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  1. Probing bacterial cell biology using image cytometry.

    Science.gov (United States)

    Cass, Julie A; Stylianidou, Stella; Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2017-03-01

    Advances in automated fluorescence microscopy have made snapshot and time-lapse imaging of bacterial cells commonplace, yet fundamental challenges remain in analysis. The vast quantity of data collected in high-throughput experiments requires a fast and reliable automated method to analyze fluorescence intensity and localization, cell morphology and proliferation as well as other descriptors. Inspired by effective yet tractable methods of population-level analysis using flow cytometry, we have developed a framework and tools for facilitating analogous analyses in image cytometry. These tools can both visualize and gate (generate subpopulations) more than 70 cell descriptors, including cell size, age and fluorescence. The method is well suited to multi-well imaging, analysis of bacterial cultures with high cell density (thousands of cells per frame) and complete cell cycle imaging. We give a brief description of the analysis of four distinct applications to emphasize the broad applicability of the tool.

  2. Patterning bacterial communities on epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammed Dwidar

    Full Text Available Micropatterning of bacteria using aqueous two phase system (ATPS enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions.

  3. Bacterial Cell Wall Growth, Shape and Division

    NARCIS (Netherlands)

    Derouaux, A.; Terrak, M.; den Blaauwen, T.; Vollmer, W.; Remaut, H.; Fronzes, R.

    2014-01-01

    The shape of a bacterial cell is maintained by its peptidoglycan sacculus that completely surrounds the cytoplasmic membrane. During growth the sacculus is enlarged by peptidoglycan synthesis complexes that are controlled by components linked to the cytoskeleton and, in Gram-negative bacteria, by ou

  4. Measuring bacterial cells size with AFM.

    Science.gov (United States)

    Osiro, Denise; Filho, Rubens Bernardes; Assis, Odilio Benedito Garrido; Jorge, Lúcio André de Castro; Colnago, Luiz Alberto

    2012-01-01

    Atomic Force Microscopy (AFM) can be used to obtain high-resolution topographical images of bacteria revealing surface details and cell integrity. During scanning however, the interactions between the AFM probe and the membrane results in distortion of the images. Such distortions or artifacts are the result of geometrical effects related to bacterial cell height, specimen curvature and the AFM probe geometry. The most common artifact in imaging is surface broadening, what can lead to errors in bacterial sizing. Several methods of correction have been proposed to compensate for these artifacts and in this study we describe a simple geometric model for the interaction between the tip (a pyramidal shaped AFM probe) and the bacterium (Escherichia coli JM-109 strain) to minimize the enlarging effect. Approaches to bacteria immobilization and examples of AFM images analysis are also described.

  5. Research on magnetic metallization of bacterial cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A multidisciplinary approach to the fabrication of biologically based magnetic monomers for biolimited forming is described. Rod-like Bacilli cereus about 0.5 μm in diameter and 3-5 μm in length, were used as templates on which the ferromagnetic material was deposited by an electroless deposition method. Different electroless plating solutions were compared in detail and CoNiP solution was selected. During the deposition process, both dispersant and mechanical stirring were used to solve the problem of aggregation of bacterial cells so as to obtain a uniform plating layer. The CoNiP film on Bacilli cereus was a mixture of crystalline and non-crystalline in the phase structure and showed a good magnetism. The magnetic metallized bacterial cells could be manipulated with a magnetic field. Parallel arrays of these micro magnetic particles were achieved and they could rotate along with the magnetic field.

