systems. As such, intracellular life requires a repertoire of adaptations to assure entry-exit from the cell, as well as to thwart innate immune mechanisms and prevent clearance. Elucidating these pathways at the cellular and molecular level may identify key steps that can be targeted to reduce parasite survival or augment immunologic responses and thereby prevent disease.

  1. Detection of intracellular phosphatidylserine in living cells.

    Science.gov (United States)

    Calderon, Frances; Kim, Hee-Yong

    2008-03-01

    To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.

  2. Screening of lactic acid bacteria from Indonesia reveals glucansucrase and fructansucrase genes in two different Weissella confusa strains from soya

    NARCIS (Netherlands)

    Malik, Amarila; Radji, Maksum; Kralj, Slavko; Dijkhuizen, Lubbert

    2009-01-01

    Homopolysaccharide (glucan and fructan) synthesis from sucrose by sucrase enzymes in lactic acid bacteria (LAB) has been well studied in the genera Leuconostoc, Streptococcus and Lactobacillus. This study aimed to identify and characterize genes encoding glucansucrase/glucosyltransferase (GTF) and

  3. Clustered Intracellular Salmonella enterica Serovar Typhimurium Blocks Host Cell Cytokinesis.

    Science.gov (United States)

    Santos, António J M; Durkin, Charlotte H; Helaine, Sophie; Boucrot, Emmanuel; Holden, David W

    2016-07-01

    Several bacterial pathogens and viruses interfere with the cell cycle of their host cells to enhance virulence. This is especially apparent in bacteria that colonize the gut epithelium, where inhibition of the cell cycle of infected cells enhances the intestinal colonization. We found that intracellular Salmonella enterica serovar Typhimurium induced the binucleation of a large proportion of epithelial cells by 14 h postinvasion and that the effect was dependent on an intact Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system. The SPI-2 effectors SseF and SseG were required to induce binucleation. SseF and SseG are known to maintain microcolonies of Salmonella-containing vacuoles close to the microtubule organizing center of infected epithelial cells. During host cell division, these clustered microcolonies prevented the correct localization of members of the chromosomal passenger complex and mitotic kinesin-like protein 1 and consequently prevented cytokinesis. Tetraploidy, arising from a cytokinesis defect, is known to have a deleterious effect on subsequent cell divisions, resulting in either chromosomal instabilities or cell cycle arrest. In infected mice, proliferation of small intestinal epithelial cells was compromised in an SseF/SseG-dependent manner, suggesting that cytokinesis failure caused by S Typhimurium delays epithelial cell turnover in the intestine.

  4. Real-Time monitoring of intracellular wax ester metabolism

    Directory of Open Access Journals (Sweden)

    Karp Matti

    2011-09-01

    Full Text Available Abstract Background Wax esters are industrially relevant molecules exploited in several applications of oleochemistry and food industry. At the moment, the production processes mostly rely on chemical synthesis from rather expensive starting materials, and therefore solutions are sought from biotechnology. Bacterial wax esters are attractive alternatives, and especially the wax ester metabolism of Acinetobacter sp. has been extensively studied. However, the lack of suitable tools for rapid and simple monitoring of wax ester metabolism in vivo has partly restricted the screening and analyses of potential hosts and optimal conditions. Results Based on sensitive and specific detection of intracellular long-chain aldehydes, specific intermediates of wax ester synthesis, bacterial luciferase (LuxAB was exploited in studying the wax ester metabolism in Acinetobacter baylyi ADP1. Luminescence was detected in the cultivation of the strain producing wax esters, and the changes in signal levels could be linked to corresponding cell growth and wax ester synthesis phases. Conclusions The monitoring system showed correlation between wax ester synthesis pattern and luminescent signal. The system shows potential for real-time screening purposes and studies on bacterial wax esters, revealing new aspects to dynamics and role of wax ester metabolism in bacteria.

  5. Targeting of a chlamydial protease impedes intracellular bacterial growth.

    Directory of Open Access Journals (Sweden)

    Jan G Christian

    2011-09-01

    Full Text Available Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole. Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus (GA into ministacks, which facilitates the acquisition of host lipids into the growing inclusion. GA fragmentation results from infection-associated cleavage of the integral GA protein, golgin-84. Golgin-84-cleavage, GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk. Here we identify the bacterial protease chlamydial protease-like activity factor (CPAF as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition. WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C. trachomatis and C. pneumoniae. The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system. Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation. GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity. These results define CPAF as the first protein that is essential for replication of Chlamydia. We suggest that this role makes CPAF a potential anti-infective therapeutic target.

  6. Quenching of fluorescence by crystal violet and its use to differentiate between surface-bound and internalized bacteria

    Science.gov (United States)

    Mathew, S.; Lim, Y. C.; Kishen, A.

    2008-06-01

    Phagocytosis is a complex process involving attachment, ingestion and intracellular processing of bacteria by phagocytes. A great difficulty in the evaluation of this process is to differentiate between attachment of the particles to the cell surface and internalization of the particles by the cells. Various techniques have been used to differentiate internalized and surface-attached bacteria in cultured cells, but only a few permit differentiations between surface-bound and internalized bacteria. In this study the quenching of fluorescence by crystal violet on acridine orange stained bacterial biofilm and planktonic bacterial cells is used to differentiate between surface-bound and internalized bacteria within macrophages. Method: One week old Enterococcus faecalis biofilm was grown on perspex and glass substrates in All-Culture medium (nutrient-rich condition) and phosphate buffered saline (nutrient-deprived condition). As model systems, human monocytic (THP-1) and histiocytic (U937) cell lines were used. These cell lines were incubated with the biofilm bacteria for 4 hrs in CO II incubator at 37 °C. The cells and bacteria were stained with acridine orange and quenched with crystal violet to distinguish between surface-bound and internalized bacteria. Results: The presence of green-fluorescing internalized bacteria was detected within the macrophages under the planktonic, nutrient-rich and nutrient-deprived biofilm conditions. All infecting bacteria take up acridine orange and fluoresced green, crystal violet quenched the fluorescence of extra-cellular adhering bacteria so that only fluorescent intracellular bacteria would be visible under fluorescent light microscopy.

  7. Strategies to improve intracellular drug delivery by targeted liposomes

    NARCIS (Netherlands)

    Fretz, M.M.

    2007-01-01

    Biotechnological advances increased the number of novel macromolecular drugs and new drug targets. The latter are mostly found intracellular. Unfortunately, most of the new macromolecular drugs rely on drug delivery tools for their intracellular delivery because their unfavourable physicochemical pr

  8. Bioinformatics analysis and detection of gelatinase encoded gene in Lysinibacillussphaericus

    Science.gov (United States)

    Repin, Rul Aisyah Mat; Mutalib, Sahilah Abdul; Shahimi, Safiyyah; Khalid, Rozida Mohd.; Ayob, Mohd. Khan; Bakar, Mohd. Faizal Abu; Isa, Mohd Noor Mat

    2016-11-01

    In this study, we performed bioinformatics analysis toward genome sequence of Lysinibacillussphaericus (L. sphaericus) to determine gene encoded for gelatinase. L. sphaericus was isolated from soil and gelatinase species-specific bacterium to porcine and bovine gelatin. This bacterium offers the possibility of enzymes production which is specific to both species of meat, respectively. The main focus of this research is to identify the gelatinase encoded gene within the bacteria of L. Sphaericus using bioinformatics analysis of partially sequence genome. From the research study, three candidate gene were identified which was, gelatinase candidate gene 1 (P1), NODE_71_length_93919_cov_158.931839_21 which containing 1563 base pair (bp) in size with 520 amino acids sequence; Secondly, gelatinase candidate gene 2 (P2), NODE_23_length_52851_cov_190.061386_17 which containing 1776 bp in size with 591 amino acids sequence; and Thirdly, gelatinase candidate gene 3 (P3), NODE_106_length_32943_cov_169.147919_8 containing 1701 bp in size with 566 amino acids sequence. Three pairs of oligonucleotide primers were designed and namely as, F1, R1, F2, R2, F3 and R3 were targeted short sequences of cDNA by PCR. The amplicons were reliably results in 1563 bp in size for candidate gene P1 and 1701 bp in size for candidate gene P3. Therefore, the results of bioinformatics analysis of L. Sphaericus resulting in gene encoded gelatinase were identified.

  9. The fecal bacteria

    Science.gov (United States)

    Sadowsky, Michael J.; Whitman, Richard L.

    2011-01-01

    The Fecal Bacteria offers a balanced, integrated discussion of fecal bacteria and their presence and ecology in the intestinal tract of mammals, in the environment, and in the food supply. This volume covers their use in examining and assessing water quality in order to offer protection from illnesses related to swimming in or ingesting contaminated water, in addition to discussing their use in engineering considerations of water quality, modeling, monitoring, and regulations. Fecal bacteria are additionally used as indicators of contamination of ready-to-eat foods and fresh produce. The intestinal environment, the microbial community structure of the gut microbiota, and the physiology and genomics of this broad group of microorganisms are explored in the book. With contributions from an internationally recognized group of experts, the book integrates medicine, public health, environmental, and microbiological topics in order to provide a unique, holistic understanding of fecal bacteria. Moreover, it shows how the latest basic science and applied research findings are helping to solve problems and develop effective management strategies. For example, readers will discover how the latest tools and molecular approaches have led to our current understanding of fecal bacteria and enabled us to improve human health and water quality. The Fecal Bacteria is recommended for microbiologists, clinicians, animal scientists, engineers, environmental scientists, food safety experts, water quality managers, and students. It will help them better understand fecal bacteria and use their knowledge to protect human and environmental health. They can also apply many of the techniques and molecular tools discussed in this book to the study of a broad range of microorganisms in a variety of habitats.

  10. Molecular mechanisms for protein-encoded inheritance.

    Science.gov (United States)

    Wiltzius, Jed J W; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R; Apostol, Marcin I; Goldschmidt, Lukasz; Soriaga, Angela B; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-09-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  11. Molecular mechanisms for protein-encoded inheritance

    Energy Technology Data Exchange (ETDEWEB)

    Wiltzius, Jed J.W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David; (Cornell); (HHMI)

    2009-12-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of {beta}-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct {beta}-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  12. Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

    OpenAIRE

    D'Amore, T; C.J. Panchal; Stewart, G G

    1988-01-01

    An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar af...

  13. Full 3D translational and rotational optical control of multiple rod-shaped bacteria.

    Science.gov (United States)

    Hörner, Florian; Woerdemann, Mike; Müller, Stephanie; Maier, Berenike; Denz, Cornelia

    2010-07-01

    The class of rod-shaped bacteria is an important example of non-spherical objects where defined alignment is desired for the observation of intracellular processes or studies of the flagella. However, all available methods for orientational control of rod-shaped bacteria are either limited with respect to the accessible rotational axes or feasible angles or restricted to one single bacterium. In this paper we demonstrate a scheme to orientate rod-shaped bacteria with holographic optical tweezers (HOT) in any direction. While these bacteria have a strong preference to align along the direction of the incident laser beam, our scheme provides for the first time full rotational control of multiple bacteria with respect to any arbitrary axis. In combination with the translational control HOT inherently provide, this enables full control of all three translational and the two important rotational degrees of freedom of multiple rod-shaped bacteria and allows one to arrange them in any desired configuration.

  14. Dynamical encoding of cursive handwriting.

    Science.gov (United States)

    Singer, Y; Tishby, N

    1994-01-01

    A model-based approach to on-line cursive handwriting analysis and recognition is presented and evaluated. In this model, on-line handwriting is considered as a modulation of a simple cycloidal pen motion, described by two coupled oscillations with a constant linear drift along the line of the writing. By slow modulations of the amplitudes and phase lags of the two oscillators, a general pen trajectory can be efficiently encoded. These parameters are then quantized into a small number of values without altering the writing intelligibility. A general procedure for the estimation and quantization of these cycloidal motion parameters for arbitrary handwriting is presented. The result is a discrete motor control representation of the continuous pen motion, via the quantized levels of the model parameters. This motor control representation enables successful word spotting and matching of cursive scripts. Our experiments clearly indicate the potential of this dynamic representation for complete cursive handwriting recognition.

  15. Current View on Phytoplasma Genomes and Encoded Metabolism

    Directory of Open Access Journals (Sweden)

    Michael Kube

    2012-01-01

    Full Text Available Phytoplasmas are specialised bacteria that are obligate parasites of plant phloem tissue and insects. These bacteria have resisted all attempts of cell-free cultivation. Genome research is of particular importance to analyse the genetic endowment of such bacteria. Here we review the gene content of the four completely sequenced ‘Candidatus Phytoplasma’ genomes that include those of ‘Ca. P. asteris’ strains OY-M and AY-WB, ‘Ca. P. australiense,’ and ‘Ca. P. mali’. These genomes are characterized by chromosome condensation resulting in sizes below 900 kb and a G + C content of less than 28%. Evolutionary adaption of the phytoplasmas to nutrient-rich environments resulted in losses of genetic modules and increased host dependency highlighted by the transport systems and limited metabolic repertoire. On the other hand, duplication and integration events enlarged the chromosomes and contribute to genome instability. Present differences in the content of membrane and secreted proteins reflect the host adaptation in the phytoplasma strains. General differences are obvious between different phylogenetic subgroups. ‘Ca. P. mali’ is separated from the other strains by its deviating chromosome organization, the genetic repertoire for recombination and excision repair of nucleotides or the loss of the complete energy-yielding part of the glycolysis. Apart from these differences, comparative analysis exemplified that all four phytoplasmas are likely to encode an alternative pathway to generate pyruvate and ATP.

  16. Bacteria as vectors for gene therapy of cancer.

    LENUS (Irish Health Repository)

    Baban, Chwanrow K

    2012-01-31

    Anti-cancer therapy faces major challenges, particularly in terms of specificity of treatment. The ideal therapy would eradicate tumor cells selectively with minimum side effects on normal tissue. Gene or cell therapies have emerged as realistic prospects for the treatment of cancer, and involve the delivery of genetic information to a tumor to facilitate the production of therapeutic proteins. However, there is still much to be done before an efficient and safe gene medicine is achieved, primarily developing the means of targeting genes to tumors safely and efficiently. An emerging family of vectors involves bacteria of various genera. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally. Furthermore, invasive species can deliver heterologous genes intra-cellularly for tumor cell expression. Here, we review the use of bacteria as vehicles for gene therapy of cancer, detailing the mechanisms of action and successes at preclinical and clinical levels.

  17. Tumour targeting with systemically administered bacteria.

    LENUS (Irish Health Repository)

    Morrissey, David

    2012-01-31

    Challenges for oncology practitioners and researchers include specific treatment and detection of tumours. The ideal anti-cancer therapy would selectively eradicate tumour cells, whilst minimising side effects to normal tissue. Bacteria have emerged as biological gene vectors with natural tumour specificity, capable of homing to tumours and replicating locally to high levels when systemically administered. This property enables targeting of both the primary tumour and secondary metastases. In the case of invasive pathogenic species, this targeting strategy can be used to deliver genes intracellularly for tumour cell expression, while non-invasive species transformed with plasmids suitable for bacterial expression of heterologous genes can secrete therapeutic proteins locally within the tumour environment (cell therapy approach). Many bacterial genera have been demonstrated to localise to and replicate to high levels within tumour tissue when intravenously (IV) administered in rodent models and reporter gene tagging of bacteria has permitted real-time visualisation of this phenomenon. Live imaging of tumour colonising bacteria also presents diagnostic potential for this approach. The nature of tumour selective bacterial colonisation appears to be tumour origin- and bacterial species- independent. While originally a correlation was drawn between anaerobic bacterial colonisation and the hypoxic nature of solid tumours, it is recently becoming apparent that other elements of the unique microenvironment within solid tumours, including aberrant neovasculature and local immune suppression, may be responsible. Here, we consider the pre-clinical data supporting the use of bacteria as a tumour-targeting tool, recent advances in the area, and future work required to develop it into a beneficial clinical tool.

  18. Magnetotactic Bacteria from Extreme Environments

    Directory of Open Access Journals (Sweden)

    Christopher T. Lefèvre

    2013-03-01

    Full Text Available Magnetotactic bacteria (MTB represent a diverse collection of motile prokaryotes that biomineralize intracellular, membrane-bounded, tens-of-nanometer-sized crystals of a magnetic mineral called magnetosomes. Magnetosome minerals consist of either magnetite (Fe3O4 or greigite (Fe3S4 and cause cells to align along the Earth’s geomagnetic field lines as they swim, a trait called magnetotaxis. MTB are known to mainly inhabit the oxic–anoxic interface (OAI in water columns or sediments of aquatic habitats and it is currently thought that magnetosomes function as a means of making chemotaxis more efficient in locating and maintaining an optimal position for growth and survival at the OAI. Known cultured and uncultured MTB are phylogenetically associated with the Alpha-, Gamma- and Deltaproteobacteria classes of the phylum Proteobacteria, the Nitrospirae phylum and the candidate division OP3, part of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC bacterial superphylum. MTB are generally thought to be ubiquitous in aquatic environments as they are cosmopolitan in distribution and have been found in every continent although for years MTB were thought to be restricted to habitats with pH values near neutral and at ambient temperature. Recently, however, moderate thermophilic and alkaliphilic MTB have been described including: an uncultured, moderately thermophilic magnetotactic bacterium present in hot springs in northern Nevada with a probable upper growth limit of about 63 °C; and several strains of obligately alkaliphilic MTB isolated in pure culture from different aquatic habitats in California, including the hypersaline, extremely alkaline Mono Lake, with an optimal growth pH of >9.0.

  19. Innate hemocyte responses of Malacosoma disstria larvae (C. Insecta) to antigens are modulated by intracellular cyclic AMP.

    Science.gov (United States)

    Gulii, Vladislav; Dunphy, Gary B; Mandato, Craig A

    2009-08-01

    Invertebrate intracellular hemocyte signaling pathways affecting cellular-antigen responses, although defined for molluscs and some arthropods including dipteran insects, is less known for lepidopterans. Hemocytic-antigen responses of the arboreal pest lepidopteran Malacosoma disstria are linked to cAMP-dependent protein kinase A implicating cAMP in cellular hemocyte immune responses. The purpose in the present study was to determine intracellular cAMP effects on larval M. disstria hemocytes adhering to slides and bacteria. Altering adenylate cyclase and phosphodiesterase activities as well as cAMP levels in vitro and in vivo changed hemocyte responses to antigens. Quiescent hemocytes had high cAMP levels due to adenylate cyclase activity and possibly low phosphodiesterase (type 4) activity. Antigen contact diminished hemocytic cAMP levels. Inhibiting adenylate cyclase increased hemocyte-antigen and hemocyte-hemocyte adhesion, the latter producing nodules in vivo without bacterial antigens. Inhibiting phosphodiesterase type 4 produced the reverse effects. Pharmacologically increasing intracellular cAMP in attached hemocytes caused many of the cells to detach. Diminished intracellular cAMP changed hemograms in vivo in bacteria-free larvae comparable to changes induced by the bacterium, Bacillus subtilis, by producing nodules. Lowering cAMP enhanced also the removal of Xenorhabdus nematophila and B. subtilisin vivo.

  20. Thiol-oxidant monochloramine mobilizes intracellular Ca2+ in parietal cells of rabbit gastric glands.

    Science.gov (United States)

    Walsh, Breda M; Naik, Haley B; Dubach, J Matthew; Beshire, Melissa; Wieland, Aaron M; Soybel, David I

    2007-11-01

    In Helicobacter pylori-induced gastritis, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the gastric mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) accumulation within the parietal cell of the gastric gland. Individual gastric glands isolated from rabbit mucosa were loaded with fluorescent reporters for Ca(2+) in the cytoplasm (fura-2 AM) or intracellular stores (mag-fura-2 AM). Conditions were adjusted to screen out contributions from metal cations such as Zn(2+), for which these reporters have affinity. Exposure to NH(2)Cl (up to 200 microM) led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), in the range of 200-400 nM above baseline levels. These alterations were prevented by pretreatment with the oxidant scavenger vitamin C or a thiol-reducing agent, dithiothreitol (DTT), which shields intracellular thiol groups from oxidation by chlorinated oxidants. Introduction of vitamin C during ongoing exposure to NH(2)Cl arrested but did not reverse accumulation of Ca(2+) in the cytoplasm. In contrast, introduction of DTT or N-acetylcysteine permitted arrest and partial reversal of the effects of NH(2)Cl. Accumulation of Ca(2+) in the cytoplasm induced by NH(2)Cl is due to release from intracellular stores, entry from the extracellular fluid, and impaired extrusion. Ca(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained increases in [Ca(2+)](i). Under certain conditions, NH(2)Cl may act not as an irritant but as an agent that activates intracellular signaling pathways. Anti-NH(2)Cl strategies should take into account different effects of oxidant scavengers and thiol-reducing agents.

  1. Amoebae-Based Screening Reveals a Novel Family of Compounds Restricting Intracellular Legionella pneumophila.

    Science.gov (United States)

    Harrison, Christopher F; Chiriano, Gianpaolo; Finsel, Ivo; Manske, Christian; Hoffmann, Christine; Steiner, Bernhard; Kranjc, Agata; Patthey-Vuadens, Ophelie; Kicka, Sébastien; Trofimov, Valentin; Ouertatani-Sakouhi, Hajer; Soldati, Thierry; Scapozza, Leonardo; Hilbi, Hubert

    2015-07-10

    The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s).

  2. Two genes encoding new carotenoid-modifying enzymes in the green sulfur bacterium Chlorobium tepidum.

    Science.gov (United States)

    Maresca, Julia A; Bryant, Donald A

    2006-09-01

    The green sulfur bacterium Chlorobium tepidum produces chlorobactene as its primary carotenoid. Small amounts of chlorobactene are hydroxylated by the enzyme CrtC and then glucosylated and acylated to produce chlorobactene glucoside laurate. The genes encoding the enzymes responsible for these modifications of chlorobactene, CT1987, and CT0967, have been identified by comparative genomics, and these genes were insertionally inactivated in C. tepidum to verify their predicted function. The gene encoding chlorobactene glucosyltransferase (CT1987) has been named cruC, and the gene encoding chlorobactene lauroyltransferase (CT0967) has been named cruD. Homologs of these genes are found in the genomes of all sequenced green sulfur bacteria and filamentous anoxygenic phototrophs as well as in the genomes of several nonphotosynthetic bacteria that produce similarly modified carotenoids. The other bacteria in which these genes are found are not closely related to green sulfur bacteria or to one another. This suggests that the ability to synthesize modified carotenoids has been a frequently transferred trait.

  3. Magnetotactic bacteria. Promising biosorbents for heavy metals

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Wei; Zhang, Yanzong; Ding, Xiaohui; Liu, Yan; Shen, Fei; Zhang, Xiaohong; Deng, Shihuai; Xiao, Hong; Yang, Gang; Peng, Hong [Sichuan Agricultural Univ., Chengdu (China). Provincial Key Lab. of Agricultural Environmental Engineering

    2012-09-15

    Magnetotactic bacteria (MTB), which can orient and migrate along a magnetic line of force due to intracellular nanosized magnetosomes, have been a subject of research in the medical field, in dating environmental changes, and in environmental remediation. This paper reviews the recent development of MTB as biosorbents for heavy metals. Ultrastructures and taxis of MTB are investigated. Adsorptions in systems of unitary and binary ions are highlighted, as well as adsorption conditions (temperature, pH value, biomass concentration, and pretreatments). The separation and desorption of MTB in magnetic separators are also discussed. A green method to produce metal nanoparticles is provided, and an energy-efficient way to recover precious metals is put forward during biosorption. (orig.)