  6. Bacterial foodborne infections after hematopoietic cell transplantation.

    Science.gov (United States)

    Boyle, Nicole M; Podczervinski, Sara; Jordan, Kim; Stednick, Zach; Butler-Wu, Susan; McMillen, Kerry; Pergam, Steven A

    2014-11-01

    Diarrhea, abdominal pain, and fever are common among patients undergoing hematopoietic cell transplantation (HCT), but such symptoms are also typical with foodborne infections. The burden of disease caused by foodborne infections in patients undergoing HCT is unknown. We sought to describe bacterial foodborne infection incidence after transplantation within a single-center population of HCT recipients. All HCT recipients who underwent transplantation from 2001 through 2011 at the Fred Hutchinson Cancer Research Center in Seattle, Washington were followed for 1 year after transplantation. Data were collected retrospectively using center databases, which include information from transplantation, on-site examinations, outside records, and collected laboratory data. Patients were considered to have a bacterial foodborne infection if Campylobacter jejuni/coli, Listeria monocytogenes, E. coli O157:H7, Salmonella species, Shigella species, Vibrio species, or Yersinia species were isolated in culture within 1 year after transplantation. Nonfoodborne infections with these agents and patients with pre-existing bacterial foodborne infection (within 30 days of transplantation) were excluded from analyses. A total of 12 of 4069 (.3%) patients developed a bacterial foodborne infection within 1 year after transplantation. Patients with infections had a median age at transplantation of 50.5 years (interquartile range [IQR], 35 to 57), and the majority were adults ≥18 years of age (9 of 12 [75%]), male gender (8 of 12 [67%]) and had allogeneic transplantation (8 of 12 [67%]). Infectious episodes occurred at an incidence rate of 1.0 per 100,000 patient-days (95% confidence interval, .5 to 1.7) and at a median of 50.5 days after transplantation (IQR, 26 to 58.5). The most frequent pathogen detected was C. jejuni/coli (5 of 12 [42%]) followed by Yersinia (3 of 12 [25%]), although Salmonella (2 of 12 [17%]) and Listeria (2 of 12 [17%]) showed equal frequencies; no cases of Shigella

  7. One bacterial cell, one complete genome.

    Directory of Open Access Journals (Sweden)

    Tanja Woyke

    Full Text Available While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200-900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA. Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs, indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  8. One Bacterial Cell, One Complete Genome

    Energy Technology Data Exchange (ETDEWEB)

    Woyke, Tanja; Tighe, Damon; Mavrommatis, Konstantinos; Clum, Alicia; Copeland, Alex; Schackwitz, Wendy; Lapidus, Alla; Wu, Dongying; McCutcheon, John P.; McDonald, Bradon R.; Moran, Nancy A.; Bristow, James; Cheng, Jan-Fang

    2010-04-26

    While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200?900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  9. Shedding light on biology of bacterial cells.

    Science.gov (United States)

    Schneider, Johannes P; Basler, Marek

    2016-11-01

    To understand basic principles of living organisms one has to know many different properties of all cellular components, their mutual interactions but also their amounts and spatial organization. Live-cell imaging is one possible approach to obtain such data. To get multiple snapshots of a cellular process, the imaging approach has to be gentle enough to not disrupt basic functions of the cell but also have high temporal and spatial resolution to detect and describe the changes. Light microscopy has become a method of choice and since its early development over 300 years ago revolutionized our understanding of living organisms. As most cellular components are indistinguishable from the rest of the cellular contents, the second revolution came from a discovery of specific labelling techniques, such as fusions to fluorescent proteins that allowed specific tracking of a component of interest. Currently, several different tags can be tracked independently and this allows us to simultaneously monitor the dynamics of several cellular components and from the correlation of their dynamics to infer their respective functions. It is, therefore, not surprising that live-cell fluorescence microscopy significantly advanced our understanding of basic cellular processes. Current cameras are fast enough to detect changes with millisecond time resolution and are sensitive enough to detect even a few photons per pixel. Together with constant improvement of properties of fluorescent tags, it is now possible to track single molecules in living cells over an extended period of time with a great temporal resolution. The parallel development of new illumination and detection techniques allowed breaking the diffraction barrier and thus further pushed the resolution limit of light microscopy. In this review, we would like to cover recent advances in live-cell imaging technology relevant to bacterial cells and provide a few examples of research that has been possible due to imaging

  10. Shedding light on biology of bacterial cells

    Science.gov (United States)

    2016-01-01

    To understand basic principles of living organisms one has to know many different properties of all cellular components, their mutual interactions but also their amounts and spatial organization. Live-cell imaging is one possible approach to obtain such data. To get multiple snapshots of a cellular process, the imaging approach has to be gentle enough to not disrupt basic functions of the cell but also have high temporal and spatial resolution to detect and describe the changes. Light microscopy has become a method of choice and since its early development over 300 years ago revolutionized our understanding of living organisms. As most cellular components are indistinguishable from the rest of the cellular contents, the second revolution came from a discovery of specific labelling techniques, such as fusions to fluorescent proteins that allowed specific tracking of a component of interest. Currently, several different tags can be tracked independently and this allows us to simultaneously monitor the dynamics of several cellular components and from the correlation of their dynamics to infer their respective functions. It is, therefore, not surprising that live-cell fluorescence microscopy significantly advanced our understanding of basic cellular processes. Current cameras are fast enough to detect changes with millisecond time resolution and are sensitive enough to detect even a few photons per pixel. Together with constant improvement of properties of fluorescent tags, it is now possible to track single molecules in living cells over an extended period of time with a great temporal resolution. The parallel development of new illumination and detection techniques allowed breaking the diffraction barrier and thus further pushed the resolution limit of light microscopy. In this review, we would like to cover recent advances in live-cell imaging technology relevant to bacterial cells and provide a few examples of research that has been possible due to imaging. This

  11. Bacterial cell curvature through mechanical control of cell growth

    DEFF Research Database (Denmark)

    Cabeen, M.; Charbon, Godefroid; Vollmer, W.

    2009-01-01

    The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure...... that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature...... can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics...

  12. Probing bacterial adhesion at the single-cell level

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Müller, Torsten; Meyer, Rikke Louise

    . Staphylococci adhere stronger on fresh glass than on hydrophilic glass, while the weaker adhesion by P. fluorescens was similar on both types of glass. These results confirmed the importance of surface hydrophobicity in bacterial adhesion. This study has demonstrated that single-cell force spectroscopy allows...... be considered. We have developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion by force spectroscopy using atomic force microscopy (AFM). A single-cell probe was readily made by picking up a bacterial cell from a glass surface by approaching a tipless AFM...... on the adhesion force, we explored the bond formation and adhesive strength of four different bacterial strains towards three abiotic substrates with variable hydrophobicity and surface roughness. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time...

  13. Bacterial cell division as a target for new antibiotics.

    Science.gov (United States)

    Sass, Peter; Brötz-Oesterhelt, Heike

    2013-10-01

    Bacterial resistance to currently applied antibiotics complicates the treatment of infections and demands the evaluation of new strategies to counteract multidrug-resistant bacteria. In recent years, the inhibition of the bacterial divisome, mainly by targeting the central cell division mediator FtsZ, has been recognized as a promising strategy for antibiotic attack. New antibiotics were shown to either interfere with the natural dynamics and functions of FtsZ during the cell cycle or to activate a bacterial protease to degrade FtsZ and thus bring about bacterial death in a suicidal manner. Their efficacy in animal models of infection together with resistance-breaking properties prove the potential of such drugs and validate the inhibition of bacterial cell division as an attractive approach for antibiotic intervention.

  14. Bacterial cell biology outside the streetlight.

    Science.gov (United States)

    Bulgheresi, Silvia

    2016-09-01

    As much as vertical transmission of microbial symbionts requires their deep integration into the host reproductive and developmental biology, symbiotic lifestyle might profoundly affect bacterial growth and proliferation. This review describes the reproductive oddities displayed by bacteria associated - more or less intimately - with multicellular eukaryotes.