  4. Magnetotactic bacteria. Magnetic navigation on the microscale

    Science.gov (United States)

    Klumpp, Stefan; Faivre, Damien

    2016-11-01

    Magnetotactic bacteria are aquatic microorganisms with the ability to swim along the field lines of a magnetic field, which in their natural environment is provided by the magnetic field of the Earth. They do so with the help of specialized magnetic organelles called magnetosomes, vesicles containing magnetic crystals. Magnetosomes are aligned along cytoskeletal filaments to give linear structures that can function as intracellular compass needles. The predominant viewpoint is that the cells passively align with an external magnetic field, just like a macroscopic compass needle, but swim actively along the field lines, propelled by their flagella. In this minireview, we give an introduction to this intriguing bacterial behavior and discuss recent advances in understanding it, with a focus on the swimming directionality, which is not only affected by magnetic fields, but also by gradients of the oxygen concentration.

  5. Chemotaxis signaling systems in model beneficial plant-bacteria associations.

    Science.gov (United States)

    Scharf, Birgit E; Hynes, Michael F; Alexandre, Gladys M

    2016-04-01

    Beneficial plant-microbe associations play critical roles in plant health. Bacterial chemotaxis provides a competitive advantage to motile flagellated bacteria in colonization of plant root surfaces, which is a prerequisite for the establishment of beneficial associations. Chemotaxis signaling enables motile soil bacteria to sense and respond to gradients of chemical compounds released by plant roots. This process allows bacteria to actively swim towards plant roots and is thus critical for competitive root surface colonization. The complete genome sequences of several plant-associated bacterial species indicate the presence of multiple chemotaxis systems and a large number of chemoreceptors. Further, most soil bacteria are motile and capable of chemotaxis, and chemotaxis-encoding genes are enriched in the bacteria found in the rhizosphere compared to the bulk soil. This review compares the architecture and diversity of chemotaxis signaling systems in model beneficial plant-associated bacteria and discusses their relevance to the rhizosphere lifestyle. While it is unclear how controlling chemotaxis via multiple parallel chemotaxis systems provides a competitive advantage to certain bacterial species, the presence of a larger number of chemoreceptors is likely to contribute to the ability of motile bacteria to survive in the soil and to compete for root surface colonization.

  6. Azithromycin effectiveness against intracellular infections of Francisella

    Directory of Open Access Journals (Sweden)

    Mann Barbara J

    2010-04-01

    Full Text Available Abstract Background Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F. tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A may have different susceptibilities to Az, a widely used and well-tolerated antibiotic. Results In vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS (Type B was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisella-infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ΔacrB and ΔacrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA were found to be less sensitive in vitro to Az compared to the wild

  7. Caspases rule the intracellular trafficking cartel.

    Science.gov (United States)

    Duclos, Catherine; Lavoie, Christine; Denault, Jean-Bernard

    2017-05-01

    During apoptosis, caspases feast on several hundreds of cellular proteins to orchestrate rapid cellular demise. Indeed, caspases are known to get a taste of every cellular process in one way or another, activating some, but most often shutting them down. Thus, it is not surprising that caspases proteolyze proteins involved in intracellular trafficking with particularly devastating consequences for this important process. This review article focuses on how caspases target the machinery responsible for smuggling goods within and outside the cell. © 2017 Federation of European Biochemical Societies.

  8. Intracellular pH in sperm physiology.

    Science.gov (United States)

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

  9. Insights into the genome of large sulfur bacteria revealed by analysis of single filaments

    DEFF Research Database (Denmark)

    Mussmann, Marc; Hu, Fen Z.; Richter, Michael

    2007-01-01

    Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical i...

  10. Insights into the genome of large sulfur bacteria revealed by analysis of single filaments.

    NARCIS (Netherlands)

    Mussmann, M.; Hu, F.Z.; Richter, M.; Beer, D. de; Preisler, A.; Jorgensen, B.B.; Huntemann, M.; Glockner, F.O.; Amann, R.; Koopman, W.J.H.; Lasken, R.S.; Janto, B.; Hogg, J.; Stoodley, P.; Boissy, R.; Ehrlich, G.D.

    2007-01-01

    Marine sediments are frequently covered by mats of the filamentous Beggiatoa and other large nitrate-storing bacteria that oxidize hydrogen sulfide using either oxygen or nitrate, which they store in intracellular vacuoles. Despite their conspicuous metabolic properties and their biogeochemical impo

  11. NMDA receptors and memory encoding.

    Science.gov (United States)

    Morris, Richard G M

    2013-11-01

    It is humbling to think that 30 years have passed since the paper by Collingridge, Kehl and McLennan showing that one of Jeff Watkins most interesting compounds, R-2-amino-5-phosphonopentanoate (d-AP5), blocked the induction of long-term potentiation in vitro at synapses from area CA3 of the hippocampus to CA1 without apparent effect on baseline synaptic transmission (Collingridge et al., 1983). This dissociation was one of the key triggers for an explosion of interest in glutamate receptors, and much has been discovered since that collectively contributes to our contemporary understanding of glutamatergic synapses - their biophysics and subunit composition, of the agonists and antagonists acting on them, and their diverse functions in different networks of the brain and spinal cord. It can be fairly said that Collingridge et al.'s (1983) observation was the stimulus that has led, on the one hand, to structural biological work at the atomic scale describing the key features of NMDA receptors that enables their coincidence function to happen; and, on the other, to work with whole animals investigating the contributions that calcium signalling via this receptor can have on rhythmical activities controlled by spinal circuits, memory encoding in the hippocampus (the topic of this article), visual cortical plasticity, sensitization in pain, and other functions. In this article, I lay out how my then interest in long-term potentiation (LTP) as a model of memory enabled me to recognise the importance of Collingridge et al.'s discovery - and how I and my colleagues endeavoured to take things forward in the area of learning and memory. This is in some respects a personal story, and I tell it as such. The idea that NMDA receptor activation is essential for memory encoding, though not for storage, took time to develop and to be accepted. Along the way, there have been confusions, challenges, and surprises surrounding the idea that activation of NMDA receptors can

  12. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development

    Science.gov (United States)

    Sørhus, Elin; Incardona, John P.; Karlsen, Ørjan; Linbo, Tiffany; Sørensen, Lisbet; Nordtug, Trond; van der Meeren, Terje; Thorsen, Anders; Thorbjørnsen, Maja; Jentoft, Sissel; Edvardsen, Rolf B.; Meier, Sonnich

    2016-08-01

    Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7-7 μg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish.

  13. Mechanisms of cellular invasion by intracellular parasites.

    Science.gov (United States)

    Walker, Dawn M; Oghumu, Steve; Gupta, Gaurav; McGwire, Bradford S; Drew, Mark E; Satoskar, Abhay R

    2014-04-01

    Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

  14. Stochastic models of intracellular calcium signals

    Energy Technology Data Exchange (ETDEWEB)

    Rüdiger, Sten, E-mail: sten.ruediger@physik.hu-berlin.de

    2014-01-10

    Cellular signaling operates in a noisy environment shaped by low molecular concentrations and cellular heterogeneity. For calcium release through intracellular channels–one of the most important cellular signaling mechanisms–feedback by liberated calcium endows fluctuations with critical functions in signal generation and formation. In this review it is first described, under which general conditions the environment makes stochasticity relevant, and which conditions allow approximating or deterministic equations. This analysis provides a framework, in which one can deduce an efficient hybrid description combining stochastic and deterministic evolution laws. Within the hybrid approach, Markov chains model gating of channels, while the concentrations of calcium and calcium binding molecules (buffers) are described by reaction–diffusion equations. The article further focuses on the spatial representation of subcellular calcium domains related to intracellular calcium channels. It presents analysis for single channels and clusters of channels and reviews the effects of buffers on the calcium release. For clustered channels, we discuss the application and validity of coarse-graining as well as approaches based on continuous gating variables (Fokker–Planck and chemical Langevin equations). Comparison with recent experiments substantiates the stochastic and spatial approach, identifies minimal requirements for a realistic modeling, and facilitates an understanding of collective channel behavior. At the end of the review, implications of stochastic and local modeling for the generation and properties of cell-wide release and the integration of calcium dynamics into cellular signaling models are discussed.

  15. Gene Therapy for HIV Infections: Intracellular Immunization

    Directory of Open Access Journals (Sweden)

    Alain Piché

    1999-01-01

    Full Text Available Despite significant advances in the treatment of human immunodeficiency virus (HIV infection in the past 10 years, it remains an incurable disease. The inability of traditional drug-based therapies to inhibit HIV replication effectively for extended periods of time has stimulated intense research to develop novel approaches for this disease. Current understanding of HIV molecular biology and pathogenesis has opened the way for the development of gene therapy strategies for HIV infections. In this context, a number of intracellular immunization-based strategies have been evaluated, and some of them have reached the stage of phase I/II human clinical trials. These strategies include the use of single-chain antibodies, capsid-targeted viral inactivation, transdominant negative mutants, ribozymes, antisense oligonucleotides and RNA decoys. While a number of issues remain to be studied before intracellular immunization can be applied to the treatment of HIV infections, the significant progress already made in this field is likely to lead to clinical applications.

  16. Intracellular accumulation of norfloxacin in Mycobacterium smegmatis.

    Science.gov (United States)

    Corti, S; Chevalier, J; Cremieux, A

    1995-11-01

    To evaluate the intracellular accumulation of norfloxacin in mycobacteria, two methods were used with Mycobacterium smegmatis. A radiometric method (K. V. Cundy, C. E. Fasching, K. E. Willard, and L. R. Peterson, J. Antimicrob. Chemother. 28:491-497, 1991) was used without great modification, but the fluorometric method (P. G. S. Mortimer and L. J. V. Piddock, J. Antimicrob. Chemother. 28:639-653, 1991) was changed considerably. Indeed, adsorption of the quinolone to the bacterial surface was characterized by measuring the level of accumulation of 0 degree C. Taking into account the adsorption, the pH of the washing buffer was increased from 7.0 to 9.0 to improve the desorption of norfloxacin from the cell surface. Both the fluorometric method, with the technical improvement, and the radiometric method could be used to estimate the intracellular accumulation of norfloxacin, which resulted from the difference between the whole uptake measured at 37 degrees C and the adsorption measured at 0 degrees C. A total of 35 ng of norfloxacin per mg of cells (dry weight) penetrated into the M. smegmatis cell, and the steady state was achieved in 5 min. Use of inhibitors of the proton motive force revealed that transport of norfloxacin was energy independent. Thus, the same mechanisms of quinolone accumulation that occur in eubacteria seem to occur in mycobacteria, at least in M. smegmatis.

  17. Bacteria-surface interactions.

    Science.gov (United States)

    Tuson, Hannah H; Weibel, Douglas B

    2013-05-14

    The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphology and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiology of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria-surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field.

  18. Antibiotic-Resistant Bacteria.

    Science.gov (United States)

    Longenecker, Nevin E.; Oppenheimer, Dan

    1982-01-01

    A study conducted by high school advanced bacteriology students appears to confirm the hypothesis that the incremental administration of antibiotics on several species of bacteria (Escherichia coli, Staphylococcus epidermis, Bacillus sublitus, Bacillus megaterium) will allow for the development of antibiotic-resistant strains. (PEB)

  19. Mycophagous soil bacteria

    NARCIS (Netherlands)

    Rudnick, M.B.

    2015-01-01

    Abstract

    Soil microorganisms evolved several strategies to compete for limited nutrients in soil. Bacteria of the genus Collimonas developed a way to exploit fungi as a source of organic nutrients. This strategy has been termed “mycophagy&r

  20. Novelty's effect on memory encoding.

    Science.gov (United States)

    Rangel-Gomez, Mauricio; Janenaite, Sigita; Meeter, Martijn

    2015-07-01

    It is often thought that novelty benefits memory formation. However, support for this idea mostly comes from paradigms that are open to alternative explanations. In the present study we manipulated novelty in a word-learning task through task-irrelevant background images. These background images were either standard (presented repeatedly), or novel (presented only once). Two types of background images were used: Landscape pictures and fractals. EEG was also recorded during encoding. Contrary to the idea that novelty aids memory formation, memory performance was not affected by the novelty of the background. In the evoked response potentials, we found evidence of distracting effects of novelty: both the N1 and P3b components were smaller to words studied with novel backgrounds, and the amplitude of the N2b component correlated negatively with subsequent retrieval. We conclude that although evidence from other studies does suggest benefits on a longer time scale, novelty has no instantaneous benefits for learning. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Multidrug efflux pumps in Gram-negative bacteria and their role in antibiotic resistance.

    Science.gov (United States)

    Blair, Jessica M A; Richmond, Grace E; Piddock, Laura J V

    2014-01-01

    Gram-negative bacteria express a plethora of efflux pumps that are capable of transporting structurally varied molecules, including antibiotics, out of the bacterial cell. This efflux lowers the intracellular antibiotic concentration, allowing bacteria to survive at higher antibiotic concentrations. Overexpression of some efflux pumps can cause clinically relevant levels of antibiotic resistance in Gram-negative pathogens. This review discusses the role of efflux in resistance of clinical isolates of Gram-negative bacteria, the regulatory mechanisms that control efflux pump expression, the recent advances in our understanding of efflux pump structure and how inhibition of efflux is a promising future strategy for tackling multidrug resistance in Gram-negative pathogens.

  2. Intracellular Transport of Plant Viruses: Finding the Door out of the Cell

    Institute of Scientific and Technical Information of China (English)

    James E. Schoelz; Phillip A. Harries; Richard S. Nelson

    2011-01-01

    Plant viruses are a class of plant pathogens that specialize in movement from cell to cell.As part of their arsenal for infection of plants,every virus encodes a movement protein (MP),a protein dedicated to enlarging the pore size of plasmodesmata (PD) and actively transporting the viral nucleic acid into the adjacent cell.As our knowledge of intercellular transport has increased,it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD.Just as viruses are too large to fit through an unmodified plasmodesma,they are also too large to be freely diffused through the cytoplasm of the cell.Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP,including viral proteins originally associated with replication or gene expression.In this review,we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD,in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread.

  3. Chlorosomes: antenna organelles in photosynthetic green bacteria

    DEFF Research Database (Denmark)

    Frigaard, N.-U.; Bryant, D. A.

    2006-01-01

    The new series "Microbiology Monographs" begins with two volumes on intracellular components in prokaryotes. In this second volume, "Complex Intracellular Structures in Prokaryotes", the components, labelled complex intracellular structures, encompass a multitude of important cellular functions. ...

  4. Computational Intelligence and Its Encoding Mechanism

    Institute of Scientific and Technical Information of China (English)

    LIU Man-dan

    2004-01-01

    The origin and characteristics of computational intelligence, and several typical computational intelligence algorithms such as genetic algorithm and DNA computing are described, and the influence of evolution strategies and convergence properties on the encoding mechanism is discussed. A novel genetic algorithm based on degressive carry number encoding is then proposed. This algorithm uses degressive carry number encoding in the evolutionary process instead of commonly used fixed carry number. Finally a novel encoding mechanism and a new algorithm are proposed, which combine modern computational intelligence with the traditional Chinese methodology.

  5. Computational Intelligence and Its Encoding Mechanism

    Institute of Scientific and Technical Information of China (English)

    LIUMan-dan

    2004-01-01

    The origin and characteristics of computational intelligence, and several typical computational intelligence algorithms such as genetic algorithm and DNA computing are described, and the influence of evolution strategies and convergence properties on the encoding mechanism is discussed. A novel genetic algorithm based on degressive carry number encoding is then proposed. This algorithm uses degressive carry number encoding in the evolutionary process instead of commonly used fixed carry number. Finally a novel encoding mechanism and a new algorithm are proposed, which combine modem computational intelligence with the traditional Chinese methodology.

  6. Encoder designed to work in harsh environments

    Energy Technology Data Exchange (ETDEWEB)

    Toop, L.

    2007-05-15

    Dynapar has developed the Acuro AX71 absolute encoder for use on offshore or land-based oil rig operations. It provides feedback on the operation of automated systems such as draw works, racking systems, rotary tables and top drives. By ensuring that automated systems function properly, this encoder responds to a need by the oil and gas industry to keep workers safe and improve efficiency, particularly for operations in rugged situations. The encoder provides feedback from motor systems to controllers, giving information about position and speed of downhole drill bits. This newly developed encoder is better than commonly used incremental encoders which are not precise in strong electrical noise environments. Rather, the absolute encoder uses a different method of reporting to the controller. A digital signal is transmitted constantly as the device operates. It is less susceptible to noise issues. It is highly accurate, tolerant of noise and is not affected by power outages. However, the absolute encoder is generally more delicate in drilling applications with high ambient temperatures and shock levels. Dynapar addressed this issue by developing compact stainless steel housing that is useful for corrosion resistance in marine applications. The AX71 absolute encoder can withstand up to 100 G of mechanical shock and ambient temperatures of up to 60 degrees C. The encoder is ATEX certified without barriers, and offers the high resolution feedback of 4,000 counts of multiturn rotation and 16,000 counts of position. 1 fig.

  7. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...

  8. Free-living amoebae and their intracellular pathogenic microorganisms: risks for water quality.

    Science.gov (United States)

    Thomas, Vincent; McDonnell, Gerald; Denyer, Stephen P; Maillard, Jean-Yves

    2010-05-01

    An increasing number of microorganisms, including bacteria but also viruses and eukaryotes, have been described as benefiting from interaction with free-living amoebae (FLA). Beneficial interaction can be due to resistance to predation conferring ecological advantage, intracellular survival and/or intracellular proliferation. This review highlights the potential risk associated with amoebae by listing all known pathogenic microbial species for which growth and/or survival promotion by FLA (mainly Acanthamoeba spp.) has been demonstrated. It focuses on the susceptibility of amoebal and intra-amoebal bacteria to various categories of biocides, the known mechanisms of action of these biocides against trophozoites and cysts and the various methods used to demonstrate efficacy of treatments against FLA. Brief descriptions of FLA ecology and prevalence in domestic/institutional water systems and their intrinsic pathogenicity are also presented. The intention is to provide an informed opinion on the environmental risks associated with the presence of FLA and on the survival of cysts following biocidal treatments, while also highlighting the need to conduct research on the roles of amoebae in aquatic ecosystems.

  9. Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

    Science.gov (United States)

    Pelc, Rebecca S; McClure, Jennifer C; Kaur, Simran J; Sears, Khandra T; Rahman, M Sayeedur; Ceraul, Shane M

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria's ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.

  10. Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

    Directory of Open Access Journals (Sweden)

    Rebecca S Pelc

    Full Text Available Peptide Nucleic Acids (PNAs are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria's ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.

  11. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.

    Science.gov (United States)

    Ishikawa, Hiroki; Ma, Zhe; Barber, Glen N

    2009-10-08

    The innate immune system is critical for the early detection of invading pathogens and for initiating cellular host defence countermeasures, which include the production of type I interferon (IFN). However, little is known about how the innate immune system is galvanized to respond to DNA-based microbes. Here we show that STING (stimulator of interferon genes) is critical for the induction of IFN by non-CpG intracellular DNA species produced by various DNA pathogens after infection. Murine embryonic fibroblasts, as well as antigen presenting cells such as macrophages and dendritic cells (exposed to intracellular B-form DNA, the DNA virus herpes simplex virus 1 (HSV-1) or bacteria Listeria monocytogenes), were found to require STING to initiate effective IFN production. Accordingly, Sting-knockout mice were susceptible to lethal infection after exposure to HSV-1. The importance of STING in facilitating DNA-mediated innate immune responses was further evident because cytotoxic T-cell responses induced by plasmid DNA vaccination were reduced in Sting-deficient animals. In the presence of intracellular DNA, STING relocalized with TANK-binding kinase 1 (TBK1) from the endoplasmic reticulum to perinuclear vesicles containing the exocyst component Sec5 (also known as EXOC2). Collectively, our studies indicate that STING is essential for host defence against DNA pathogens such as HSV-1 and facilitates the adjuvant activity of DNA-based vaccines.

  12. A novel intracellular nitrogen-fixing symbiosis made by Ustilago maydis and Bacillus spp.

    Science.gov (United States)

    Ruiz-Herrera, José; León-Ramírez, Claudia; Vera-Nuñez, Antonio; Sánchez-Arreguín, Alejandro; Ruiz-Medrano, Roberto; Salgado-Lugo, Holjes; Sánchez-Segura, Lino; Peña-Cabriales, Juan José

    2015-08-01

    We observed that the maize pathogenic fungus Ustilago maydis grew in nitrogen (N)-free media at a rate similar to that observed in media containing ammonium nitrate, suggesting that it was able to fix atmospheric N2 . Because only prokaryotic organisms have the capacity to reduce N2 , we entertained the possibility that U. maydis was associated with an intracellular bacterium. The presence of nitrogenase in the fungus was analyzed by acetylene reduction, and capacity to fix N2 by use of (15) N2 . Presence of an intracellular N2 -fixing bacterium was analyzed by PCR amplification of bacterial 16S rRNA and nifH genes, and by microscopic observations. Nitrogenase activity and (15) N incorporation into the cells proved that U. maydis fixed N2 . Light and electron microscopy, and fluorescence in situ hybridization (FISH) experiments revealed the presence of intracellular bacteria related to Bacillus pumilus, as evidenced by sequencing of the PCR-amplified fragments. These observations reveal for the first time the existence of an endosymbiotic N2 -fixing association involving a fungus and a bacterium.

  13. Toxins from Bacteria

    OpenAIRE

    Henkel, James S.; Baldwin, Michael R.; Barbieri, Joseph T.

    2010-01-01

    Bacterial toxins damage the host at the site of bacterial infection or distanced from the site of infections. Bacterial toxins can be single proteins or organized as oligomeric protein complexes and are organized with distinct AB structure-function properties. The A domain encodes a catalytic activity; ADP-ribosylation of host proteins is the earliest post-translational modification determine to be performed by bacterial toxin, and now include glucosylation and proteolysis among other s. Bact...

  14. Cytoskeletal network morphology regulates intracellular transport dynamics

    CERN Document Server

    Ando, David; Huang, Kerwyn Casey; Gopinathan, Ajay

    2016-01-01

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable time scales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that r...

  15. [Measurement of intracellular pH].

    Science.gov (United States)

    Hanaoka, K; Imai, M; Yoshitomi, K

    1992-09-01

    Since various cellular processes depend on changes in pH, the regulation of intracellular pH (pHi) is important both for the individual cell and for the organism. The mechanisms of the regulation of pHi can be investigated by monitoring pHi. In this report, we discuss the four major techniques available for measuring pHi, which are 1) Distribution of weak acids and bases, 2) pH-sensitive microelectrodes, 3) pH-sensitive dyes, and 4) Nuclear magnetic resonance. Among four techniques, the advantage of the microelectrode approach is that it can monitor membrane potential at the same time and be applied to a single cell. The dye technique is a relative new developing technique, which has lots of advantages. It is easy to use, and is capable of monitoring rapid pHi changes, and being applied to a smaller cell, or a single cell.

  16. Myometrial oxytocin receptor expression and intracellular pathways.

    Science.gov (United States)

    Yulia, A; Johnson, M R

    2014-06-01

    Oxytocin (OT) signalling plays a fundamental role in the mechanisms of parturition. OT is one of the most frequently used drugs in obstetrics, promoting uterine contractions for labor induction and augmentation and to prevent postpartum hemorrhage (PPH). Expression of the oxytocin receptor (OTR) in the human myometrium is tightly regulated during pregnancy and its levels have been shown to peak upon labour onset and to fall sharply in advanced labour and the postpartum period, when the uterus become refractive to OT. However, uterine sensitivity to OT varies between pregnant women, probably reflecting differences in their myometrial OTR expression. Control of OTR expression is mediated by a combination of steroid hormone stimulation, stretch, and inflammation. This review summarises current knowledge regarding the complex regulation of myometrial OTR expression and its associated intracellular signaling pathways.

  17. Differential Translation Tunes Uneven Production of Operon-Encoded Proteins

    Directory of Open Access Journals (Sweden)

    Tessa E.F. Quax

    2013-09-01

    Full Text Available Clustering of functionally related genes in operons allows for coregulated gene expression in prokaryotes. This is advantageous when equal amounts of gene products are required. Production of protein complexes with an uneven stoichiometry, however, requires tuning mechanisms to generate subunits in appropriate relative quantities. Using comparative genomic analysis, we show that differential translation is a key determinant of modulated expression of genes clustered in operons and that codon bias generally is the best in silico indicator of unequal protein production. Variable ribosome density profiles of polycistronic transcripts correlate strongly with differential translation patterns. In addition, we provide experimental evidence that de novo initiation of translation can occur at intercistronic sites, allowing for differential translation of any gene irrespective of its position on a polycistronic messenger. Thus, modulation of translation efficiency appears to be a universal mode of control in bacteria and archaea that allows for differential production of operon-encoded proteins.

  18. Recognition of extracellular bacteria by NLRs and its role in the development of adaptive immunity

    Directory of Open Access Journals (Sweden)

    Jonathan eFerrand

    2013-10-01

    Full Text Available Innate immune recognition of bacteria is the first requirement for mounting an effective immune response able to control infection. Over the previous decade, the general paradigm was that extracellular bacteria were only sensed by cell surface-expressed Toll-like receptors (TLRs, whereas cytoplasmic sensors, including members of the Nod-like receptor (NLR family, were specific to pathogens capable of breaching the host cell membrane. It has become apparent, however, that intracellular innate immune molecules, such as the NLRs, play key roles in the sensing of not only intracellular, but also extracellular bacterial pathogens or their components. In this review, we will discuss the various mechanisms used by bacteria to activate NLR signaling in host cells. These mechanisms include bacterial secretion systems, pore-forming toxins and outer membrane vesicles. We will then focus on the influence of NLR activation on the development of adaptive immune responses in different cell types.

  19. 'Candidatus Odyssella thessalonicensis' gen. nov., sp. nov., an obligate intracellular parasite of Acanthamoeba species.

    Science.gov (United States)

    Birtles, R J; Rowbotham, T J; Michel, R; Pitcher, D G; Lascola, B; Alexiou-Daniel, S; Raoult, D

    2000-01-01

    An intracellular bacterium, strain L13, was observed infecting an environmental isolate of an Acanthamoeba species. The bacterium could not be recovered on axenic medium but was recovered and cultivated in vitro using cultures of Acanthamoeba polyphaga. The 16S rRNA gene sequence of L13 was found to be new, sharing less than 84% similarity with other sequences in the GenBank/EMBL database. L13 was found to be a member of the alpha-Proteobacteria, sharing an evolutionary line of descent with a group of uniquely obligate intracellular organisms comprised of Caedibacter and Holospora species and the NHP bacterium. Viable bacteria appeared to be highly motile within amoebae. Ultrastructural analysis of the bacterium demonstrated that it is rod-shaped and possesses a typical Gram-negative cell wall, but has no other outstanding features except small vesicle-like structures often associated with the outer surface of each bacterium. The host range of L13 was found to be limited to the genus Acanthamoeba. In A. polyphaga, L13 infection was slow to manifest when cultures were incubated below 30 degrees C, but at higher temperatures bacteria multiplied prolifically and induced host cell lysis. The protein profile of the bacterium purified from the amoebae was assessed by SDS-PAGE and its G+C content was estimated to be 41 mol%. Although these results support the proposal of L13 as a new species, its obligate intracellular nature prevented isolation of a definitive type strain. L13 is therefore proposed as 'Candidatus Odyssella thessalonicensis' gen. nov., sp. nov.

  20. Intracellular mechanisms of lymphoid cell activation.

    Science.gov (United States)

    Fresa, K; Hameed, M; Cohen, S

    1989-01-01

    Activation of lymphocytes for proliferation is associated with the appearance of an intracellular factor (ADR) that can induce DNA synthesis in isolated quiescent nuclei. ADR plays a role in the sequence of intracellular events leading to activation for IL-2-mediated proliferation. Because of the nature of the defining assay, the locus of ADR action appears to be near the terminal end of the transduction pathway. Interestingly, although lymphocytes from aged individuals respond poorly to proliferative stimuli, they appear to produce normal to above-normal levels of ADR. In contrast, their nuclei are only poorly responsive to stimulation by ADR. Preparations rich in ADR activity have proteolytic activity as well. In addition, aprotinin, as well as a variety of other protease inhibitors, suppresses ADR-induced DNA synthesis in a dose-dependent manner. ADR activity can be removed from active extracts by absorption with aprotinin-conjugated agarose beads, and can be removed from the beads by elution at pH 5.0. This latter suggests that ADR itself is a protease. However, its endogenous substrate is not yet known. We have also detected an inhibitor of ADR activity in the cytoplasm of resting lymphocytes. This is a heat-stable protein of approximately 60,000 Da. In addition to suppressing the interaction of ADR with quiescent nuclei, the inhibitor can suppress DNA synthetic activity of replicative nuclei isolated from mitogen-activated lymphocytes. Interestingly, these preparations had little or no activity on replicative nuclei derived from several neoplastic cell lines. The resistance of tumor cell nuclei to spontaneously occurring cytoplasmic inhibitory factors such as the one described here may provide one explanation for the loss of growth control in neoplastic cells.

  1. Cellular encoding for interactive evolutionary robotics

    NARCIS (Netherlands)

    Gruau, F.C.; Quatramaran, K.

    1996-01-01

    This work reports experiments in interactive evolutionary robotics. The goal is to evolve an Artificial Neural Network (ANN) to control the locomotion of an 8-legged robot. The ANNs are encoded using a cellular developmental process called cellular encoding. In a previous work similar experiments ha

  2. A METHOD OF SHAPE ENCODING AND RETRIEVAL

    Institute of Scientific and Technical Information of China (English)

    Huang Xianglin; Song Lei; Shen Lansun

    2002-01-01

    A method of shape encoding and retrieval is proposed in this letter, which uses centripetal code to encode shape and extracts shape's convex for retrieval. For the rotation invariance and translation invariance of the centripetal code and the normalization of convex,the proposed retrieval method is similarity transform resistant, Experimental results confirm this capability.

  3. Is Your ATM Dispensing Bacteria?

    Science.gov (United States)

    ... news/fullstory_162067.html Is Your ATM Dispensing Bacteria? Study in New York City found most of ... keypads in New York City were covered in bacteria, researchers reported, with most of the microbes coming ...

  4. Exopolysaccharides from Marine Bacteria

    Institute of Scientific and Technical Information of China (English)

    CHI Zhenming; FANG Yan

    2005-01-01

    Microbial polysaccharides represent a class of important products of growing interest for many sectors of industry. In recent years, there has been a growing interest in isolating new exopolysaccharides (EPSs)-producing bacteria from marine environments, particularly from various extreme marine environments. Many new marine microbial EPSs with novel chemical compositions, properties and structures have been found to have potential applications in fields such as adhesives,textiles, pharmaceuticals and medicine for anti-cancer, food additives, oil recovery and metal removal in mining and industrial waste treatments, etc This paper gives a brief summary of the information about the EPSs produced by marine bacteria,including their chemical compositions, properties and structures, together with their potential applications in industry.

  5. Genomics of oral bacteria.

    Science.gov (United States)

    Duncan, Margaret J

    2003-01-01

    Advances in bacterial genetics came with the discovery of the genetic code, followed by the development of recombinant DNA technologies. Now the field is undergoing a new revolution because of investigators' ability to sequence and assemble complete bacterial genomes. Over 200 genome projects have been completed or are in progress, and the oral microbiology research community has benefited through projects for oral bacteria and their non-oral-pathogen relatives. This review describes features of several oral bacterial genomes, and emphasizes the themes of species relationships, comparative genomics, and lateral gene transfer. Genomics is having a broad impact on basic research in microbial pathogenesis, and will lead to new approaches in clinical research and therapeutics. The oral microbiota is a unique community especially suited for new challenges to sequence the metagenomes of microbial consortia, and the genomes of uncultivable bacteria.

  6. Manufacture of Probiotic Bacteria

    Science.gov (United States)

    Muller, J. A.; Ross, R. P.; Fitzgerald, G. F.; Stanton, C.

    Lactic acid bacteria (LAB) have been used for many years as natural biopreservatives in fermented foods. A small group of LAB are also believed to have beneficial health effects on the host, so called probiotic bacteria. Probiotics have emerged from the niche industry from Asia into European and American markets. Functional foods are one of the fastest growing markets today, with estimated growth to 20 billion dollars worldwide by 2010 (GIA, 2008). The increasing demand for probiotics and the new food markets where probiotics are introduced, challenges the industry to produce high quantities of probiotic cultures in a viable and stable form. Dried concentrated probiotic cultures are the most convenient form for incorporation into functional foods, given the ease of storage, handling and transport, especially for shelf-stable functional products. This chapter will discuss various aspects of the challenges associated with the manufacturing of probiotic cultures.

  7. Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

    Science.gov (United States)

    Meza, Beatriz; de-Bashan, Luz E; Bashan, Yoav

    2015-01-01

    Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris.

  8. Bacteriome-localized intracellular symbionts in pollen-feeding beetles of the genus Dasytes (Coleoptera, Dasytidae

    Directory of Open Access Journals (Sweden)

    Benjamin Weiss

    2016-09-01

    Full Text Available Several insect taxa are associated with intracellular symbionts that provision limiting nutrients to their hosts. Such tightly integrated symbioses are especially common in insects feeding on nutritionally challenging diets like phloem sap or vertebrate blood, but also occur in seed-eating and omnivorous taxa. Here, we characterize an intracellular symbiosis in pollen-feeding beetles of the genus Dasytes (Coleoptera, Dasytidae. High-throughput tag-encoded 16S amplicon pyrosequencing of adult D. plumbeus and D. virens revealed a single gamma-proteobacterial symbiont that amounts to 52.4-98.7% of the adult beetles’ entire microbial community. Almost complete 16S rRNA sequences phylogenetically placed the symbiont into a clade comprising Buchnera and other insect endosymbionts, but sequence similarities to these closest relatives were surprisingly low (83.4 to 87.4%. Using histological examination, three-dimensional reconstructions, and fluorescence in situ hybridization, we localized the symbionts in three mulberry-shaped bacteriomes that are associated with the mid- to hind-gut transition in adult male and female beetles. Given the specialized pollen-feeding habits of the adults that contrasts with the larvae’s carnivorous lifestyle, the symbionts may provision limiting essential amino acids or vitamins as in other intracellular symbioses, or they might produce digestive enzymes that break up the fastidious pollen walls and thereby contribute to the host’s nutrition. In either case, the presence of gamma-proteobacterial symbionts in pollen-feeding beetles indicates that intracellular mutualists are more widely distributed across insects with diverse feeding habits than previously recognized.

  9. Type IV pili in Francisella – A virulence trait in an intracellular pathogen

    Directory of Open Access Journals (Sweden)

    Emelie eNäslund Salomonsson

    2011-02-01

    Full Text Available Francisella tularensis is a highly virulent intracellular human pathogen that is capable of rapid proliferation in the infected host. Mutants affected in intracellular survival and growth are highly attenuated which highlights the importance of the intracellular phase of the infection. Genomic analysis has revealed that Francisella encodes all genes required for expression of functional type IV pili (Tfp, and in this focused review we summarise recent findings regarding this system in the pathogenesis of tularemia. Tfp are dynamic adhesive structures that have been identified as major virulence determinants in several human pathogens, but it is not obvious what role these structures could have in an intracellular pathogen like Francisella. In the human pathogenic strains, genes required for secretion and assembly of Tfp and one pilin, PilA, have shown to be required for full virulence. Importantly, specific genetic differences have been identified between the different Francisella subspecies where in the most pathogenic type A variants all genes are intact while several Tfp genes are pseudogenes in the less pathogenic type B strains. This suggests that there has been a selection for expression of Tfp with different properties in the different subspecies. There is also a possibility that the genetic differences reflect adaption to different environmental niches of the subspecies and plays a role in transmission of tularemia. This is also in line with recent findings where Tfp pilins are found to be glycosylated which could reflect a role for Tfp in the environment to promote survival and transmission. We are still far from understanding the role of Tfp in virulence and transmission of tularemia, but with the genomic information and genetic tools available we are in a good position to address these issues in the future.

  10. The Arabic Diatessaron Project: Digitalizing, Encoding, Lemmatization

    Directory of Open Access Journals (Sweden)

    Giuliano Lancioni

    2016-04-01

    Full Text Available The Arabic Diatessaron Project (henceforth ADP is an international research project in Digital Humanities that aims to collect, digitalise and encode all known manuscripts of the Arabic Diatessaron (henceforth AD, a text that has been relatively neglected in scholarly research. ADP’s final goal is to provide a number of tools that can enable scholars to effectively query, compare and investigate all known variants of the text that will be encoded as far as possible in compliance with the Text Encoding Initiative (TEI guidelines. The paper addresses a number of issues involved in the process of digitalising manuscripts included in the two existing editions (Ciasca 1888 and Marmardji 1935, adding variants in unedited manuscripts, encoding and lemmatising the text. Issues involved in the design of the ADP include presentation of variants, choice of the standard text, applicability of TEI guidelines, automatic translation between different encodings, cross-edition concordances and principles of lemmatisation.

  11. Bacteria-Targeting Nanoparticles for Managing Infections

    Science.gov (United States)

    Radovic-Moreno, Aleksandar Filip

    Bacterial infections continue to be a significant concern particularly in healthcare settings and in the developing world. Current challenges include the increasing spread of drug resistant (DR) organisms, the side effects of antibiotic therapy, the negative consequences of clearing the commensal bacterial flora, and difficulties in developing prophylactic vaccines. This thesis was an investigation of the potential of a class of polymeric nanoparticles (NP) to contribute to the management of bacterial infections. More specifically, steps were taken towards using these NPs (1) to achieve greater spatiotemporal control over drug therapy by more targeted antibiotic delivery to bacteria, and (2) to develop a prophylactic vaccine formulation against the common bacterial sexually transmitted disease (STD) caused by Chlamydia trachomatis. In the first part, we synthesized polymeric NPs containing poly(lactic-co-glycolic acid)-block-poly(L-histidine)-block-poly(ethylene glycol) (PLGA-PLH-PEG). We show that these NPs are able to bind to bacteria under model acidic infection conditions and are able to encapsulate and deliver vancomycin to inhibit the growth of Staphylococcus aureus bacteria in vitro. Further work showed that the PLGA-PLH-PEG-based NPs demonstrated the potential for competition for binding bacteria at a site of infection from soluble protein and model phagocytic and tissue-resident cells in a NP composition dependent manner. The NPs demonstrated low toxicity in vitro, were well tolerated by mice in vivo, and circulated in the blood on timescales comparable to control PLGA-PEG NPs. In the second part, we used PLGA-PLH-PEG-based NPs to design a prophylactic vaccine against the obligate intracellular bacterium Chlamydia trachomatis, the most common cause of bacterial STD in the world. Currently, no vaccines against this pathogen are approved for use in humans. We first formulated NPs encapsulating the TLR7 agonist R848 conjugated to poly(lactic acid) (R848-PLA

  12. South-Seeking Magnetic Bacteria

    OpenAIRE

    Kirschvink, J.L.

    1980-01-01

    Magnetotactic bacteria, originally discovered by Blakemore (1975), are by far the most convincing and abundant example of magnetically sensitive organisms in existence. Their magnetite crystals passively align the bacteria with the earth's magnetic field like a 3-dimensional compass (Frankel et al. 1979). These microaerophilic bacteria normally live in the soupy, oxygen-poor mud/water transition zone in many freshwater and marine environments. If the mud is disturbed so that the bacteria are ...

  13. Cable Bacteria in Freshwater Sediments

    OpenAIRE

    Risgaard-Petersen, Nils; Kristiansen, Michael; Frederiksen, Rasmus B.; Dittmer, Anders Lindequist; Bjerg, Jesper Tataru; Trojan, Daniela; Schreiber, Lars; Damgaard, Lars Riis; Schramm, Andreas; Nielsen, Lars Peter

    2015-01-01

    In marine sediments cathodic oxygen reduction at the sediment surface can be coupled to anodic sulfide oxidation in deeper anoxic layers through electrical currents mediated by filamentous, multicellular bacteria of the Desulfobulbaceae family, the so-called cable bacteria. Until now, cable bacteria have only been reported from marine environments. In this study, we demonstrate that cable bacteria also occur in freshwater sediments. In a first step, homogenized sediment collected from the fre...

  14. Traffic jams II: an update of diseases of intracellular transport.

    Science.gov (United States)

    Aridor, Meir; Hannan, Lisa A

    2002-11-01

    As more details emerge on the mechanisms that mediate and control intracellular transport, the molecular basis for variety of human diseases has been revealed. In turn, disease pathology and physiology shed light on the intricate controls that regulate intracellular transport to assure proper cellular and tissue function and homeostasis. We previously listed a number of diseases that are the result of defects in intracellular transport, or cause defects in intracellular transport. (Aridor M, Hannan LA. Traffic Jam: A compendium of human diseases that affect intracellular transport processes. Traffic 2000; 1: 836-851). This Toolbox updates the previous list to include additional disorders that were recently identified to be related to intracellular trafficking. In the time since we have published our first list there have been significant advances in understanding of the molecular basis of these defects. Such advances will pave the way to future effective therapeutics.

  15. Intracellular facilitated diffusion: searchers, crowders and blockers

    CERN Document Server

    Brackley, C A; Marenduzzo, D

    2013-01-01

    In bacteria, regulatory proteins search for a specific DNA binding target via "facilitated diffusion": a series of rounds of 3D diffusion in the cytoplasm, and 1D linear diffusion along the DNA contour. Using large scale Brownian dynamics simulations we find that each of these steps is affected differently by crowding proteins, which can either be bound to the DNA acting as a road block to the 1D diffusion, or freely diffusing in the cytoplasm. Macromolecular crowding can strongly affect mechanistic features such as the balance between 3D and 1D diffusion, but leads to surprising robustness of the total search time.

  16. A model for visual memory encoding.

    Directory of Open Access Journals (Sweden)

    Rodolphe Nenert

    Full Text Available Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA. All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN. Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.

  17. A model for visual memory encoding.

    Science.gov (United States)

    Nenert, Rodolphe; Allendorfer, Jane B; Szaflarski, Jerzy P

    2014-01-01

    Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA) with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA). All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions) and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN). Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s) of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.

  18. Human Neutrophil’S Chemotaxis and Intracellular Killing in Response to Type 1 Piliated Uropathogenic Escherichia Coli

    Directory of Open Access Journals (Sweden)

    N Nooritalab

    2007-06-01

    Full Text Available Introduction: Uropathogenic Escherichia coli (UPEC, the commonest cause of urinary tract infections, bind to target cells and phagocytes via several distinct pairs of adhesins and receptors. In some cases bacterial binding to phagocytes ends to bacterial elimination. The survival and spread of bacteria in infected tissues are determined by the resistance of bacteria to elimination by phagocytic cells like neutrophils. The aim of this study was to determine the role of type 1 pili in interaction of UPEC with human neutrophils and its effect on bacterial killing. Methods: We used 3 clinical and 1 standard strains of type 1 piliated and 1 unpiliated standard strain of UPEC. Type 1 piliated and unpiliated strains (obtained by growth at a pilus-restrictive temperature of UPEC were used for determining the effect of this pili on migration of neutrophils towards bacteria in Boyden chamber. Also intracellular killing of bacteria by human neutrophils was estimated by counting of the number of viable bacteria in 45 minutes after incubation of piliated and unpiliated strains with purified neutrophils.The results were analyzed with t-test. Results: In chemotaxis assay, PMN migration towards piliated strains was 46-73% of that observed with FMLP, but it was 34-41% in unpiliated strains.The results obtained showed that type 1 piliated UPEC stimulated significantly greater chemotaxis than did unpiliated ones(P<0.05.In phagocytic killing assay, 40-70% of piliated strains were killed in 30 min after incubation with PMN, but the number of viable unpiliated strains was increased in this period of time .There was a significant difference between the intracellular killing of piliated and unpiliated strains with neutrophils (P<0.05. Discusion: Human granulocytes recognize type 1 piliated UPEC via α-mannose-containing structures. So the existence of this adhesin on UPEC strains can leads to increase of neutrophil chemotaxis towards bacteria, phagocytosis and

  19. Naloxonazine, an Amastigote-Specific Compound, Affects Leishmania Parasites through Modulation of Host-Encoded Functions

    Science.gov (United States)

    Vanhollebeke, Benoit; Caljon, Guy; Wolfe, Alan R.; McKerrow, James; Dujardin, Jean-Claude

    2016-01-01

    Host-directed therapies (HDTs) constitute promising alternatives to traditional therapy that directly targets the pathogen but is often hampered by pathogen resistance. HDT could represent a new treatment strategy for leishmaniasis, a neglected tropical disease caused by the obligate intracellular parasite Leishmania. This protozoan develops exclusively within phagocytic cells, where infection relies on a complex molecular interplay potentially exploitable for drug targets. We previously identified naloxonazine, a compound specifically active against intracellular but not axenic Leishmania donovani. We evaluated here whether this compound could present a host cell-dependent mechanism of action. Microarray profiling of THP-1 macrophages treated with naloxonazine showed upregulation of vATPases, which was further linked to an increased volume of intracellular acidic vacuoles. Treatment of Leishmania-infected macrophages with the vATPase inhibitor concanamycin A abolished naloxonazine effects, functionally demonstrating that naloxonazine affects Leishmania amastigotes indirectly, through host cell vacuolar remodeling. These results validate amastigote-specific screening approaches as a powerful way to identify alternative host-encoded targets. Although the therapeutic value of naloxonazine itself is unproven, our results further demonstrate the importance of intracellular acidic compartments for host defense against Leishmania, highlighting the possibility of targeting this host cell compartment for anti-leishmanial therapy. PMID:28036391

  20. White Spot Syndrome Virus Orf514 Encodes a Bona Fide DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Rogerio R. Sotelo-Mundo

    2011-01-01

    Full Text Available White spot syndrome virus (WSSV is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514, which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication.

  1. Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer

    OpenAIRE

    Baker, Erin L.; Bonnecaze, Roger T.; Zaman, Muhammad H.

    2009-01-01

    Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating E...

  2. Unlocking the proteomic information encoded in MALDI-TOF-MS data used for microbial identification and characterization

    Science.gov (United States)

    Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)is increasingly utilized as a rapid technique to identify microorganisms including pathogenic bacteria. However, little attention has been paid to the significant proteomic information encoded in ...

  3. High constitutive activity of a virus-encoded seven transmembrane receptor in the absence of the conserved DRY motif (Asp-Arg-Tyr) in transmembrane helix 3

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Kledal, Thomas N; Schwartz, Thue W

    2005-01-01

    The highly conserved Arg in the so-called DRY motif (Asp-Arg-Tyr) at the intracellular end of transmembrane helix 3 is in general considered as an essential residue for G protein coupling in rhodopsin-like seven transmembrane (7TM) receptors. In the open reading frame 74 (ORF74) receptor encoded ...

  4. Transcriptome reprogramming during developmental switching in Physarum polycephalum involves extensive remodeling of intracellular signaling networks.

    Science.gov (United States)

    Glöckner, Gernot; Marwan, Wolfgang

    2017-09-26

    Activation of a phytochrome photoreceptor triggers a program of Physarum polycephalum plasmodial cell differentiation through which a mitotic multinucleate protoplasmic mass synchronously develops into haploid spores formed by meiosis and rearrangement of cellular components. We have performed a transcriptome-wide RNAseq study of cellular reprogramming and developmental switching. RNAseq analysis revealed extensive remodeling of intracellular signaling and regulation in switching the expression of sets of genes encoding transcription factors, kinases, phosphatases, signal transduction proteins, RNA-binding proteins, ubiquitin ligases, regulators of the mitotic and meiotic cell cycle etc. in conjunction with the regulation of genes encoding metabolic enzymes and cytoskeletal proteins. About 15% of the differentially expressed genes shared similarity with members of the evolutionary conserved set of core developmental genes of social amoebae. Differential expression of genes encoding regulators that act at the transcriptional, translational, and post-translational level indicates the establishment of a new state of cellular function and reveals evolutionary deeply conserved molecular changes involved in cellular reprogramming and differentiation in a prototypical eukaryote.

  5. A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

    Science.gov (United States)

    Weber, Stephen; Stirnimann, Christian U; Wieser, Mara; Frey, Daniel; Meier, Roger; Engelhardt, Sabrina; Li, Xiaodan; Capitani, Guido; Kammerer, Richard A; Hilbi, Hubert

    2014-12-05

    The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ∼300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 Å resolution revealed a mainly α-helical globular protein stabilized by four antiparallel β-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.

  6. Strategies of Intracellular Pathogens for Obtaining Iron from the Environment

    Directory of Open Access Journals (Sweden)

    Nidia Leon-Sicairos

    2015-01-01

    Full Text Available Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed.

  7. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian L.; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    Amplitude- and frequency-modulated waves of Ca(2+) ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...... of the physical and biochemical conditions in plant cells. As model system, we use a H(2)O(2) signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits...... which diffusion-mediated signaling is possible. We show that purely diffusive transmission of intracellular information by H(2)O(2) over a distance of 1 μm (typical distance between organelles, which may function as relay stations) is possible at frequencies well above 1 Hz, which is the highest...

  8. Intracellular signaling by diffusion: can waves of hydrogen peroxide transmit intracellular information in plant cells?

    DEFF Research Database (Denmark)

    Vestergaard, Christian Lyngby; Flyvbjerg, Henrik; Møller, Ian Max

    2012-01-01

    Amplitude- and frequency-modulated waves of Ca2+ ions transmit information inside cells. Reactive Oxygen Species (ROS), specifically hydrogen peroxide, have been proposed to have a similar role in plant cells. We consider the feasibility of such an intracellular communication system in view...... of the physical and biochemical conditions in plant cells. As model system, we use a H2O2 signal originating at the plasma membrane (PM) and spreading through the cytosol. We consider two maximally simple types of signals, isolated pulses and harmonic oscillations. First we consider the basic limits...... diffusion-mediated signaling is possible. We show that purely diffusive transmission of intracellular information by H2O2 over a distance of 1 μm (typical distance between organelles, which may function as relay stations) is possible at frequencies well above 1 Hz, which is the highest frequency observed...

  9. pTAR-encoded proteins in plasmid partitioning.

    Science.gov (United States)

    Kalnin, K; Stegalkina, S; Yarmolinsky, M

    2000-04-01

    Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent cis-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of Agrobacterium spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465-478, 1987). However, resequencing of the region revealed two small downstream genes, parB and orf-84, of which only parB was found to be essential for partitioning in A. tumefaciens. Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA. ParB bound in vitro to repeated sequences present in a region, parS, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension. In certain respects the Par proteins behave normally in the foreign host Escherichia coli. In E. coli, as in A. tumefaciens, ParB repressed the partition operon; ParA, inactive alone, augmented this repression. Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.

  10. Evolution of the Kdo2-lipid A Biosynthesis in Bacteria

    Energy Technology Data Exchange (ETDEWEB)

    S Opiyo; R Pardy; H Moriyama; E Moriyama

    2011-12-31

    BACKGROUND: Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps. RESULTS: In order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genes only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria. CONCLUSIONS: The nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.

  11. In vitro fermentation of fructooligosaccharides with human gut bacteria.

    Science.gov (United States)

    Mao, Bingyong; Li, Dongyao; Zhao, Jianxin; Liu, Xiaoming; Gu, Zhennan; Chen, Yong Q; Zhang, Hao; Chen, Wei

    2015-03-01

    Fructooligosaccharides (FOS) are one of the most studied prebiotics, selectively stimulating the growth of health-promoting bacteria in the host. However, there is increasing evidence that commensal gut bacteria, such as Bacteroides fragilis, Clostridium butyricum, Enterobacter cloacae, and even the pathogenic Escherichia coli BEN2908, are also able to metabolize FOS in vitro, and in some cases, FOS displayed adverse effects. Therefore, it is necessary to identify FOS-metabolizing species that are present in the gut. Unlike previous studies focusing on individual strains, this study used the traditional culture method combined with an alignment search on the gut bacteria database established from the Human Microbiome Project (HMP). The alignment results showed that homologous proteins for FOS transporters and glycosidases were distributed in 237 of the 453 strains of gut bacteria. La506 msmK encoding the ATP-binding protein and Aec45 fosGH1 encoding glycoside hydrolase were most widely distributed, in 155 and 55 strains, respectively. Seven of eight strains with both transporters and glycosidases were proven to be capable of metabolizing FOS, while five strains without either transporters or glycosidases were not. Fifteen species isolated from human feces and 11 species from the alignment search were identified to be FOS-metabolizing, of which Cronobacter sakazakii, Marvinbryantia formatexigens, Ruminococcus gnavus, and Weissella paramesenteroides are reported here for the first time. Thus, alignment search combined with the culture method is an effective method for obtaining a global view of the FOS-metabolizing bacteria in the gut and will be helpful in further investigating the relationship between FOS and human gut bacteria.

  12. Structure and function of a cyanophage-encoded peptide deformylase.

    Science.gov (United States)

    Frank, Jeremy A; Lorimer, Don; Youle, Merry; Witte, Pam; Craig, Tim; Abendroth, Jan; Rohwer, Forest; Edwards, Robert A; Segall, Anca M; Burgin, Alex B

    2013-06-01

    Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 Å resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities.

  13. Bacteria counting method based on polyaniline/bacteria thin film.

    Science.gov (United States)

    Zhihua, Li; Xuetao, Hu; Jiyong, Shi; Xiaobo, Zou; Xiaowei, Huang; Xucheng, Zhou; Tahir, Haroon Elrasheid; Holmes, Mel; Povey, Malcolm

    2016-07-15

    A simple and rapid bacteria counting method based on polyaniline (PANI)/bacteria thin film was proposed. Since the negative effects of immobilized bacteria on the deposition of PANI on glass carbon electrode (GCE), PANI/bacteria thin films containing decreased amount of PANI would be obtained when increasing the bacteria concentration. The prepared PANI/bacteria film was characterized with cyclic voltammetry (CV) technique to provide quantitative index for the determination of the bacteria count, and electrochemical impedance spectroscopy (EIS) was also performed to further investigate the difference in the PANI/bacteria films. Good linear relationship of the peak currents of the CVs and the log total count of bacteria (Bacillus subtilis) could be established using the equation Y=-30.413X+272.560 (R(2)=0.982) over the range of 5.3×10(4) to 5.3×10(8)CFUmL(-1), which also showed acceptable stability, reproducibility and switchable ability. The proposed method was feasible for simple and rapid counting of bacteria. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Metabolic Requirements of Escherichia coli in Intracellular Bacterial Communities during Urinary Tract Infection Pathogenesis.

    Science.gov (United States)

    Conover, Matt S; Hadjifrangiskou, Maria; Palermo, Joseph J; Hibbing, Michael E; Dodson, Karen W; Hultgren, Scott J

    2016-04-12

    Uropathogenic Escherichia coli (UPEC) is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs). Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs) of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly, lacZ was highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZ strain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, a galK mutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated gene yeaR revealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies. Urinary tract infections (UTIs) are one of the most common bacterial infections, impacting mostly women. Every year, millions of UTIs occur in the U.S. with most being caused by uropathogenic E. coli(UPEC). During a UTI, UPEC invade bladder cells and form an intracellular bacterial community

  15. The olive fly endosymbiont, "Candidatus Erwinia dacicola," switches from an intracellular existence to an extracellular existence during host insect development.

    Science.gov (United States)

    Estes, Anne M; Hearn, David J; Bronstein, Judith L; Pierson, Elizabeth A

    2009-11-01

    As polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitat for endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine the endosymbiont of the olive fly [Bactrocera oleae (Rossi) (Diptera: Tephritidae)], a tephritid of great economic importance. "Candidatus Erwinia dacicola" was found in the digestive systems of all life stages of wild olive flies from the southwestern United States. PCR and microscopy demonstrated that "Ca. Erwinia dacicola" resided intracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. "Ca. Erwinia dacicola" is one of the few nonpathogenic endosymbionts that transitions between intracellular and extracellular lifestyles during specific stages of the host's life cycle. Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagous insects, "Ca. Erwinia dacicola" has a G+C nucleotide composition similar to those of closely related plant-pathogenic and free-living bacteria. These two characteristics of "Ca. Erwinia dacicola," the ability to transition between intracellular and extracellular lifestyles and a G+C nucleotide composition similar to those of free-living relatives, may facilitate survival in a changing environment during the development of a polyphagous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on the environment that the host provides to the endosymbiont (the endosymbiont environment).

  16. The C-terminus of IcmT is essential for pore formation and for intracellular trafficking of Legionella pneumophila within Acanthamoeba polyphaga.

    Science.gov (United States)

    Molmeret, Maëlle; Alli, O A Terry; Radulic, Marina; Susa, Milorad; Doric, Miljenko; Kwaik, Yousef Abu

    2002-03-01

    We have shown previously that the five rib (release of intracellular bacteria) mutants of Legionella pneumophila are competent for intracellular replication but defective in pore formation-mediated cytolysis and egress from protozoan and mammalian cells. The rib phenotype results from a point mutation (deletion) DeltaG544 in icmT that is predicted to result in the expression of a protein truncated by 32 amino acids from the C-terminus. In contrast to the rib mutants that are capable of intracellular replication, an icmT null mutant was completely defective in intracellular replication within mammalian and protozoan cells, in addition to its defect in pore formation-mediated cytolysis. The icmT wild-type allele complemented the icmT null mutant for both defects of intracellular replication and pore formation-mediated cytolysis and egress from mammalian cells. In contrast, the icmTDeltaG544 allele complemented the icmT null mutant for intracellular growth, but not for the pore-forming activity. Consistent with their defect in pore formation-mediated cytotoxicity in vitro, both mutants failed to cause pulmonary inflammation in A/J mice. Interestingly, the rib mutant was severely defective in intracellular growth within Acanthamoeba polyphaga. Confocal laser scanning and electron microscopy confirmed that the rib mutant and the icmT null mutant were severely and completely defective, respectively, in intracellular growth in A. polyphaga, and the respective defects correlated with fusion of the bacterial phagosomes to lysosomes. Taken together, the data showed that the C-terminus domain of IcmT is essential for the pore-forming activity and is required for intracellular trafficking and replication within A. polyphaga, but not within mammalian cells.

  17. Formation of Nodular Structures and Nitrogen Fixation by Rhizobia on Oilseed Rape Roots Following Treatment with Pectionolytic Bacteria

    Institute of Scientific and Technical Information of China (English)

    HUXIAOJIA; ZHANGXUEJIANG

    1996-01-01

    Nodular structures were formed by rhizobia on oilseed rape oilseed rape roots following treatment with pectinolytic bacteria.Nodules developed within 50 days.Photomicrograph of nodule cells showed that the capsulated bacteria were intracellular.Rhizobia resolated from the root nodules retained not only the ability of nodulation but also the characteristic of resistance to 100μg neomycin mL-1,A low nitrogenase activity of the nodules was determined by the method of acetylene reduction.

  18. Duodenal Intracellular Bicarbonate and the 'CF Paradox'

    Directory of Open Access Journals (Sweden)

    Kaunitz JD

    2001-07-01

    Full Text Available HCO(3(- secretion, which is believed to neutralize acid within the mucus gel, is the most studied duodenal defense mechanism. In general, HCO(3(- secretion rate and mucosal injury susceptibility correlate closely. Recent studies suggest that luminal acid can lower intracellular pH (pH(i of duodenal epithelial cells and that HCO(3(- secretion is unchanged during acid stress. Furthermore, peptic ulcers are rare in cystic fibrosis (CF, although, with impaired HCO(3(- secretion, increased ulcer prevalence is predicted, giving rise to the 'CF Paradox'. We thus tested the hypothesis that duodenal epithelial cell protection occurs as the result of pH(i regulation rather than by neutralization of acid by HCO(3(- in the pre-epithelial mucus. Cellular acidification during luminal acid perfusion, and unchanged HCO(3(- secretion during acid stress are inconsistent with pre-epithelial acid neutralization by secreted HCO(3(-. Furthermore, inhibition of HCO(3(- secretion by 5-nitro-2-(3-phenylpropylamino benzoic acid (NPPB despite preservation of pH(i and protection from acid-induced injury further question the pre-epithelial acid neutralization hypothesis. This decoupling of HCO(3(- secretion and injury susceptibility by NPPB (and possibly by CF further suggest that cellular buffering, rather than HCO(3(- exit into the mucus, is of primary importance for duodenal mucosal protection, and may account for the lack of peptic ulceration in CF patients.

  19. Intracellular Signals of T Cell Costimulation

    Institute of Scientific and Technical Information of China (English)

    Jianxun Song; Fengyang Tylan Lei; Xiaofang Xiong; Rizwanul Haque

    2008-01-01

    Ligation of T cell receptor (TCR) alone is insufficient to induce full activation of T lymphocytes. Additional ligand-receptor interactions (costimulation) on antigen presenting cells (APCs) and T cells are required. T cell costimulation has been shown to be essential for eliciting efficient T cell responses, involving all phases during T cell development. However, the mechanisms by which costimulation affects the function of T cells still need to be elucidated. In recent years, advances have been made in studies of costimulation as potential therapies in cancer, infectious disease as well as autoimmune disease. In this review, we discussed intracellular costimulation signals that regulate T cell proliferation, cell cycle progression, cytokine production, survival, and memory development. In general, the pathway of phosphoinositide-3 kinase (PBK)/protein kinase B (PKB, also known as Akt)/nuclear factor κB (NF-κB) might be central to many costimulatory effects. Through these pathways, costimulation controls T-cell expansion and proliferation by maintenance of survivin and aurora B expression, and sustains long-term T-cell survival and memory development by regulating the expression of bci-2 family members. Cellular & Molecular Immunology.2008;5(4):239-247.

  20. On the Computing Potential of Intracellular Vesicles.

    Science.gov (United States)

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  1. On the Computing Potential of Intracellular Vesicles.

    Directory of Open Access Journals (Sweden)

    Richard Mayne

    Full Text Available Collision-based computing (CBC is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing.

  2. Brotes germinados y bacterias

    OpenAIRE

    García Olmedo, Francisco

    2011-01-01

    Ante la confusión y el revuelo asociados al último incidente causado por una cepa de la bacteria Escherichia coli (E. coli) en Alemania, tal vez no esté de más esta carta para recordar y actualizar escritos míos anteriores aparecidos en Revista de Libros sobre los riesgos alimentarios en general y sobre los peligros de dicho microorganismo en particular. 1 . Aunque es cierto que la proporción de cepas peligrosas de E. coli es quizás inferior a la de delincuentes entre los humanos, exi...

  3. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

    Science.gov (United States)

    Ericson, Megan E.; Frank, Matthew W.

    2016-01-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

  4. Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

    Science.gov (United States)

    Yao, Jiangwei; Ericson, Megan E; Frank, Matthew W; Rock, Charles O

    2016-12-01

    Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, T.; Fukuda, H. [Kobe University, Kobe (Japan). Graduate School of Science and Technology, Division of Molecular Science; Takahashi, S.; Ueda, M.; Tanaka, A. [Kyoto University, Kyoto (Japan). Dept. of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering; Kaieda, M.; Kondo, A. [Kobe University, Kobe (Japan). Faculty of Engineering, Dept. of Chemical Science and Engineering

    2001-07-01

    Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomyces cerevisiae MT8-1. The gene encoding lipase from R. oryzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rPRoROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization. (orig.)

  6. Cellobiohydrolase variants and polynucleotides encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Wogulis, Mark

    2017-04-04

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  7. Chemical Space of DNA-Encoded Libraries.

    Science.gov (United States)

    Franzini, Raphael M; Randolph, Cassie

    2016-07-28

    In recent years, DNA-encoded chemical libraries (DECLs) have attracted considerable attention as a potential discovery tool in drug development. Screening encoded libraries may offer advantages over conventional hit discovery approaches and has the potential to complement such methods in pharmaceutical research. As a result of the increased application of encoded libraries in drug discovery, a growing number of hit compounds are emerging in scientific literature. In this review we evaluate reported encoded library-derived structures and identify general trends of these compounds in relation to library design parameters. We in particular emphasize the combinatorial nature of these libraries. Generally, the reported molecules demonstrate the ability of this technology to afford hits suitable for further lead development, and on the basis of them, we derive guidelines for DECL design.

  8. Clustering of polarization-encoded images.

    Science.gov (United States)

    Zallat, Jihad; Collet, Christophe; Takakura, Yoshitate

    2004-01-10

    Polarization-encoded imaging consists of the distributed measurements of polarization parameters for each pixel of an image. We address clustering of multidimensional polarization-encoded images. The spatial coherence of polarization information is considered. Two methods of analysis are proposed: polarization contrast enhancement and a more-sophisticated image-processing algorithm based on a Markovian model. The proposed algorithms are applied and validated with two different Mueller images acquired by a fully polarimetric imaging system.

  9. Using XML to encode TMA DES metadata

    Directory of Open Access Journals (Sweden)

    Oliver Lyttleton

    2011-01-01

    Full Text Available Background: The Tissue Microarray Data Exchange Specification (TMA DES is an XML specification for encoding TMA experiment data. While TMA DES data is encoded in XML, the files that describe its syntax, structure, and semantics are not. The DTD format is used to describe the syntax and structure of TMA DES, and the ISO 11179 format is used to define the semantics of TMA DES. However, XML Schema can be used in place of DTDs, and another XML encoded format, RDF, can be used in place of ISO 11179. Encoding all TMA DES data and metadata in XML would simplify the development and usage of programs which validate and parse TMA DES data. XML Schema has advantages over DTDs such as support for data types, and a more powerful means of specifying constraints on data values. An advantage of RDF encoded in XML over ISO 11179 is that XML defines rules for encoding data, whereas ISO 11179 does not. Materials and Methods: We created an XML Schema version of the TMA DES DTD. We wrote a program that converted ISO 11179 definitions to RDF encoded in XML, and used it to convert the TMA DES ISO 11179 definitions to RDF. Results: We validated a sample TMA DES XML file that was supplied with the publication that originally specified TMA DES using our XML Schema. We successfully validated the RDF produced by our ISO 11179 converter with the W3C RDF validation service. Conclusions: All TMA DES data could be encoded using XML, which simplifies its processing. XML Schema allows datatypes and valid value ranges to be specified for CDEs, which enables a wider range of error checking to be performed using XML Schemas than could be performed using DTDs.

  10. Using XML to encode TMA DES metadata.

    Science.gov (United States)

    Lyttleton, Oliver; Wright, Alexander; Treanor, Darren; Lewis, Paul

    2011-01-01

    The Tissue Microarray Data Exchange Specification (TMA DES) is an XML specification for encoding TMA experiment data. While TMA DES data is encoded in XML, the files that describe its syntax, structure, and semantics are not. The DTD format is used to describe the syntax and structure of TMA DES, and the ISO 11179 format is used to define the semantics of TMA DES. However, XML Schema can be used in place of DTDs, and another XML encoded format, RDF, can be used in place of ISO 11179. Encoding all TMA DES data and metadata in XML would simplify the development and usage of programs which validate and parse TMA DES data. XML Schema has advantages over DTDs such as support for data types, and a more powerful means of specifying constraints on data values. An advantage of RDF encoded in XML over ISO 11179 is that XML defines rules for encoding data, whereas ISO 11179 does not. We created an XML Schema version of the TMA DES DTD. We wrote a program that converted ISO 11179 definitions to RDF encoded in XML, and used it to convert the TMA DES ISO 11179 definitions to RDF. We validated a sample TMA DES XML file that was supplied with the publication that originally specified TMA DES using our XML Schema. We successfully validated the RDF produced by our ISO 11179 converter with the W3C RDF validation service. All TMA DES data could be encoded using XML, which simplifies its processing. XML Schema allows datatypes and valid value ranges to be specified for CDEs, which enables a wider range of error checking to be performed using XML Schemas than could be performed using DTDs.

  11. Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria

    DEFF Research Database (Denmark)

    Batchelor, Miranda; Hopkins, Katie L; Liebana, Ernesto

    2008-01-01

    We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended-spectrum ......We describe the development of a miniaturised microarray for the detection of antimicrobial resistance genes in Gram-negative bacteria. Included on the array are genes encoding resistance to aminoglycosides, trimethoprim, sulphonamides, tetracyclines and beta-lactams, including extended...

  12. Beneficial bacteria inhibit cachexia.

    Science.gov (United States)

    Varian, Bernard J; Goureshetti, Sravya; Poutahidis, Theofilos; Lakritz, Jessica R; Levkovich, Tatiana; Kwok, Caitlin; Teliousis, Konstantinos; Ibrahim, Yassin M; Mirabal, Sheyla; Erdman, Susan E

    2016-03-15

    Muscle wasting, known as cachexia, is a debilitating condition associated with chronic inflammation such as during cancer. Beneficial microbes have been shown to optimize systemic inflammatory tone during good health; however, interactions between microbes and host immunity in the context of cachexia are incompletely understood. Here we use mouse models to test roles for bacteria in muscle wasting syndromes. We find that feeding of a human commensal microbe, Lactobacillus reuteri, to mice is sufficient to lower systemic indices of inflammation and inhibit cachexia. Further, the microbial muscle-building phenomenon extends to normal aging as wild type animals exhibited increased growth hormone levels and up-regulation of transcription factor Forkhead Box N1 [FoxN1] associated with thymus gland retention and longevity. Interestingly, mice with a defective FoxN1 gene (athymic nude) fail to inhibit sarcopenia after L. reuteri therapy, indicating a FoxN1-mediated mechanism. In conclusion, symbiotic bacteria may serve to stimulate FoxN1 and thymic functions that regulate inflammation, offering possible alternatives for cachexia prevention and novel insights into roles for microbiota in mammalian ontogeny and phylogeny.

  13. Chemical communication in bacteria

    Science.gov (United States)

    Suravajhala, Srinivasa Sandeep; Saini, Deepak; Nott, Prabhu

    Luminescence in Vibrio fischeri is a model for quorum-sensing-gene-regulation in bacteria. We study luminescence response of V. fischeri to both internal and external cues at the single cell and population level. Experiments with ES114, a wild-type strain, and ainS mutant show that luminescence induction in cultures is not always proportional to cell-density and there is always a basal level of luminescence. At any given concentration of the exogenously added signals, C6-HSL and C8-HSL, luminescence per cell reaches a maximum during the exponential phase and decreases thereafter. We hypothesize that (1) C6-HSL production and LuxR activity are not proportional to cell-density, and (2) there is a shift in equilibrium from C6-HSL to C8-HSL during the later stages of growth of the culture. RT-PCR analysis of luxI and luxR shows that the expression of these genes is maximum corresponding to the highest level of luminescence. The shift in equilibrium is shown by studying competitive binding of C6-HSL and C8-HSL to LuxR. We argue that luminescence is a unicellular behaviour, and an intensive property like per cell luminescence is more important than gross luminescence of the population in understanding response of bacteria to chemical signalling. Funding from the Department of Science and Technology, India is acknowledged.

  14. Biotechnical Microbiology, yeast and bacteria

    DEFF Research Database (Denmark)

    Villadsen, Ingrid Stampe

    1999-01-01

    This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular...

  15. Biotechnical Microbiology, yeast and bacteria

    DEFF Research Database (Denmark)

    Villadsen, Ingrid Stampe

    1999-01-01

    This section contains the following single lecture notes: Eukaryotic Cell Biology. Kingdom Fungi. Cell Division. Meiosis and Recombination. Genetics of Yeast. Organisation of the Chromosome. Organization and genetics of the mitochondrial Geneme. Regulatio of Gene Expression. Intracellular Compart...

  16. Recombinant differential anchorage probes that tower over the spatial dimension of intracellular signals for high content screening and analysis.

    Science.gov (United States)

    Schembri, Laura; Zanese, Marion; Depierre-Plinet, Gaelle; Petit, Muriel; Elkaoukabi-Chaibi, Assia; Tauzin, Loic; Florean, Cristina; Lartigue, Lydia; Medina, Chantal; Rey, Christophe; Belloc, Francis; Reiffers, Josy; Ichas, François; De Giorgi, Francesca

    2009-12-01

    Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.

  17. Neurally Encoding Time for Olfactory Navigation.

    Directory of Open Access Journals (Sweden)

    In Jun Park

    2016-01-01

    Full Text Available Accurately encoding time is one of the fundamental challenges faced by the nervous system in mediating behavior. We recently reported that some animals have a specialized population of rhythmically active neurons in their olfactory organs with the potential to peripherally encode temporal information about odor encounters. If these neurons do indeed encode the timing of odor arrivals, it should be possible to demonstrate that this capacity has some functional significance. Here we show how this sensory input can profoundly influence an animal's ability to locate the source of odor cues in realistic turbulent environments-a common task faced by species that rely on olfactory cues for navigation. Using detailed data from a turbulent plume created in the laboratory, we reconstruct the spatiotemporal behavior of a real odor field. We use recurrence theory to show that information about position relative to the source of the odor plume is embedded in the timing between odor pulses. Then, using a parameterized computational model, we show how an animal can use populations of rhythmically active neurons to capture and encode this temporal information in real time, and use it to efficiently navigate to an odor source. Our results demonstrate that the capacity to accurately encode temporal information about sensory cues may be crucial for efficient olfactory navigation. More generally, our results suggest a mechanism for extracting and encoding temporal information from the sensory environment that could have broad utility for neural information processing.

  18. The bimodal lifestyle of intracellular Salmonella in epithelial cells: replication in the cytosol obscures defects in vacuolar replication.

    Directory of Open Access Journals (Sweden)

    Preeti Malik-Kale

    Full Text Available Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens.

  19. GENOMIC ANALYSIS OF PLANT-ASSOCIATED BACTERIA AND THEIR POTENTIAL IN ENHANCING PHYTOREMEDIATION EFFICIENCY

    Directory of Open Access Journals (Sweden)

    Artur Piński

    2017-07-01

    Full Text Available Phytoremediation is an emerging technology that uses plants in order to cleanup pollutants including xenobiotics and heavy metals from soil, water and air. Inoculation of plants with plant growth promoting endophytic and rhizospheric bacteria can enhance efficiency of phytoremediation. Genomic analysis of four plant-associated strains belonging to the Stenotrophomonas maltophilia species revealed the presence of genes encoding proteins involved in plant growth promotion, biocontrol of phytopathogens, biodegradation of xenobiotics, heavy metals resistance and plant-bacteria-environment interaction. The results of this analysis suggest great potential of bacteria belonging to Stenotrophomonas maltophilia species in enhancing phytoremediation efficiency.

  20. [Acanthamoeba, naturally intracellularly infected with Pseudomonas aeruginosa, after their isolation from a microbiologically contaminated drinking water system in a hospital].

    Science.gov (United States)

    Michel, R; Burghardt, H; Bergmann, H

    1995-03-01

    The drinking water system of a new hospital building that was highly contaminated with bacteria before opening was investigated too for the prevalence of small free living amoebae. Germ counts resulted in > 100 CFU/ml in 100% of the cold water samples, that showed also growth of P. aeruginosa, whereas E. coli and coliforme bacteria could not be identified. The investigation of 37 water samples for protozoa revealed growth of small freeliving amoebae in 20 samples (54%) belonging to 10 species of the genus Acanthamoeba, Naegleria, Hartmannella, Echinamoeba among others. In addition 2 Ciliate- and 2 Microflagellate-species could be observed. While all Naegleria strains isolated belonged to the N. gruberi-complex two of 16 Acanthamoeba-isolates proved to be pathogenic for laboratory mice. From 7 watersamples positive with P. aeruginosa 5 Acanthamoeba- and 2 Echinamoeba strains could be isolated which revealed intracellular multiplication of P. aeruginosa. Because of their well known resistances against chlorine, the amoebae and their cysts are considered to be vectors for these intracellular bacteria. A complete sanitation of the incriminated drinking water system was accomplished by combined chemical and thermic disinfection measures.

  1. Antibiotic-resistant soil bacteria in transgenic plant fields.

    Science.gov (United States)

    Demanèche, Sandrine; Sanguin, Hervé; Poté, John; Navarro, Elisabeth; Bernillon, Dominique; Mavingui, Patrick; Wildi, Walter; Vogel, Timothy M; Simonet, Pascal

    2008-03-11

    Understanding the prevalence and polymorphism of antibiotic resistance genes in soil bacteria and their potential to be transferred horizontally is required to evaluate the likelihood and ecological (and possibly clinical) consequences of the transfer of these genes from transgenic plants to soil bacteria. In this study, we combined culture-dependent and -independent approaches to study the prevalence and diversity of bla genes in soil bacteria and the potential impact that a 10-successive-year culture of the transgenic Bt176 corn, which has a blaTEM marker gene, could have had on the soil bacterial community. The bla gene encoding resistance to ampicillin belongs to the beta-lactam antibiotic family, which is widely used in medicine but is readily compromised by bacterial antibiotic resistance. Our results indicate that soil bacteria are naturally resistant to a broad spectrum of beta-lactam antibiotics, including the third cephalosporin generation, which has a slightly stronger discriminating effect on soil isolates than other cephalosporins. These high resistance levels for a wide range of antibiotics are partly due to the polymorphism of bla genes, which occur frequently among soil bacteria. The blaTEM116 gene of the transgenic corn Bt176 investigated here is among those frequently found, thus reducing any risk of introducing a new bacterial resistance trait from the transgenic material. In addition, no significant differences were observed in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn fields, although the bacterial populations were different.

  2. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    Science.gov (United States)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  3. Immunomodulatory properties of probiotic bacteria

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen

    2007-01-01

    Certain lactic acid bacteria (LAB) are part of the commensal intestinal flora and considered beneficial for health, as they compete with pathogens for adhesion sites in the intestine and ferment otherwise indigestible compounds. Another important property of these so-called probiotic bacteria...... with bacteria, and the cytokine pattern induced by specific bacteria resembled the pattern induced in MoDC, except for TNF-alpha and IL-6, which were induced in response to different bacteria in blood DC/monocytes and monocyte-derived DC. Autologous NK cells produced IFN-gamma when cultured with blood DC......, monocytes and monocyte-derived DC and IL-12-inducing bacteria, whereas only DC induced IFN-gamma production in allogeneic T cells. In vitro-generated DC is a commonly used model of tissue DC, but they differ in certain aspects from intestinal DC, which are in direct contact with the intestinal microbiota...

  4. Cable Bacteria in Freshwater Sediments

    DEFF Research Database (Denmark)

    Risgaard-Petersen, Nils; Kristiansen, Michael; Frederiksen, Rasmus

    2015-01-01

    In marine sediments cathodic oxygen reduction at the sediment surface can be coupled to anodic sulfide oxidation in deeper anoxic layers through electrical currents mediated by filamentous, multicellular bacteria of the Desulfobulbaceae family, the so-called cable bacteria. Until now, cable...... marine cable bacteria, with the genus Desulfobulbus as the closest cultured lineage. The results of the present study indicate that electric currents mediated by cable bacteria could be important for the biogeochemistry in many more environments than anticipated thus far and suggest a common evolutionary...... bacteria have only been reported from marine environments. In this study, we demonstrate that cable bacteria also occur in freshwater sediments. In a first step, homogenized sediment collected from the freshwater stream Giber Å, Denmark, was incubated in the laboratory. After 2 weeks, pH signatures...

  5. Visually Improved Image Compression by Combining EZW Encoding with Texture Modeling using Huffman Encoder

    Directory of Open Access Journals (Sweden)

    Vinay U. Kale

    2010-05-01

    Full Text Available This paper proposes a technique for image compression which uses the Wavelet-based Image/Texture Coding Hybrid (WITCH scheme [1] in combination with Huffman encoder. It implements a hybrid coding approach, while nevertheless preserving the features of progressive and lossless coding. The hybrid scheme was designed to encode the structural image information by Embedded Zerotree Wavelet (EZW encoding algorithm [2] and the stochastic texture in a model-based manner and this encoded data is then compressed using Huffman encoder. The scheme proposed here achieves superior subjective quality while increasing the compression ratio by more than a factor of three or even four. With this technique, it is possible to achieve compression ratios as high as 10 to 12 but with some minor distortions in the encoded image.

  6. Delivery of a Salmonella Typhi exotoxin from a host intracellular compartment.

    Science.gov (United States)

    Spanò, Stefania; Ugalde, Juan E; Galán, Jorge E

    2008-01-17

    Salmonella Typhi, an exclusive human pathogen and the cause of typhoid fever, expresses a functional cytolethal distending toxin for which only the active subunit, CdtB, has been identified. Here, we show that PltA and PltB, which are encoded in the same pathogenicity islet as cdtB, associate with CdtB to form a multipartite toxin. PltA and PltB are homologs of components of the pertussis toxin, including its ADP-ribosyl transferase subunit. We also show that PltA and PltB are required for the delivery of CdtB from an intracellular compartment to target cells via autocrine and paracrine pathways. We hypothesize that this toxin, which we have named "typhoid toxin," and its delivery mechanism may contribute to S. Typhi's unique virulence properties.

  7. Hydrophilic trans-Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling.

    Science.gov (United States)

    Kozma, Eszter; Nikić, Ivana; Varga, Balázs R; Aramburu, Iker Valle; Kang, Jun Hee; Fackler, Oliver T; Lemke, Edward A; Kele, Péter

    2016-08-17

    Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.

  8. Mycobacterium smegmatis MSMEG_3705 encodes a selective major facilitator superfamily efflux pump with multiple roles.

    Science.gov (United States)

    Zhang, Zhen; Wang, Rui; Xie, Jianping

    2015-06-01

    Mycobacterium smegmatis mc(2)155 MSMEG_3705 gene was annotated to encode a transporter protein that contains 12 alpha-helical transmembrane domains. We predicted MSMEG_3705 encoding a major facilitator superfamily (MFS) member. To confirm the prediction, the M. smegmatis mc(2)155 MSMEG_3705 gene was deleted. The MSMEG_3705 deletion mutant strain M. smegmatis mc(2)155 ∆MSMEG_3705 was more sensitive to capreomycin. Moreover, M. smegmatis mc(2)155 ∆MSMEG_3705 strain accumulated more ethidium bromide intracellular than wild-type M. smegmatis mc(2)155. Quite unexpectedly, M. smegmatis mc(2)155 ∆MSMEG_3705 grew faster than the wild-type M. smegmatis mc(2)155. The upregulation of the expression of MSMEG_3706, a gene encoding isocitrate lyase downstream MSMEG_3705, in the deletion mutant, might underlie such faster growth in the mutant. The study showed that MSMEG_3705 encodes a genuine MFS member and plays significant role in bacterial growth and antibiotics resistance.

  9. Bacteria transport under unsaturated conditions

    OpenAIRE

    Gargiulo, Grazia

    2007-01-01

    The aim of this work was to study the bacteria transport behaviour in different conditions using an unsaturated porous media. A column based system able to keep the unsaturated conditions was designed and developed to perform the experiments. Two bacteria strains Deinococcus radiodurans and Rhodococcus rhodochrous strongly different in hydrophobicity were employed. During the experiments the bacteria concentration in the outflow was continuously on-line measured and after the experiment the c...

  10. Social behavior of bacteria: from physics to complex organization

    Science.gov (United States)

    Ben-Jacob, E.

    2008-10-01

    I describe how bacteria develop complex colonial patterns by utilizing intricate communication capabilities, such as quorum sensing, chemotactic signaling and exchange of genetic information (plasmids) Bacteria do not store genetically all the information required for generating the patterns for all possible environments. Instead, additional information is cooperatively generated as required for the colonial organization to proceed. Each bacterium is, by itself, a biotic autonomous system with its own internal cellular informatics capabilities (storage, processing and assessments of information). These afford the cell certain plasticity to select its response to biochemical messages it receives, including self-alteration and broadcasting messages to initiate alterations in other bacteria. Hence, new features can collectively emerge during self-organization from the intra-cellular level to the whole colony. Collectively bacteria store information, perform decision make decisions (e.g. to sporulate) and even learn from past experience (e.g. exposure to antibiotics)-features we begin to associate with bacterial social behavior and even rudimentary intelligence. I also take Schrdinger’s’ “feeding on negative entropy” criteria further and propose that, in addition organisms have to extract latent information embedded in the environment. By latent information we refer to the non-arbitrary spatio-temporal patterns of regularities and variations that characterize the environmental dynamics. In other words, bacteria must be able to sense the environment and perform internal information processing for thriving on latent information embedded in the complexity of their environment. I then propose that by acting together, bacteria can perform this most elementary cognitive function more efficiently as can be illustrated by their cooperative behavior.

  11. Bacteriophages of methanotrophic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Tyutikow, F.M. (All-Union Research Inst. for Genetics and Selection of Industrial Microorganisms, Moscow, USSR); Bespalova, I.A.; Rebentish, B.A.; Aleksandrushkina, N.N.; Krivisky, A.S.

    1980-10-01

    Bacteriophages of methanotrophic bacteria have been found in 16 out of 88 studied samples (underground waters, pond water, soil, gas and oil installation waters, fermentor cultural fluids, bacterial paste, and rumen of cattle) taken in different geographic zones of the Soviet Union. Altogether, 23 phage strains were isolated. By fine structure, the phages were divided into two types (with very short or long noncontractile tails); by host range and serological properties, they fell into three types. All phages had guanine- and cytosine-rich double-stranded deoxyribonucleic acid consisting of common nitrogen bases. By all of the above-mentioned properties, all phages within each of the groups were completely identical to one another, but differed from phages of other groups.

  12. Bacteria, phages and septicemia.

    Directory of Open Access Journals (Sweden)

    Ausra Gaidelyte

    Full Text Available The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.

  13. Acoustofluidic bacteria separation

    Science.gov (United States)

    Li, Sixing; Ma, Fen; Bachman, Hunter; Cameron, Craig E.; Zeng, Xiangqun; Huang, Tony Jun

    2017-01-01

    Bacterial separation from human blood samples can help with the identification of pathogenic bacteria for sepsis diagnosis. In this work, we report an acoustofluidic device for label-free bacterial separation from human blood samples. In particular, we exploit the acoustic radiation force generated from a tilted-angle standing surface acoustic wave (taSSAW) field to separate Escherichia coli from human blood cells based on their size difference. Flow cytometry analysis of the E. coli separated from red blood cells shows a purity of more than 96%. Moreover, the label-free electrochemical detection of the separated E. coli displays reduced non-specific signals due to the removal of blood cells. Our acoustofluidic bacterial separation platform has advantages such as label-free separation, high biocompatibility, flexibility, low cost, miniaturization, automation, and ease of in-line integration. The platform can be incorporated with an on-chip sensor to realize a point-of-care sepsis diagnostic device.

  14. Multichannel compressive sensing MRI using noiselet encoding.

    Directory of Open Access Journals (Sweden)

    Kamlesh Pawar

    Full Text Available The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS. In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding.

  15. Multichannel compressive sensing MRI using noiselet encoding.

    Science.gov (United States)

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding.

  16. An encyclopedia of mouse DNA elements (Mouse ENCODE).

    Science.gov (United States)

    Stamatoyannopoulos, John A; Snyder, Michael; Hardison, Ross; Ren, Bing; Gingeras, Thomas; Gilbert, David M; Groudine, Mark; Bender, Michael; Kaul, Rajinder; Canfield, Theresa; Giste, Erica; Johnson, Audra; Zhang, Mia; Balasundaram, Gayathri; Byron, Rachel; Roach, Vaughan; Sabo, Peter J; Sandstrom, Richard; Stehling, A Sandra; Thurman, Robert E; Weissman, Sherman M; Cayting, Philip; Hariharan, Manoj; Lian, Jin; Cheng, Yong; Landt, Stephen G; Ma, Zhihai; Wold, Barbara J; Dekker, Job; Crawford, Gregory E; Keller, Cheryl A; Wu, Weisheng; Morrissey, Christopher; Kumar, Swathi A; Mishra, Tejaswini; Jain, Deepti; Byrska-Bishop, Marta; Blankenberg, Daniel; Lajoie, Bryan R; Jain, Gaurav; Sanyal, Amartya; Chen, Kaun-Bei; Denas, Olgert; Taylor, James; Blobel, Gerd A; Weiss, Mitchell J; Pimkin, Max; Deng, Wulan; Marinov, Georgi K; Williams, Brian A; Fisher-Aylor, Katherine I; Desalvo, Gilberto; Kiralusha, Anthony; Trout, Diane; Amrhein, Henry; Mortazavi, Ali; Edsall, Lee; McCleary, David; Kuan, Samantha; Shen, Yin; Yue, Feng; Ye, Zhen; Davis, Carrie A; Zaleski, Chris; Jha, Sonali; Xue, Chenghai; Dobin, Alex; Lin, Wei; Fastuca, Meagan; Wang, Huaien; Guigo, Roderic; Djebali, Sarah; Lagarde, Julien; Ryba, Tyrone; Sasaki, Takayo; Malladi, Venkat S; Cline, Melissa S; Kirkup, Vanessa M; Learned, Katrina; Rosenbloom, Kate R; Kent, W James; Feingold, Elise A; Good, Peter J; Pazin, Michael; Lowdon, Rebecca F; Adams, Leslie B

    2012-08-13

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

  17. Heterologous expression of leader-less pga gene in Pichia pastoris: intracellular production of prokaryotic enzyme

    Directory of Open Access Journals (Sweden)

    Kyslík Pavel

    2010-02-01

    Full Text Available Abstract Background Penicillin G acylase of Escherichia coli (PGAEc is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of β-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage, continues at crossing the cytoplasmic membrane (signal sequence removing, and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. Results Leader-less pga gene encoding PGAEcunder the control of AOX1 promoter was cloned in Pichia pastoris X-33. The intracellular overproduction of heterologous PGAEc(hPGAEc was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l-1 was reached. The hPGAEc was purified, characterized and compared with the wild-type PGAEc. The α-subunit of the hPGAEc formed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin. Conclusions Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGAEc

  18. Bacteria versus selenium: A view from the inside out

    Science.gov (United States)

    Staicu, Lucian; Oremland, Ronald S.; Tobe, Ryuta; Mihara, Hisaaki

    2017-01-01

    Bacteria and selenium (Se) are closely interlinked as the element serves both essential nutrient requirements and energy generation functions. However, Se can also behave as a powerful toxicant for bacterial homeostasis. Conversely, bacteria play a tremendous role in the cycling of Se between different environmental compartments, and bacterial metabolism has been shown to participate to all valence state transformations undergone by Se in nature. Bacteria possess an extensive molecular repertoire for Se metabolism. At the end of the 1980s, a novel mode of anaerobic respiration based on Se oxyanions was experimentally documented for the first time. Following this discovery, specific enzymes capable of reducing Se oxyanions and harvesting energy were found in a number of anaerobic bacteria. The genes involved in the expression of these enzymes have later been identified and cloned. This iterative approach undertaken outside-in led to the understanding of the molecular mechanisms of Se transformations in bacteria. Based on the extensive knowledge accumulated over the years, we now have a full(er) view from the inside out, from DNA-encoding genes to enzymes and thermodynamics. Bacterial transformations of Se for assimilatory purposes have been the object of numerous studies predating the investigation of Se respiration. Remarkable contributions related to the understating of the molecular picture underlying seleno-amino acid biosynthesis are reviewed herein. Under certain circumstances, Se is a toxicant for bacterial metabolism and bacteria have evolved strategies to counteract this toxicity, most notably by the formation of elemental Se (nano)particles. Several biotechnological applications, such as the production of functional materials and the biofortification of crop species using Se-utilizing bacteria, are presented in this chapter.

  19. Gene clusters involved in isethionate degradation by terrestrial and marine bacteria.

    KAUST Repository

    Weinitschke, Sonja

    2010-01-01

    Ubiquitous isethionate (2-hydroxyethanesulfonate) is dissimilated by diverse bacteria. Growth of Cupriavidus necator H16 with isethionate was observed, as was inducible membrane-bound isethionate dehydrogenase (IseJ) and inducible transcription of the genes predicted to encode IseJ and a transporter (IseU). Biodiversity in isethionate transport genes was observed and investigated by transcription experiments.

  20. Use of the alr gene as a food-grade selection marker in lactic acid bacteria

    NARCIS (Netherlands)

    Bron, P.A.; Benchimol, M.G.; Lambert, J.; Palumbo, E.; Deghorain, M.; Delcour, J.; Vos, de W.M.; Kleerebezem, M.; Hols, P.

    2002-01-01

    Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of D-alanine and L-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection marker

  1. Extracellular matrix stiffness and architecture govern intracellular rheology in cancer.

    Science.gov (United States)

    Baker, Erin L; Bonnecaze, Roger T; Zaman, Muhammad H

    2009-08-19

    Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of beta1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells.

  2. Metabolic Requirements of Escherichia coli in Intracellular Bacterial Communities during Urinary Tract Infection Pathogenesis

    Directory of Open Access Journals (Sweden)

    Matt S. Conover

    2016-04-01

    Full Text Available Uropathogenic Escherichia coli (UPEC is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs. Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly, lacZ was highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZ strain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, a galK mutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated gene yeaR revealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies.

  3. Growth ability of Gram negative bacteria in free-living amoebae.

    Science.gov (United States)

    Zeybek, Zuhal; Binay, Ali Rıza

    2014-11-01

    When bacteria and free-living amoebae (FLAs) live both in natural waters and man-made aquatic systems, they constantly interact with each other. Some bacteria can survive and grow within FLAs. Therefore, it has recently been thought that FLAs play an important role in spreading pathogenic bacteria in aquatic systems. In this study we investigated the intracellular growing ability of 7 different Gram-negative bacteria (Pseudomonas fluorescens, Pseudomonas putida, Pasteurella pneumotropica, Aeromonas salmonicida, Legionella pneumophila serogroup 1, L. pneumophila serogroup 3, L. pneumophila serogroup 6) in four different FLA isolates (A1-A4). Among these, four bacterial isolates (P. fluorescens, P.putida, P.pneumotropica, A.salmonicida) and two free-living amoebae isolates (A3, A4) were isolated from the tap water in our city (Istanbul). It was found that 4 different Gram-negative bacteria could grow in A1, 2 different Gram-negative bacteria could grow in A2, 4 different Gram-negative bacteria could grow in A3, 1 Gram-negative bacterium could grow in A4. In conclusion, we think that this ability of growth could vary according to the characteristics of both bacteria and FLA isolates. Also, other factors such as environmental temperature, bacterial concentration, and extended incubation period may play a role in these interactions. This situation can be clarified with future studies.

  4. Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila neuromuscular junction.

    Directory of Open Access Journals (Sweden)

    Cathy I Ramos

    2015-04-01

    Full Text Available The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs requires Neto (Neuropillin and Tolloid-like, a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK, a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes.

  5. Tissue architecture and function: dynamic reciprocity via extra- and intra-cellular matrices

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ren; Boudreau, Aaron; Bissell, Mina J

    2008-12-23

    Mammary gland development, functional differentiation, and homeostasis are orchestrated and sustained by a balance of biochemical and biophysical cues from the organ's microenvironment. The three-dimensional microenvironment of the mammary gland, predominantly 'encoded' by a collaboration between the extracellular matrix (ECM), hormones, and growth factors, sends signals from ECM receptors through the cytoskeletal intracellular matrix to nuclear and chromatin structures resulting in gene expression; the ECM in turn is regulated and remodeled by signals from the nucleus. In this chapter, we discuss how coordinated ECM deposition and remodeling is necessary for mammary gland development, how the ECM provides structural and biochemical cues necessary for tissue-specific function, and the role of the cytoskeleton in mediating the extra - to intracellular dialogue occurring between the nucleus and the microenvironment. When operating normally, the cytoskeletal-mediated dynamic and reciprocal integration of tissue architecture and function directs mammary gland development, tissue polarity, and ultimately, tissue-specific gene expression. Cancer occurs when these dynamic interactions go awry for an extended time.

  6. 3D Spatially Resolved Models of the Intracellular Dynamics of the Hepatitis C Genome Replication Cycle

    KAUST Repository

    Knodel, Markus

    2017-10-02

    Mathematical models of virus dynamics have not previously acknowledged spatial resolution at the intracellular level despite substantial arguments that favor the consideration of intracellular spatial dependence. The replication of the hepatitis C virus (HCV) viral RNA (vRNA) occurs within special replication complexes formed from membranes derived from endoplasmatic reticulum (ER). These regions, termed membranous webs, are generated primarily through specific interactions between nonstructural virus-encoded proteins (NSPs) and host cellular factors. The NSPs are responsible for the replication of the vRNA and their movement is restricted to the ER surface. Therefore, in this study we developed fully spatio-temporal resolved models of the vRNA replication cycle of HCV. Our simulations are performed upon realistic reconstructed cell structures-namely the ER surface and the membranous webs-based on data derived from immunostained cells replicating HCV vRNA. We visualized 3D simulations that reproduced dynamics resulting from interplay of the different components of our models (vRNA, NSPs, and a host factor), and we present an evaluation of the concentrations for the components within different regions of the cell. Thus far, our model is restricted to an internal portion of a hepatocyte and is qualitative more than quantitative. For a quantitative adaption to complete cells, various additional parameters will have to be determined through further in vitro cell biology experiments, which can be stimulated by the results deccribed in the present study.

  7. The intracellular transport and secretion of calumenin-1/2 in living cells.

    Directory of Open Access Journals (Sweden)

    Qiao Wang

    Full Text Available Calumenin isoforms 1 and 2 (calu-1/2, encoded by the CALU gene, belong to the CREC protein family. Calu-1/2 proteins are secreted into the extracellular space, but the secretory process and regulatory mechanism are largely unknown. Here, using a time-lapse imaging system, we visualized the intracellular transport and secretory process of calu-1/2-EGFP after their translocation into the ER lumen. Interestingly, we observed that an abundance of calu-1/2-EGFP accumulated in cellular processes before being released into the extracellular space, while only part of calu-1/2-EGFP proteins were secreted directly after attaching to the cell periphery. Moreover, we found the secretion of calu-1/2-EGFP required microtubule integrity, and that calu-1/2-EGFP-containing vesicles were transported by the motor proteins Kif5b and cytoplasmic dynein. Finally, we determined the export signal of calu-1/2-EGFP (amino acid positions 20-46 and provided evidence that the asparagine at site 131 was indispensable for calu-1/2-EGFP stabilization. Taken together, we provide a detailed picture of the intracellular transport of calu-1/2-EGFP, which facilitates our understanding of the secretory mechanism of calu-1/2.

  8. Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters.

    Science.gov (United States)

    DeSouza, Sunita; Fu, Jie; States, Bradley A; Ziff, Edward B

    2002-05-01

    Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.

  9. In vitro intracellular trafficking of virulence antigen during infection by Yersinia pestis.

    Directory of Open Access Journals (Sweden)

    Tracy L DiMezzo

    Full Text Available Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops and a multifunctional virulence antigen (V. V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s of V, time-course experiments were performed with macrophages (MPhis infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MPhis were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs] in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MPhis were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1 endosomal proteins between 10-45 min of infection, (2 lysosomal protein(s between 1-2 h of infection, (3 mitochondrial proteins between 2.5-3 h infection, and (4 Golgi protein(s between 4-6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.

  10. Intracellular protein transport to the thyrocyte plasma membrane: potential implications for thyroid physiology.

    Science.gov (United States)

    Arvan, P; Kim, P S; Kuliawat, R; Prabakaran, D; Muresan, Z; Yoo, S E; Abu Hossain, S

    1997-02-01

    We present a snapshot of developments in epithelial biology that may prove helpful in understanding cellular aspects of the machinery designed for the synthesis of thyroid hormones on the thyroglobulin precursor. The functional unit of the thyroid gland is the follicle, delimited by a monolayer of thyrocytes. Like the cells of most simple epithelia, thyrocytes exhibit specialization of the cell surface that confronts two different extracellular environments-apical and basolateral, which are separated by tight junctions. Specifically, the basolateral domain faces the interstitium/bloodstream, while the apical domain is in contact with the lumen that is the primary target for newly synthesized thyroglobulin secretion and also serves as a storage depot for previously secreted protein. Thyrocytes use their polarity in several important ways, such as for maintaining basolaterally located iodide uptake and T4 deiodination, as well apically located iodide efflux and iodination machinery. The mechanisms by which this organization is established, fall in large part under the more general cell biological problem of intracellular sorting and trafficking of different proteins en route to the cell surface. Nearly all exportable proteins begin their biological life after synthesis in an intracellular compartment known as the endoplasmic reticulum (ER), upon which different degrees of difficulty may be encountered during nascent polypeptide folding and initial export to the Golgi complex. In these initial stages, ER molecular chaperones can assist in monitoring protein folding and export while themselves remaining as resident proteins of the thyroid ER. After export from the ER, most subsequent sorting for protein delivery to apical or basolateral surfaces of thyrocytes occurs within another specialized intracellular compartment known as the trans-Golgi network. Targeting information encoded in secretory proteins and plasma membrane proteins can be exposed or buried at different

  11. The large intracellular loop of hZIP4 is an intrinsically disordered zinc binding domain†

    Science.gov (United States)

    Bafaro, Elizabeth M.; Antala, Sagar; Nguyen, Tuong-Vi; Dzul, Stephen P.; Doyon, Brian; Stemmler, Timothy L.; Dempski, Robert E.

    2015-01-01

    The human (h) ZIP4 transporter is a plasma membrane protein which functions to increase the cytosolic concentration of zinc. hZIP4 transports zinc into intestinal cells and therefore has a central role in the absorption of dietary zinc. hZIP4 has eight transmembrane domains and encodes a large intracellular loop between transmembrane domains III and IV, M3M4. Previously, it has been postulated that this domain regulates hZIP4 levels in the plasma membrane in a zinc-dependent manner. The objective of this research was to examine the zinc binding properties of the large intracellular loop of hZIP4. Therefore, we have recombineantly expressed and purified M3M4 and showed that this domain binds two zinc ions. Using a combination of site-directed mutagenesis, metal binding affinity assays, and X-ray absorption spectroscopy, we demonstrated that the two Zn2+ ions bind sequentially, with the first Zn2+ binding to a CysHis3 site with a nanomolar binding affinity, and the second Zn2+ binding to a His4 site with a weaker affinity. Circular dichroism spectroscopy revealed that the M3M4 domain is intrinsically disordered, with only a small structural change induced upon Zn2+ coordination. Our data supports a model in which the intracellular M3M4 domain senses high cytosolic Zn2+ concentrations and regulates the plasma membrane levels of the hZIP4 transporter in response to Zn2+ binding. PMID:25882556

  12. Histological evaluation of the presence of bacteria in induced periapical lesions in monkeys.

    Science.gov (United States)

    Walton, R E; Ardjmand, K

    1992-05-01

    In endodontic periapical lesions, both presence and location of bacteria are controversial. Various experimental techniques have produced differing results perhaps related to potential artifacts such as contamination during specimen recovery. Our objective was to examine for bacteria in uncontaminated, undisturbed periapical lesions in an animal model. Pulp necrosis was induced by exposing molars in nonhuman primates and closing the exposure after 1 week with amalgam. Lesions developed at 18 apices. After 7 months, block sections including tooth and surrounding tissues were removed, processed histologically, and Gram stained. Bacteria, primarily Gram positive, were consistently identified in necrotic tissue in canals. Two canals demonstrated bacterial masses to the apical foramen. No bacterial colonies, only intracellular microorganisms, were seen periapically. Inflammatory lesions seemed to resist the spread of bacteria, confining them to the canal space. Bacterial masses at the apical foramen could contaminate periapical tissues during surgery or extraction and give a false positive upon microbiological sampling.

  13. Simultaneous transcriptional profiling of bacteria and their host cells.

    Directory of Open Access Journals (Sweden)

    Michael S Humphrys

    Full Text Available We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness. Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.

  14. Chaperone receptors: guiding proteins to intracellular compartments.

    Science.gov (United States)

    Kriechbaumer, Verena; von Löffelholz, Ottilie; Abell, Ben M

    2012-01-01

    Despite mitochondria and chloroplasts having their own genome, 99% of mitochondrial proteins (Rehling et al., Nat Rev Mol Cell Biol 5:519-530, 2004) and more than 95% of chloroplast proteins (Soll, Curr Opin Plant Biol 5:529-535, 2002) are encoded by nuclear DNA, synthesised in the cytosol and imported post-translationally. Protein targeting to these organelles depends on cytosolic targeting factors, which bind to the precursor, and then interact with membrane receptors to deliver the precursor into a translocase. The molecular chaperones Hsp70 and Hsp90 have been widely implicated in protein targeting to mitochondria and chloroplasts, and receptors capable of recognising these chaperones have been identified at the surface of both these organelles (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007). The role of these chaperone receptors is not fully understood, but they have been shown to increase the efficiency of protein targeting (Young et al., Cell 112:41-50, 2003; Qbadou et al., EMBO J 25:1836-1847, 2006). Whether these receptors contribute to the specificity of targeting is less clear. A class of chaperone receptors bearing tetratricopeptide repeat domains is able to specifically bind the highly conserved C terminus of Hsp70 and/or Hsp90. Interestingly, at least of one these chaperone receptors can be found on each organelle (Schlegel et al., Mol Biol Evol 24:2763-2774, 2007), which suggests a universal role in protein targeting for these chaperone receptors. This review will investigate the role that chaperone receptors play in targeting efficiency and specificity, as well as examining recent in silico approaches to find novel chaperone receptors.

  15. New perspective in the assessment of total intracellular magnesium

    Directory of Open Access Journals (Sweden)

    Azzurra Sargenti

    2014-01-01

    Full Text Available Magnesium (Mg is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ, that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.

  16. Human neutrophils dump Candida glabrata after intracellular killing.

    Science.gov (United States)

    Essig, Fabian; Hünniger, Kerstin; Dietrich, Stefanie; Figge, Marc Thilo; Kurzai, Oliver

    2015-11-01

    Interaction between fungal pathogens and human phagocytes can lead to remarkably variable outcomes, ranging from intracellular killing to prolonged survival and replication of the pathogen in the host cell. Using live cell imaging we observed primary human neutrophils that release phagocytosed Candida glabrata yeast cells after intracellular killing. This process, for which we propose the name "dumping", adds a new outcome to phagocyte-fungus interaction which may be of potential immunological importance as it allows professional antigen presenting cells to take up and process neutrophil-inactivated pathogens that in their viable state are able to evade intracellular degradation in these cells.

  17. Intracellular Acidosis Enhances the Excitability of Working Muscle

    Science.gov (United States)

    Pedersen, Thomas H.; Nielsen, Ole B.; Lamb, Graham D.; Stephenson, D. George

    2004-08-01

    Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.

  18. Intracellular acidosis enhances the excitability of working muscle.

    Science.gov (United States)

    Pedersen, Thomas H; Nielsen, Ole B; Lamb, Graham D; Stephenson, D George

    2004-08-20

    Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.

  19. Chlorosomes: antenna organelles in photosynthetic green bacteria

    DEFF Research Database (Denmark)

    Frigaard, N.-U.; Bryant, D. A.

    2006-01-01

    that cover the spectrum of the complex intracellular structures of prokaryotes: proteasomes, phycobilisomes, chlorosomes, gas vesicles, carboxysomes, magnetosomes, intracytoplasmic membranes, membrane-bound nucleoids, anammoxosomes, and cytoarchitecture of Epulopiscium spp. Cameos of selected additional......The new series "Microbiology Monographs" begins with two volumes on intracellular components in prokaryotes. In this second volume, "Complex Intracellular Structures in Prokaryotes", the components, labelled complex intracellular structures, encompass a multitude of important cellular functions....... Continuing and newly initiated research will provide a clearer understanding of the complex intracellular structures known at present and will bring to light surprising new ones as well. "Complex Intracellular Structures in Prokaryotes" provides historical background and comprehensive reviews of ten topics...

  20. Construction of mobilizable mini-Tn7 vectors for bioluminescent detection of gram-negative bacteria and single-copy promoter lux reporter analysis.

    Science.gov (United States)

    Damron, F Heath; McKenney, Elizabeth S; Barbier, Mariette; Liechti, George W; Schweizer, Herbert P; Goldberg, Joanna B

    2013-07-01

    We describe the construction of mini-Tn7-based broad-host-range vectors encoding lux genes as bioluminescent reporters. These constructs can be mobilized into the desired host(s) by conjugation for chromosomal mini-Tn7-lux integration and are useful for localization of bacteria during infections or for characterizing regulation of promoters of interest in Gram-negative bacteria.

  1. Enteropathogenic Escherichia coli, Samonella, Shigella and Yersinia: cellular aspects of host-bacteria interactions in enteric diseases

    Directory of Open Access Journals (Sweden)

    Reis Roberta

    2010-07-01

    Full Text Available Abstract A successful infection of the human intestine by enteropathogenic bacteria depends on the ability of bacteria to attach and colonize the intestinal epithelium and, in some cases, to invade the host cell, survive intracellularly and disseminate from cell to cell. To accomplish these processes bacteria have evolved an arsenal of molecules that are mostly secreted by dedicated type III secretion systems, and that interact with the host, subverting normal cellular functions. Here we overview the most important molecular strategies developed by enteropathogenic Escherichia coli, Salmonella enterica, Shigella flexneri, and Yersinia enterocolitica to cause enteric infections. Despite having evolved different effectors, these four microorganisms share common host cellular targets.

  2. Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing.

    Science.gov (United States)

    Pumphrey, Graham M; Ranchou-Peyruse, Anthony; Spain, Jim C

    2011-07-01

    Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of (13)CO(2) was H(2) dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from (13)C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H(2) concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.

  3. RNA chaperones encoded by RNA viruses

    Institute of Scientific and Technical Information of China (English)

    Jie Yang; Hongjie Xia; Qi Qian; Xi Zhou

    2015-01-01

    RNAs are functionally diverse macromolecules whose proper functions rely strictly upon their correct tertiary structures. However, because of their high structural flexibility, correct folding of RNAs is challenging and slow. Therefore, cells and viruses encode a variety of RNA remodeling proteins, including helicases and RNA chaperones. In RNA viruses, these proteins are believed to play pivotal roles in all the processes involving viral RNAs during the life cycle. RNA helicases have been studied extensively for decades, whereas RNA chaperones, particularly virus-encoded RNA chaperones, are often overlooked. This review describes the activities of RNA chaperones encoded by RNA viruses, particularly the ones identified and characterized in recent years, and the functions of these proteins in different steps of viral life cycles, and presents an overview of this unique group of proteins.

  4. Challenges in Decomposing Encodings of Verification Problems

    Directory of Open Access Journals (Sweden)

    Peter Schrammel

    2016-07-01

    Full Text Available Modern program verifiers use logic-based encodings of the verification problem that are discharged by a back end reasoning engine. However, instances of such encodings for large programs can quickly overwhelm these back end solvers. Hence, we need techniques to make the solving process scale to large systems, such as partitioning (divide-and-conquer and abstraction. In recent work, we showed how decomposing the formula encoding of a termination analysis can significantly increase efficiency. The analysis generates a sequence of logical formulas with existentially quantified predicates that are solved by a synthesis-based program analysis engine. However, decomposition introduces abstractions in addition to those required for finding the unknown predicates in the formula, and can hence deteriorate precision. We discuss the challenges associated with such decompositions and their interdependencies with the solving process.

  5. An information theoretic characterisation of auditory encoding.

    Directory of Open Access Journals (Sweden)

    Tobias Overath

    2007-10-01

    Full Text Available The entropy metric derived from information theory provides a means to quantify the amount of information transmitted in acoustic streams like speech or music. By systematically varying the entropy of pitch sequences, we sought brain areas where neural activity and energetic demands increase as a function of entropy. Such a relationship is predicted to occur in an efficient encoding mechanism that uses less computational resource when less information is present in the signal: we specifically tested the hypothesis that such a relationship is present in the planum temporale (PT. In two convergent functional MRI studies, we demonstrated this relationship in PT for encoding, while furthermore showing that a distributed fronto-parietal network for retrieval of acoustic information is independent of entropy. The results establish PT as an efficient neural engine that demands less computational resource to encode redundant signals than those with high information content.

  6. Cluster parallel rendering based on encoded mesh

    Institute of Scientific and Technical Information of China (English)

    QIN Ai-hong; XIONG Hua; PENG Hao-yu; LIU Zhen; SHI Jiao-ying

    2006-01-01

    Use of compressed mesh in parallel rendering architecture is still an unexplored area, the main challenge of which is to partition and sort the encoded mesh in compression-domain. This paper presents a mesh compression scheme PRMC (Parallel Rendering based Mesh Compression) supplying encoded meshes that can be partitioned and sorted in parallel rendering system even in encoded-domain. First, we segment the mesh into submeshes and clip the submeshes' boundary into Runs, and then piecewise compress the submeshes and Runs respectively. With the help of several auxiliary index tables, compressed submeshes and Runs can serve as rendering primitives in parallel rendering system. Based on PRMC, we design and implement a parallel rendering architecture. Compared with uncompressed representation, experimental results showed that PRMC meshes applied in cluster parallel rendering system can dramatically reduce the communication requirement.

  7. Swimming bacteria in liquid crystal

    Science.gov (United States)

    Sokolov, Andrey; Zhou, Shuang; Aranson, Igor; Lavrentovich, Oleg

    2014-03-01

    Dynamics of swimming bacteria can be very complex due to the interaction between the bacteria and the fluid, especially when the suspending fluid is non-Newtonian. Placement of swimming bacteria in lyotropic liquid crystal produces a new class of active materials by combining features of two seemingly incompatible constituents: self-propelled live bacteria and ordered liquid crystals. Here we present fundamentally new phenomena caused by the coupling between direction of bacterial swimming, bacteria-triggered flows and director orientations. Locomotion of bacteria may locally reduce the degree of order in liquid crystal or even trigger nematic-isotropic phase transition. Microscopic flows generated by bacterial flagella disturb director orientation. Emerged birefringence patterns allow direct optical observation and quantitative characterization of flagella dynamics. At high concentration of bacteria we observed the emergence of self-organized periodic texture caused by bacteria swimming. Our work sheds new light on self-organization in hybrid bio-mechanical systems and can lead to valuable biomedical applications. Was supported by the US DOE, Office of Basic Energy Sciences, Division of Materials Science and Engineering, under the Contract No. DE AC02-06CH11357.

  8. Ecophysiology of the anammox bacteria

    NARCIS (Netherlands)

    Kartal, Mustafa Boran

    2008-01-01

    Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium to dinitrogen gas with nitrite as the electron acceptor. These bacteria are the key players in the global nitrogen cycle, responsible for the most of nitrogen production in natural ecosystems. The anammox process is also a cost-effecti

  9. Money and transmission of bacteria

    NARCIS (Netherlands)

    Gedik, H.; Voss, T.A.; Voss, A.

    2013-01-01

    Money is one of the most frequently passed items in the world. The aim of this study was to ascertain the survival status of bacteria including Staphylococcus aureus, Escherichia coli, and Vancomycin- Resistant Enterococci (VRE) on banknotes from different countries and the transmission of bacteria

  10. Characterization of intracellular palladium nanoparticles synthesized by Desulfovibrio desulfuricans and Bacillus benzeovorans

    Science.gov (United States)

    Omajali, Jacob B.; Mikheenko, Iryna P.; Merroun, Mohamed L.; Wood, Joseph; Macaskie, Lynne E.

    2015-06-01

    Early studies have focused on the synthesis of palladium nanoparticles within the periplasmic layer or on the outer membrane of Desulfovibrio desulfuricans and on the S-layer protein of Bacillus sphaericus. However, it has remained unclear whether the synthesis of palladium nanoparticles also takes place in the bacterial cell cytoplasm. This study reports the use of high-resolution scanning transmission electron microscopy with a high-angle annular dark field detector and energy dispersive X-ray spectrometry attachment to investigate the intracellular synthesis of palladium nanoparticles (Pd NPs). We show the intracellular synthesis of Pd NPs within cells of two anaerobic strains of D. desulfuricans and an aerobic strain of B. benzeovorans using hydrogen and formate as electron donors. The Pd nanoparticles were small and largely monodispersed, between 0.2 and 8 nm, occasionally from 9 to 12 nm with occasional larger nanoparticles. With D. desulfuricans NCIMB 8307 (but not D. desulfuricans NCIMB 8326) and with B. benzeovorans NCIMB 12555, the NPs were larger when made at the expense of formate, co-localizing with phosphate in the latter, and were crystalline, but were amorphous when made with H2, with no phosphorus association. The intracellular Pd nanoparticles were mainly icosahedrons with surfaces comprising {111} facets and about 5 % distortion when compared with that of bulk palladium. The particles were more concentrated in the cell cytoplasm than the cell wall, outer membrane, or periplasm. We provide new evidence for synthesis of palladium nanoparticles within the cytoplasm of bacteria, which were confirmed to maintain cellular integrity during this synthesis.

  11. Characterization of intracellular palladium nanoparticles synthesized by Desulfovibrio desulfuricans and Bacillus benzeovorans

    Energy Technology Data Exchange (ETDEWEB)

    Omajali, Jacob B., E-mail: JBO037@bham.ac.uk, E-mail: jbomajali@gmail.com; Mikheenko, Iryna P. [University of Birmingham, Unit of Functional Bionanomaterials, School of Biosciences, Institute of Microbiology and Infection (United Kingdom); Merroun, Mohamed L. [University of Granada, Department of Microbiology, Faculty of Sciences (Spain); Wood, Joseph [University of Birmingham, School of Chemical Engineering (United Kingdom); Macaskie, Lynne E. [University of Birmingham, Unit of Functional Bionanomaterials, School of Biosciences, Institute of Microbiology and Infection (United Kingdom)

    2015-06-15

    Early studies have focused on the synthesis of palladium nanoparticles within the periplasmic layer or on the outer membrane of Desulfovibrio desulfuricans and on the S-layer protein of Bacillus sphaericus. However, it has remained unclear whether the synthesis of palladium nanoparticles also takes place in the bacterial cell cytoplasm. This study reports the use of high-resolution scanning transmission electron microscopy with a high-angle annular dark field detector and energy dispersive X-ray spectrometry attachment to investigate the intracellular synthesis of palladium nanoparticles (Pd NPs). We show the intracellular synthesis of Pd NPs within cells of two anaerobic strains of D. desulfuricans and an aerobic strain of B. benzeovorans using hydrogen and formate as electron donors. The Pd nanoparticles were small and largely monodispersed, between 0.2 and 8 nm, occasionally from 9 to 12 nm with occasional larger nanoparticles. With D. desulfuricans NCIMB 8307 (but not D. desulfuricans NCIMB 8326) and with B. benzeovorans NCIMB 12555, the NPs were larger when made at the expense of formate, co-localizing with phosphate in the latter, and were crystalline, but were amorphous when made with H{sub 2,} with no phosphorus association. The intracellular Pd nanoparticles were mainly icosahedrons with surfaces comprising {111} facets and about 5 % distortion when compared with that of bulk palladium. The particles were more concentrated in the cell cytoplasm than the cell wall, outer membrane, or periplasm. We provide new evidence for synthesis of palladium nanoparticles within the cytoplasm of bacteria, which were confirmed to maintain cellular integrity during this synthesis.

  12. Identification of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth.

    Directory of Open Access Journals (Sweden)

    Sarah A Stanley

    2014-02-01

    Full Text Available Mycobacterium tuberculosis remains a significant threat to global health. Macrophages are the host cell for M. tuberculosis infection, and although bacteria are able to replicate intracellularly under certain conditions, it is also clear that macrophages are capable of killing M. tuberculosis if appropriately activated. The outcome of infection is determined at least in part by the host-pathogen interaction within the macrophage; however, we lack a complete understanding of which host pathways are critical for bacterial survival and replication. To add to our understanding of the molecular processes involved in intracellular infection, we performed a chemical screen using a high-content microscopic assay to identify small molecules that restrict mycobacterial growth in macrophages by targeting host functions and pathways. The identified host-targeted inhibitors restrict bacterial growth exclusively in the context of macrophage infection and predominantly fall into five categories: G-protein coupled receptor modulators, ion channel inhibitors, membrane transport proteins, anti-inflammatories, and kinase modulators. We found that fluoxetine, a selective serotonin reuptake inhibitor, enhances secretion of pro-inflammatory cytokine TNF-α and induces autophagy in infected macrophages, and gefitinib, an inhibitor of the Epidermal Growth Factor Receptor (EGFR, also activates autophagy and restricts growth. We demonstrate that during infection signaling through EGFR activates a p38 MAPK signaling pathway that prevents macrophages from effectively responding to infection. Inhibition of this pathway using gefitinib during in vivo infection reduces growth of M. tuberculosis in the lungs of infected mice. Our results support the concept that screening for inhibitors using intracellular models results in the identification of tool compounds for probing pathways during in vivo infection and may also result in the identification of new anti

  13. Requirement of the RNA-binding protein SmpB during intracellular growth of Listeria monocytogenes.

    Science.gov (United States)

    Mraheil, Mobarak Abu; Frantz, Renate; Teubner, Lisa; Wendt, Heiko; Linne, Uwe; Wingerath, Jessica; Wirth, Thomas; Chakraborty, Trinad

    2017-04-01

    Bacterial trans-translation is the main quality control mechanism employed to relieve stalled ribosomes. Trans-translation is mediated by the small protein B (SmpB) and transfer-mRNA (tmRNA) ribonucleoprotein complex, which interacts with translational complexes stalled at the 3' end of non-stop mRNAs to release the stalled ribosomes thereby targeting the nascent polypeptides and truncated mRNAs for degradation. The trans-translation system exists with a few exceptions in all bacteria. In the present study, we assessed the contribution of SmpB to the growth and virulence of Listeria monocytogenes, a human intracellular food-borne pathogen that colonizes host tissues to cause severe invasive infections. A smpB knockout significantly decreased the intracellular growth rate of L. monocytogenes during infection of murine macrophages. In addition, the mutant strain was attenuated for virulence when examined with the Galleria mellonella larvae killing assay and the organ colonisation model of mice following infection. Proteomic analysis of whole cell extracts of ΔsmpB deletion mutant revealed elevated protein levels of several proteins involved in ribosome assembly and interaction with tRNA substrates. These included the elongation factor Tu [EF-Tu] which promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis as well as the CysK which is known to interact with bacterial toxins that cleave tRNA substrates. The data presented here shed light on the role of SmpB and trans-translation during intracellular growth of L. monocytogenes. Copyright © 2017 Elsevier GmbH. All rights reserved.

  14. Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza

    Science.gov (United States)

    Moleres, Javier; Sinha, Sunita; Fernández-Calvet, Ariadna; Porsch, Eric A.; St. Geme, Joseph W.; Nislow, Corey; Redfield, Rosemary J.; Garmendia, Junkal

    2016-01-01

    Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe “transformed recombinant enrichment profiling” (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation. PMID:27124727

  15. Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

    Science.gov (United States)

    Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

    2015-06-01

    Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host.

  16. Clinical Concentrations of Thioridazine Kill Intracellular Multidrug-Resistant Mycobacterium tuberculosis

    Science.gov (United States)

    Ordway, Diane; Viveiros, Miguel; Leandro, Clara; Bettencourt, Rosário; Almeida, Josefina; Martins, Marta; Kristiansen, Jette E.; Molnar, Joseph; Amaral, Leonard

    2003-01-01

    The phenothiazines chlorpromazine (CPZ) and thioridazine (TZ) have equal in vitro activities against antibiotic-sensitive and -resistant Mycobacterium tuberculosis. These compounds have not been used as anti-M. tuberculosis agents because their in vitro activities take place at concentrations which are beyond those that are clinically achievable. In addition, chronic administration of CPZ produces frequent severe side effects. Because CPZ has been shown to enhance the killing of intracellular M. tuberculosis at concentrations in the medium that are clinically relevant, we have investigated whether TZ, a phenothiazine whose negative side effects are less frequent and serious than those associated with CPZ, kills M. tuberculosis organisms that have been phagocytosed by human macrophages, which have nominal killing activities against these bacteria. Both CPZ and TZ killed intracellular antibiotic-sensitive and -resistant M. tuberculosis organisms when they were used at concentrations in the medium well below those present in the plasma of patients treated with these agents. These concentrations in vitro were not toxic to the macrophage, nor did they affect in vitro cellular immune processes. TZ thus appears to be a serious candidate for the management of a freshly diagnosed infection of pulmonary tuberculosis or as an adjunct to conventional antituberculosis therapy if the patient originates from an area known to have a high prevalence of multidrug-resistant M. tuberculosis isolates. Nevertheless, we must await the outcomes of clinical trials to determine whether TZ itself may be safely and effectively used as an antituberculosis agent. PMID:12604522

  17. Host Intracellular Signaling Events and Pro-inflammatory Cytokine Production in African Trypanosomiasis.

    Science.gov (United States)

    Kuriakose, Shiby M; Singh, Rani; Uzonna, Jude E

    2016-01-01

    Pathogens, such as bacteria, viruses, and parasites, possess specific molecules or proteins that are recognized by several host innate immune receptors, leading to the activation of several intracellular signaling molecules and pathways. The magnitude and quality of these events significantly affect the outcome of infection. African trypanosomes, including Trypanosoma congolense, are capable of manipulating the host immune response, including the activity of macrophages, which are the key immune cells that contribute to the immunopathogenesis of African trypanosomiasis. Although it is known that immune hyperactivation and excessive pro-inflammatory cytokine production are the hallmarks of African trypanosomiasis, the mechanisms through which these events are triggered are poorly defined. However, it is known that macrophages may play a significant role in these processes, because phagocytosis of trypanosomes by macrophages initiates intracellular signal transduction cascades that lead to the release of pro-inflammatory cytokines and alteration in cell function. This review highlights recent progress in our understanding of the innate immune receptors, signaling pathways, and transcription factors involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. It will reveal the existence of complex signaling events through which the parasite modulates the host immune response, thus identifying novel targets that could aid in designing strategies to effectively control the disease.

  18. Increasing intracellular trehalose is sufficient to confer desiccation tolerance to Saccharomyces cerevisiae.

    Science.gov (United States)

    Tapia, Hugo; Young, Lindsey; Fox, Douglas; Bertozzi, Carolyn R; Koshland, Douglas

    2015-05-12

    Diverse organisms capable of surviving desiccation, termed anhydrobiotes, include species from bacteria, yeast, plants, and invertebrates. However, most organisms are sensitive to desiccation, likely due to an assortment of different stresses such as protein misfolding and aggregation, hyperosmotic stress, membrane fracturing, and changes in cell volume and shape leading to an overcrowded cytoplasm and metabolic arrest. The exact stress(es) that cause lethality in desiccation-sensitive organisms and how the lethal stresses are mitigated in desiccation-tolerant organisms remain poorly understood. The presence of trehalose in anhydrobiotes has been strongly correlated with desiccation tolerance. In the yeast Saccharomyces cerevisiae, trehalose is essential for survival after long-term desiccation. Here, we establish that the elevation of intracellular trehalose in dividing yeast by its import from the media converts yeast from extreme desiccation sensitivity to a high level of desiccation tolerance. This trehalose-induced tolerance is independent of utilization of trehalose as an energy source, de novo synthesis of other stress effectors, or the metabolic effects of trehalose biosynthetic intermediates, indicating that a chemical property of trehalose is directly responsible for desiccation tolerance. Finally, we demonstrate that elevated intracellular maltose can also make dividing yeast tolerant to short-term desiccation, indicating that other disaccharides have stress effector activity. However, trehalose is much more effective than maltose at conferring tolerance to long-term desiccation. The effectiveness and sufficiency of trehalose as an antagonizer of desiccation-induced damage in yeast emphasizes its potential to confer desiccation tolerance to otherwise sensitive organisms.

  19. Motility of electric cable bacteria

    DEFF Research Database (Denmark)

    Bjerg, Jesper Tataru; Damgaard, Lars Riis; Holm, Simon Agner

    2016-01-01

    Cable bacteria are filamentous bacteria that electrically couple sulfide oxidation and oxygen reduction at centimeter distances, and observations in sediment environments have suggested that they are motile. By time-lapse microscopy, we found that cable bacteria used gliding motility on surfaces...... with a highly variable speed of 0.50.3 ms1 (meanstandard deviation) and time between reversals of 155108 s. They frequently moved forward in loops, and formation of twisted loops revealed helical rotation of the filaments. Cable bacteria responded to chemical gradients in their environment, and around the oxic......-anoxic interface, they curled and piled up, with straight parts connecting back to the source of sulfide. Thus, it appears that motility serves the cable bacteria in establishing and keeping optimal connections between their distant electron donor and acceptors in a dynamic sediment environment....

  20. Enhanced double patterning decomposition using lines encoding

    Directory of Open Access Journals (Sweden)

    Khaled M. Soradi

    2016-09-01

    Full Text Available Double patterning photolithography (DPL is considered one of the best solutions used for enabling 32 nm/22 nm technology. In this paper, we propose a new technique for double patterning post decomposition conflict resolution. The algorithm is based on lines positions encoding followed by code pattern matching. Experimental results show that the usage of encoded patterns decreases the time needed for pattern matching and increases the matching accuracy. The overall manual problem solution time is reduced to about 1%.

  1. Fidelity enhancement by logical qubit encoding.

    Science.gov (United States)

    Henry, Michael K; Ramanathan, Chandrasekhar; Hodges, Jonathan S; Ryan, Colm A; Ditty, Michael J; Laflamme, Raymond; Cory, David G

    2007-11-30

    We demonstrate coherent control of two logical qubits encoded in a decoherence free subspace (DFS) of four dipolar-coupled protons in an NMR quantum information processor. A pseudopure fiducial state is created in the DFS, and a unitary logical qubit entangling operator evolves the system to a logical Bell state. The four-spin molecule is partially aligned by a liquid crystal solvent, which introduces strong dipolar couplings among the spins. Although the system Hamiltonian is never fully specified, we demonstrate high fidelity control over the logical degrees of freedom. In fact, the DFS encoding leads to higher fidelity control than is available in the full four-spin Hilbert space.

  2. Comparing mannose binding lectin genetic diversity in intracellular ...

    African Journals Online (AJOL)

    SERVER

    2007-09-05

    Sep 5, 2007 ... mannose binding lectin level for susceptibility to intracellular pathogens. Key words: .... The distribution of alleles and genotypes between groups were compared using ... Frequency of promoter variants and position +4 (Table.

  3. Intracellular Ca(2+) and Zn(2+) signals during monochloramine-induced oxidative stress in isolated rat colon crypts.

    Science.gov (United States)

    Cima, Robert R; Dubach, J Matthew; Wieland, Aaron M; Walsh, Breda M; Soybel, David I

    2006-02-01

    During acute exacerbations of inflammatory bowel diseases, oxidants are generated through the interactions of bacteria in the lumen, activated granulocytes, and cells of the colon mucosa. In this study we explored the ability of one such class of oxidants, represented by monochloramine (NH(2)Cl), to serve as agonists of Ca(2+) and Zn(2+) accumulation within the colonocyte. Individual colon crypts prepared from Sprague-Dawley rats were mounted in perfusion chambers after loading with fluorescent reporters fura 2-AM and fluozin 3-AM. These reporters were characterized, in situ, for responsiveness to Ca(2+) and Zn(2+) in the cytoplasm. Responses to different concentrations of NH(2)Cl (50, 100, and 200 microM) were monitored. Subsequent studies were designed to identify the sources and mechanisms of NH(2)Cl-induced increases in Ca(2+) and Zn(2+) in the cytoplasm. Exposure to NH(2)Cl led to dose-dependent increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in the range of 200-400 nM above baseline levels. Further studies indicated that NH(2)Cl-induced accumulation of Ca(2+) in the cytoplasm is the result of release from intracellular stores and basolateral entry of extracellular Ca(2+) through store-operated channels. In addition, exposure to NH(2)Cl resulted in dose-dependent and sustained increases in intracellular Zn(2+) concentration ([Zn(2+)](i)) in the nanomolar range. These alterations were neutralized by dithiothreitol, which shields intracellular thiol groups from oxidation. We conclude that Ca(2+)- and Zn(2+)-handling proteins are susceptible to oxidation by chloramines, leading to sustained, but not necessarily toxic, increases in [Ca(2+)](i) and [Zn(2+)](i). Under certain conditions, NH(2)Cl may act not as a toxin but as an agent that activates intracellular signaling pathways.

  4. Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

    Science.gov (United States)

    Ashour, Joseph; Hondalus, Mary K

    2003-04-01

    Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

  5. FLIPR assays of intracellular calcium in GPCR drug discovery

    DEFF Research Database (Denmark)

    Hansen, Kasper Bø; Bräuner-Osborne, Hans

    2009-01-01

    Fluorescent dyes sensitive to changes in intracellular calcium have become increasingly popular in G protein-coupled receptor (GPCR) drug discovery for several reasons. First of all, the assays using the dyes are easy to perform and are of low cost compared to other assays. Second, most non-Galph...... making them obtainable even for academic groups. Here, we present a protocol for measuring changes in intracellular calcium levels in living mammalian cells based on the fluorescent calcium binding dye, fluo-4....

  6. Study of neurotoxic intracellular calcium signalling triggered by amyloids.

    Science.gov (United States)

    Villalobos, Carlos; Caballero, Erica; Sanz-Blasco, Sara; Núñez, Lucía

    2012-01-01

    Neurotoxicity in Alzheimer's disease (AD) is associated to dishomeostasis of intracellular Ca(2+) induced by amyloid β peptide (Aβ) species. Understanding of the effects of Aβ on intracellular Ca(2+) homeostasis requires preparation of the different Aβ assemblies including oligomers and fibrils and the testing of their effects on cytosolic and mitochondrial Ca(2+) in neurons. Procedures for cerebellar granule cell culture, preparation of Aβ species as well as fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca(2+) in neurons are described.

  7. Intracellular concentrations determine the cytotoxicity of adefovir, cidofovir and tenofovir.

    Science.gov (United States)

    Zhang, Xun; Wang, Ruduan; Piotrowski, Mary; Zhang, Hui; Leach, Karen L

    2015-02-01

    Lack of in vitro to in vivo translation is a major challenge in safety prediction during early drug discovery.One of the most common in vitro assays to evaluate the probability of a compound to cause adverse effects is a cytotoxicity assay. Cytotoxicity of a compound is often measured by dose–response curves assuming the administered doses and intracellular exposures are equal at the time of measurement.However, this may not be true for compounds with low membrane permeability or those which are substrates for drug transporters as intracellular concentrations are determined both by passive permeability and active uptake through drug transporters. We show here that three antiviral drugs, adefovir, cidofovir and tenofovir exhibit significantly increased cytotoxicity in HEK293 cells transfected with organic anion transporter (OAT) 1 and 3 compared to a lack of cytotoxicity in HEK293 wildtype cells. A further look at the media and intracellular drug concentrations showed that 24 h after dosing, all three drugs had higher intracellular drug concentrations than that of media in the HEK-OAT1 cells whereas the intracellular drug concentrations in the wildtype cells were much lower than the administered doses. Comparing cytotoxicity IC(50) values of adefovir, cidofovir and tenofovir based on administered doses and measured intracellular concentrations in HEK-OAT1 cells revealed that intracellular drug concentrations have significant impact on calculated IC(50) values. Tenofovir showed much less intrinsic cytotoxicity than adefovir and cidofovir using intracellular concentrations rather than media concentration. Our data suggest that for low permeable drugs or drugs that are substrates for drug transporters, the choice of cellular model is critical for providing an accurate determination of cytotoxicity.

  8. In vivo metabolite compartmentalization probed using intracellular GdDTPA

    DEFF Research Database (Denmark)

    Peters, David Alberg; Rowland, Ian

    Fast trans-membrane water exchange enables in- tracellular relaxation enhancement of water by contrast agents in the extracellular space. For me- tabolites not in fast exchange across membranes, intracellular metabolite relaxation enhancement will only occur if the contrast agent and metabolite a...... are in the same compartment. Extracellular contrast has utilized electroporation methods to deliver GdDTPA into the cytosol of rat muscle in vivo in order to probe the intracellular compart- mentalization of MR-visible metabolites....

  9. Intra-cellular Staphylococcus aureus alone causes infection in vivo

    Directory of Open Access Journals (Sweden)

    T Hamza

    2013-07-01

    Full Text Available Chronic and recurrent bone infections occur frequently but have not been explained. Staphylococcus aureus (S. aureus is often found among chronic and recurrent infections and may be responsible for such infections. One possible reason is that S. aureus can internalize and survive within host cells and by doing so, S. aureus can evade both host defense mechanisms and most conventional antibiotic treatments. In this study, we hypothesized that intra-cellular S. aureus could induce infections in vivo. Osteoblasts were infected with S. aureus and, after eliminating extra-cellular S. aureus, inoculated into an open fracture rat model. Bacterial cultures and radiographic observations at post-operative day 21 confirmed local bone infections in animals inoculated with intra-cellular S. aureus within osteoblasts alone. We present direct in vivo evidence that intra-cellular S. aureus could be sufficient to induce bone infection in animals; we found that intra-cellular S. aureus inoculation of as low as 102 colony forming units could induce severe bone infections. Our data may suggest that intra-cellular S. aureus can “hide” in host cells during symptom-free periods and, under certain conditions, they may escape and lead to infection recurrence. Intra-cellular S. aureus therefore could play an important role in the pathogenesis of S. aureus infections, especially those chronic and recurrent infections in which disease episodes may be separated by weeks, months, or even years.

  10. Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the phosphoethanolamine transferase is encoded by opgE.

    Science.gov (United States)

    Bontemps-Gallo, Sébastien; Cogez, Virginie; Robbe-Masselot, Catherine; Quintard, Kevin; Dondeyne, Jacqueline; Madec, Edwige; Lacroix, Jean-Marie

    2013-01-01

    Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity.

  11. Biosynthesis of Osmoregulated Periplasmic Glucans in Escherichia coli: The Phosphoethanolamine Transferase Is Encoded by opgE

    Directory of Open Access Journals (Sweden)

    Sébastien Bontemps-Gallo

    2013-01-01

    Full Text Available Osmoregulated periplasmic glucans (OPGs are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity.

  12. Evidence for the presence of cell-surface-bound and intracellular bactericidal toxins in the dinoflagellate Heterocapsa circularisquama.

    Science.gov (United States)

    Cho, Kichul; Wencheng, Li; Takeshita, Satoshi; Seo, Jung-Kil; Chung, Young-Ho; Kim, Daekyung; Oda, Tatsuya

    2017-08-01

    Heterocapsa circularisquama, a harmful dinoflagellate, has multiple haemolytic toxins that are considered to be involved in the toxic mechanism against shellfish and certain species of zooplankton. To evaluate the further nature of the toxins of H. circularisquama, we investigated its effects on several species of bacteria. By colony formation assay, we found that H. circularisquama had antibacterial activity toward the marine bacterium Vibrio alginolyticus in a cell density-dependent manner. When the inoculated bacterial cells were co-cultured with H. circularisquama under dinoflagellate cell culture conditions, the bacterial growth was significantly suppressed, whereas the number of live bacterial cells increased when cultured in the medium alone. Since the cell-free culture supernatant and the ruptured dinoflagellate cell suspension showed no toxic effects on V. alginolyticus, it is speculated that direct cell-to-cell contact mediated by the live dinoflagellate cells may be the major toxic mechanism. The decrease in bactericidal activity of theca-removed dinoflagellate cells may further support this speculation. H. circularisquama also showed bactericidal activities towards Escherichia coli and Staphylococcus aureus. In the dinoflagellate/bacteria co-culture system, the number of live bacterial cells declined with increasing incubation time. Light-dependent antibacterial activity of the ruptured dinoflagellate cells against S. aureus was observed, whereas no such activity was detected against E. coli. These results suggest that intracellular photosensitising bactericidal toxins, which were previously found to be porphyrin derivatives, may have specificity towards gram-positive bacteria. Based on these results together with previous studies, it is obvious that H. circularisquama possesses antibacterial activity, which may be mediated through toxins located on its cell surface. It is likely that such toxins play a role in the defence mechanism against predators

  13. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids.

    Science.gov (United States)

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Bamford, Jaana K H; Buckling, Angus

    2011-12-23

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.

  14. Phosphate solubilizing bacteria and their role in plant growth promotion.

    Science.gov (United States)

    Rodríguez, H; Fraga, R

    1999-10-01

    The use of phosphate solubilizing bacteria as inoculants simultaneously increases P uptake by the plant and crop yield. Strains from the genera Pseudomonas, Bacillus and Rhizobium are among the most powerful phosphate solubilizers. The principal mechanism for mineral phosphate solubilization is the production of organic acids, and acid phosphatases play a major role in the mineralization of organic phosphorous in soil. Several phosphatase-encoding genes have been cloned and characterized and a few genes involved in mineral phosphate solubilization have been isolated. Therefore, genetic manipulation of phosphate-solubilizing bacteria to improve their ability to improve plant growth may include cloning genes involved in both mineral and organic phosphate solubilization, followed by their expression in selected rhizobacterial strains. Chromosomal insertion of these genes under appropriate promoters is an interesting approach.

  15. Encoded Archival Description as a Halfway Technology

    Science.gov (United States)

    Dow, Elizabeth H.

    2009-01-01

    In the mid 1990s, Encoded Archival Description (EAD) appeared as a revolutionary technology for publishing archival finding aids on the Web. The author explores whether or not, given the advent of Web 2.0, the archival community should abandon EAD and look for something to replace it. (Contains 18 notes.)

  16. 47 CFR 11.32 - EAS Encoder.

    Science.gov (United States)

    2010-10-01

    ... codes shall be retained even with the power removed. (7) Indicator. An aural or visible means that it... levels of the mark or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal that complies with the following: (i) Tone Frequencies. The audio tones shall have...

  17. Encoding and Decoding Procedures for Arrangements

    Directory of Open Access Journals (Sweden)

    Alexander A. Babaev

    2012-05-01

    Full Text Available This article discusses an algorithm based on the encoding procedure for representing a set of arrangement elements as a single number. Also the author provides the procedure for the inverse transformation of the code into arrangement elements. In addition the Article includes recommendations on the use of the above procedures in combinatorial algorithms of optimization.

  18. Design Primer for Reed-Solomon Encoders

    Science.gov (United States)

    Perlman, M.; Lee, J. J.

    1985-01-01

    Design and operation of Reed-Solomon (RS) encoders discussed in document prepared as instruction manual for computer designers and others in dataprocessing field. Conventional and Berlekamp architectures compared. Engineers who equip computer memory chips with burst-error and dropout detection and correction find report especially useful.

  19. How Attention Modulates Encoding of Dynamic Stimuli

    Science.gov (United States)

    Oren, Noga; Shapira-Lichter, Irit; Lerner, Yulia; Tarrasch, Ricardo; Hendler, Talma; Giladi, Nir; Ash, Elissa L.

    2016-01-01

    When encoding a real-life, continuous stimulus, the same neural circuits support processing and integration of prior as well as new incoming information. This ongoing interplay is modulated by attention, and is evident in regions such as the prefrontal cortex section of the task positive network (TPN), and in the posterior cingulate cortex (PCC), a hub of the default mode network (DMN). Yet the exact nature of such modulation is still unclear. To investigate this issue, we utilized an fMRI task that employed movies as the encoded stimuli and manipulated attentional load via an easy or hard secondary task that was performed simultaneously with encoding. Results showed increased intersubject correlation (inter-SC) levels when encoding movies in a condition of high, as compared to low attentional load. This was evident in bilateral ventrolateral and dorsomedial prefrontal cortices and the dorsal PCC (dPCC). These regions became more attuned to the combination of the movie and the secondary task as the attentional demand of the latter increased. Activation analyses revealed that at higher load the prefrontal TPN regions were more activated, whereas the dPCC was more deactivated. Attentional load also influenced connectivity within and between the networks. At high load the dPCC was anti-correlated to the prefrontal regions, which were more functionally coherent amongst themselves. Finally and critically, greater inter-SC in the dPCC at high load during encoding predicted lower memory strength when that information was retrieved. This association between inter-SC levels and memory strength suggest that as attentional demands increased, the dPCC was more attuned to the secondary task at the expense of the encoded stimulus, thus weakening memory for the encoded stimulus. Together, our findings show that attentional load modulated the function of core TPN and DMN regions. Furthermore, the observed relationship between memory strength and the modulation of the dPCC points

  20. Differences in protein synthesis between wild type and intracellular growth-deficient strains of Legionella pneumophila in U937 and Acanthamoeba polyphaga.

    Science.gov (United States)

    Miyake, Masaki; Fukui, Takashi; Imai, Yasuyuki

    2006-04-01

    An important aspect of Legionnaires' disease is the growth of the causative agent, Legionella pneumophila, within infected host cells. Many proteins including stress proteins of L. pneumophila were strongly induced in a wild type strain that had been used to infect U937 human macrophage-like cells. In contrast, the expression of the proteins was much weaker within a protozoan host, Acanthamoeba polyphaga. The results suggested that active bacterial protein synthesis is required more within macrophages than within protozoa for adaptation of L. pneumophila to intracellular environments. The synthesis of these proteins was not observed in intracellular growth-deficient strains after infection in either type of host cells. The inability of protein synthesis in these strains is correlated with their inability of intracellular growth. Furthermore, on U937 infection, the synthesis of beta-galactosidase encoded in an inducible reporter construct immediately ceased in the in intracellular growth-deficient strains after infection, while the wild type strain was able to synthesize it during the course of infection. These results suggested that the intracellular growth of Legionella pneumophila within macrophages requires active protein synthesis from an earlier stage of bacterial infection.

  1. Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga.

    Science.gov (United States)

    Molmeret, Maëlle; Bitar, Dina M; Han, Lihui; Kwaik, Yousef Abu

    2004-07-01

    Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion

  2. Stoichiometry and kinetics of mercury uptake by photosynthetic bacteria.

    Science.gov (United States)

    Kis, Mariann; Sipka, Gábor; Maróti, Péter

    2017-05-01

    Mercury adsorption on the cell surface and intracellular uptake by bacteria represent the key first step in the production and accumulation of highly toxic mercury in living organisms. In this work, the biophysical characteristics of mercury bioaccumulation are studied in intact cells of photosynthetic bacteria by use of analytical (dithizone) assay and physiological photosynthetic markers (pigment content, fluorescence induction, and membrane potential) to determine the amount of mercury ions bound to the cell surface and taken up by the cell. It is shown that the Hg(II) uptake mechanism (1) has two kinetically distinguishable components, (2) includes co-opted influx through heavy metal transporters since the slow component is inhibited by Ca(2+) channel blockers, (3) shows complex pH dependence demonstrating the competition of ligand binding of Hg(II) ions with H(+) ions (low pH) and high tendency of complex formation of Hg(II) with hydroxyl ions (high pH), and (4) is not a passive but an energy-dependent process as evidenced by light activation and inhibition by protonophore. Photosynthetic bacteria can accumulate Hg(II) in amounts much (about 10(5)) greater than their own masses by well-defined strong and weak binding sites with equilibrium binding constants in the range of 1 (μM)(-1) and 1 (mM)(-1), respectively. The strong binding sites are attributed to sulfhydryl groups as the uptake is blocked by use of sulfhydryl modifying agents and their number is much (two orders of magnitude) smaller than the number of weak binding sites. Biofilms developed by some bacteria (e.g., Rvx. gelatinosus) increase the mercury binding capacity further by a factor of about five. Photosynthetic bacteria in the light act as a sponge of Hg(II) and can be potentially used for biomonitoring and bioremediation of mercury-contaminated aqueous cultures.

  3. Advantages and limitations of the use of optogenetic approach in studying fast-scale spike encoding.

    Directory of Open Access Journals (Sweden)

    Aleksey Malyshev

    Full Text Available Understanding single-neuron computations and encoding performed by spike-generation mechanisms of cortical neurons is one of the central challenges for cell electrophysiology and computational neuroscience. An established paradigm to study spike encoding in controlled conditions in vitro uses intracellular injection of a mixture of signals with fluctuating currents that mimic in vivo-like background activity. However this technique has two serious limitations: it uses current injection, while synaptic activation leads to changes of conductance, and current injection is technically most feasible in the soma, while the vast majority of synaptic inputs are located on the dendrites. Recent progress in optogenetics provides an opportunity to circumvent these limitations. Transgenic expression of light-activated ionic channels, such as Channelrhodopsin2 (ChR2, allows induction of controlled conductance changes even in thin distant dendrites. Here we show that photostimulation provides a useful extension of the tools to study neuronal encoding, but it has its own limitations. Optically induced fluctuating currents have a low cutoff (~70 Hz, thus limiting the dynamic range of frequency response of cortical neurons. This leads to severe underestimation of the ability of neurons to phase-lock their firing to high frequency components of the input. This limitation could be worked around by using short (2 ms light stimuli which produce membrane potential responses resembling EPSPs by their fast onset and prolonged decay kinetics. We show that combining application of short light stimuli to different parts of dendritic tree for mimicking distant EPSCs with somatic injection of fluctuating current that mimics fluctuations of membrane potential in vivo, allowed us to study fast encoding of artificial EPSPs photoinduced at different distances from the soma. We conclude that dendritic photostimulation of ChR2 with short light pulses provides a powerful tool to

  4. Amygdala neurons differentially encode motivation and reinforcement.

    Science.gov (United States)

    Tye, Kay M; Janak, Patricia H

    2007-04-11

    Lesion studies demonstrate that the basolateral amygdala complex (BLA) is important for assigning motivational significance to sensory stimuli, but little is known about how this information is encoded. We used in vivo electrophysiology procedures to investigate how the amygdala encodes motivating and reinforcing properties of cues that induce reinstatement of reward-seeking behavior. Two groups of rats were trained to respond to a sucrose reward. The "paired" group was trained with a reward-predictive cue, whereas the "unpaired" group was trained with a randomly presented cue. Both groups underwent identical extinction and reinstatement procedures during which the reward was withheld. The proportion of neurons that were phasically cue responsive during reinstatement was significantly higher in the paired group (46 of 100) than in the unpaired group (8 of 112). Cues that induce reward-seeking behavior can do so by acting as incentives or reinforcers. Distinct populations of neurons responded to the cue in trials in which the cue acted as an incentive, triggering a motivated reward-seeking state, or as a reinforcer, supporting continued instrumental responding. The incentive motivation-encoding population of neurons (34 of 46 cue-responsive neurons; 74%) extinguished in temporal agreement with a decrease in the rate of instrumental responding. The conditioned reinforcement-encoding population of neurons (12 of 46 cue-responsive neurons; 26%) maintained their response for the duration of cue-reinforced instrumental responding. These data demonstrate that separate populations of cue-responsive neurons in the BLA encode the motivating or reinforcing properties of a cue previously associated with a reward.

  5. Role of bacteria in the etiopathogenesis of inflammatory bowel disease

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Increased numbers of mucosa-associated Escherichia coli are observed in both of the major inflammatory bowel diseases, Crohn's disease (CD) and ulcerative colitis (DC). A potential pathophysiological link between the presence of pathogenic invasive bacteria and genetic host susceptibility of patients with ileal CD is suspected. In CD patients, with increased ileal expression of the CEACAM6 molecule acting as a receptor recognized by type 1 pilus bacterial adhesin, and with the identification of mutations in the NOD2-encoding gene, the presence of pathogenic invasive bacteria could be the link between abnormal ileal bacterial colonization and innate immune responses to invasive bacteria. In a susceptible host, the sequential etiological steps of the disease induced by adherent-invasive E. Coli (AIEC) are: (1) abnormal colonization via binding to the CEACAM6 receptor, which is overexpressed in the ileal mucosa of CD patients; (2) ability to adhere to and to invade intestinal epithelial cells, which allows bacteria to cross the mucosal barrier; (3) survival and replication within infected macrophages in the lamina propria; and (4) induction of tumor necrosis factor-a secretion and granuloma formation.

  6. Emerging pathogens of gilthead seabream: characterisation and genomic analysis of novel intracellular β-proteobacteria.

    Science.gov (United States)

    Seth-Smith, Helena M B; Dourala, Nancy; Fehr, Alexander; Qi, Weihong; Katharios, Pantelis; Ruetten, Maja; Mateos, José M; Nufer, Lisbeth; Weilenmann, Roseline; Ziegler, Urs; Thomson, Nicholas R; Schlapbach, Ralph; Vaughan, Lloyd

    2016-07-01

    New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

  7. Sampling bacteria with a laser

    Science.gov (United States)

    Schwarzwälder, Kordula; Rutschmann, Peter

    2014-05-01

    Water quality is a topic of high interest and it's getting more and more important due to climate change and the implementation of European Water Framework Directive (WFD). One point of interest here is the inflow of bacteria into a river caused by combined sewer overflows which lead untreated wastewater including bacteria directly into a river. These bacteria remain in the river for a certain time, they settle down and can be remobilised again. In our study we want to investigate these processes of sedimentation and resuspension and use the results for the development of a software module coupled with the software Flow3D. Thereby we should be able to simulate and therefore predict the water quality influenced by combined sewer overflows. Hence we need to get information about the bacteria transport and fate. We need to know about the size of the bacteria or of the bacteria clumps and the size of the particles the bacteria are attached to. The agglomerates lead to different characteristics and velocities of settlement. The timespan during this bacteria can be detected in the bulk phase depends on many factors like the intensity of UV light, turbidity of the water, the temperature of the water, if there are grazers and a lot more. The size, density and composition of the agglomerates is just a part of all these influencing factors, but it is extremely difficult to differ between the other effects if we have no information about the simple sedimentation in default of these basic information. However we have a big problem getting the data. The chaining between bacteria or bacteria and particles is not too strong, so filtering the water to get a sieving curve may destroy these connections. We did some experiments similar to PIV (particle image velocimetry) measurements and evaluated the pictures with a macro written for the software ImageJ. Doing so we were able to get the concentration of bacteria in the water and collect information about the size of the bacteria. We

  8. Motility of Electric Cable Bacteria

    OpenAIRE

    Bjerg, Jesper Tataru; Damgaard, Lars Riis; Holm, Simon Agner; Schramm, Andreas; Nielsen, Lars Peter

    2016-01-01

    Cable bacteria are filamentous bacteria that electrically couple sulfide oxidation and oxygen reduction at centimeter distances, and observations in sediment environments have suggested that they are motile. By time-lapse microscopy, we found that cable bacteria used gliding motility on surfaces with a highly variable speed of 0.5 ± 0.3 μm s−1 (mean ± standard deviation) and time between reversals of 155 ± 108 s. They frequently moved forward in loops, and formation of twisted loops revealed ...

  9. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria.

    OpenAIRE

    Diorio, C.; Cai, J.; Marmor, J; Shinder, R; DuBow, M S

    1995-01-01

    Arsenic is a known toxic metalloid, whose trivalent and pentavalent ions can inhibit many biochemical processes. Operons which encode arsenic resistance have been found in multicopy plasmids from both gram-positive and gram-negative bacteria. The resistance mechanism is encoded from a single operon which typically consists of an arsenite ion-inducible repressor that regulates expression of an arsenate reductase and inner membrane-associated arsenite export system. Using a lacZ transcriptional...

  10. Beer spoilage bacteria and hop resistance

    NARCIS (Netherlands)

    Sakamoto, K; Konings, WN

    2003-01-01

    For brewing industry, beer spoilage bacteria have been problematic for centuries. They include some lactic acid bacteria such as Lactobacillus brevis, Lactobacillus lindneri and Pediococcus damnosus, and some Gram-negative bacteria such as Pectinatus cerevisiiphilus, Pectinatus frisingensis and

  11. Beer spoilage bacteria and hop resistance

    NARCIS (Netherlands)

    Sakamoto, K; Konings, WN

    2003-01-01

    For brewing industry, beer spoilage bacteria have been problematic for centuries. They include some lactic acid bacteria such as Lactobacillus brevis, Lactobacillus lindneri and Pediococcus damnosus, and some Gram-negative bacteria such as Pectinatus cerevisiiphilus, Pectinatus frisingensis and Mega

  12. Impact of Microalgae-Bacteria Interactions on the Production of Algal Biomass and Associated Compounds.

    Science.gov (United States)

    Fuentes, Juan Luis; Garbayo, Inés; Cuaresma, María; Montero, Zaida; González-Del-Valle, Manuel; Vílchez, Carlos

    2016-05-19

    A greater insight on the control of the interactions between microalgae and other microorganisms, particularly bacteria, should be useful for enhancing the efficiency of microalgal biomass production and associated valuable compounds. Little attention has been paid to the controlled utilization of microalgae-bacteria consortia. However, the studies of microalgal-bacterial interactions have revealed a significant impact of the mutualistic or parasitic relationships on algal growth. The algal growth, for instance, has been shown to be enhanced by growth promoting factors produced by bacteria, such as indole-3-acetic acid. Vitamin B12 produced by bacteria in algal cultures and bacterial siderophores are also known to be involved in promoting faster microalgal growth. More interestingly, enhancement in the intracellular levels of carbohydrates, lipids and pigments of microalgae coupled with algal growth stimulation has also been reported. In this sense, massive algal production might occur in the presence of bacteria, and microalgae-bacteria interactions can be beneficial to the massive production of microalgae and algal products. This manuscript reviews the recent knowledge on the impact of the microalgae-bacteria interactions on the production of microalgae and accumulation of valuable compounds, with an emphasis on algal species having application in aquaculture.

  13. ENOD40 encodes a peptide growth factor.

    NARCIS (Netherlands)

    Sande, van de G.P.C.M.

    1997-01-01

    Rhizobium bacteria induce the formation of nodules on the roots of leguminous plants. The nodules create the right biological niche for the rhizobia to carry out biological nitrogen fixation by which atmospheric nitrogen is reduced to ammonia. The nodule is a new organ that provides the plant with a

  14. An Encoder/Decoder Scheme of OCDMA Based on Waveguide

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new encoder/decoder scheme of OCDMA based on waveguide isproposed in this paper. The principle as well as the structure of waveguide encoder/decoder is given. It can be seen that all-optical OCDMA encoder/decoder can be realized by the proposed scheme of the waveguide encoder/decoder. It can also make the OCDMA encoder/decoder integrated easily and the access controlled easily. The system based on this scheme can work under the entirely asynchronous condition.

  15. Atypical Enteropathogenic Escherichia coli Secretes Plasmid Encoded Toxin

    Directory of Open Access Journals (Sweden)

    Rita C. Ruiz

    2014-01-01

    Full Text Available Plasmid encoded toxin (Pet is a serine protease originally described in enteroaggregative Escherichia coli (EAEC prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.

  16. A genetically encoded, high-signal-to-noise maltose sensor

    Energy Technology Data Exchange (ETDEWEB)

    Marvin, Jonathan S.; Schreiter, Eric R.; Echevarría, Ileabett M.; Looger, Loren L. (Puerto Rico); (HHMI)

    2012-10-23

    We describe the generation of a family of high-signal-to-noise single-wavelength genetically encoded indicators for maltose. This was achieved by insertion of circularly permuted fluorescent proteins into a bacterial periplasmic binding protein (PBP), Escherichia coli maltodextrin-binding protein, resulting in a four-color family of maltose indicators. The sensors were iteratively optimized to have sufficient brightness and maltose-dependent fluorescence increases for imaging, under both one- and two-photon illumination. We demonstrate that maltose affinity of the sensors can be tuned in a fashion largely independent of the fluorescent readout mechanism. Using literature mutations, the binding specificity could be altered to moderate sucrose preference, but with a significant loss of affinity. We use the soluble sensors in individual E. coli bacteria to observe rapid maltose transport across the plasma membrane, and membrane fusion versions of the sensors on mammalian cells to visualize the addition of maltose to extracellular media. The PBP superfamily includes scaffolds specific for a number of analytes whose visualization would be critical to the reverse engineering of complex systems such as neural networks, biosynthetic pathways, and signal transduction cascades. We expect the methodology outlined here to be useful in the development of indicators for many such analytes.

  17. Vertical distribution of phosphorus and P-dissolving/decomposing bacteria in the sediment of Guanting reservoir

    Institute of Scientific and Technical Information of China (English)

    LI; Cui; YUAN; Hongli; HUANG; Huaizeng

    2005-01-01

    In this study, we determined pH, phosphorus content and the number of P-dis- solving/decomposing bacteria in relation to the depth in the sediment of Guanting reservoir in Beijing. The pH value was slightly increased from 8.0 in the surface to 8.5 in the bottle (69 cm in depth) of sediment. The highest P content, 1269 mg/kg, was detected in the 35 cm layer, followed by the 5 cm (993 mg/kg) and 69 cm (580 mg/kg) layers. The number of inorganic P-dissolving bacteria varied from 6(102 to 8(104 and the organic P-discomposing bacteria were from 1.9(103 to 6.3(104 per gram sediment in different depths, which were counted under 28℃ and 20℃ ( pH7.5 and 8.5. The number of P-dissolving/discomposing bacteria was directly correlated to the P content in each layer of sediments. The analysis of P-dissolving/decomposing ability of bacteria showed that some of these bacteria were also capable of accumulating P intracellularly. The intracellular P-accumulation was more efficient at lower temperature; in contrast, the activity of P-dissolving/decomposing was str