  15. Beyond growth: novel functions for bacterial cell wall hydrolases.

    Science.gov (United States)

    Wyckoff, Timna J; Taylor, Jennifer A; Salama, Nina R

    2012-11-01

    The peptidoglycan cell wall maintains turgor pressure and cell shape of most bacteria. Cell wall hydrolases are essential, together with synthases, for growth and daughter cell separation. Recent work in diverse organisms has uncovered new cell wall hydrolases that act autonomously or on neighboring cells to modulate invasion of prey cells, cell shape, innate immune detection, intercellular communication, and competitor lysis. The hydrolases involved in these processes catalyze the cleavage of bonds throughout the sugar and peptide moities of peptidoglycan. Phenotypes associated with these diverse hydrolases reveal new functions of the bacterial cell wall beyond growth and division.

  16. Conductivity and Dielectric Dispersion of Gram-Positive Bacterial Cells

    Science.gov (United States)

    van der Wal A; Minor; Norde; Zehnder; Lyklema

    1997-02-01

    The conductivity of bacterial cell suspensions has been studied over a wide range of ionic strengths and is interpreted in terms of their cell wall properties. The experimental data have been analyzed after improving the high kappaa double-layer theory of Fixman, by accounting for ionic mobility in the hydrodynamically stagnant layer, i.e., in the bacterial wall. Static conductivity and dielectric dispersion measurements both show that the counterions in the porous gel-like cell wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the ions in the bacterial wall are of the same order but somewhat lower than those in the bulk electrolyte solution. The occurrence of surface conductance reduces the electrophoretic mobility in electrophoresis studies. If this effect is not taken into account, the zeta-potential will be underestimated, especially at low electrolyte concentrations.

  17. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    Science.gov (United States)

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  18. Micro-magnet arrays for specific single bacterial cell positioning

    Energy Technology Data Exchange (ETDEWEB)

    Pivetal, Jérémy, E-mail: jeremy.piv@netcmail.com [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Royet, David [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Ciuta, Georgeta [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Frenea-Robin, Marie [Université de Lyon, Université Lyon 1, CNRS UMR 5005, Laboratoire Ampère, F-69622 Villeurbanne (France); Haddour, Naoufel [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France); Dempsey, Nora M. [Univ. Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Dumas-Bouchiat, Frédéric [Univ Limoges, CNRS, SPCTS UMR 7513, 12 Rue Atlantis, F-87068 Limoges (France); Simonet, Pascal [Ecole Centrale de Lyon, CNRS UMR 5005, Laboratoire Ampère, F-69134 Écully (France)

    2015-04-15

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications. - Highlights: 1.We report a new approach to selectively micropattern bacterial cells individually upon micro-magnet arrays. 2.Permanent micro-magnets of a size approaching that of bacteria could be fabricated using a Thermo-Magnetic Patterning process. 3.Bacterial cells were labeled using two different magnetic labeling strategies providing flexible approach adaptable to several applications in the field of microbiology.

  19. Studying biomolecule localization by engineering bacterial cell wall curvature.

    Directory of Open Access Journals (Sweden)

    Lars D Renner

    Full Text Available In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.

  20. Mathematical Modeling of the Induced Mutation Process in Bacterial Cells

    Science.gov (United States)

    Belov, Oleg V.; Krasavin, Evgeny A.; Parkhomenko, Alexander Yu.

    2010-01-01

    A mathematical model of the ultraviolet (UV) irradiation-induced mutation process in bacterial cells Escherichia coli is developed. Using mathematical approaches, the whole chain of events is tracked from a cell exposure to the damaging factor to mutation formation in the DNA chain. An account of the key special features of the regulation of this genetic network allows predicting the effects induced by the cell exposure to certain UV energy fluence.

  1. Isolation of biologically active nanomaterial (inclusion bodies from bacterial cells

    Directory of Open Access Journals (Sweden)

    Peternel Špela

    2010-09-01

    Full Text Available Abstract Background In recent years bacterial inclusion bodies (IBs were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry. To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process. To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. Results In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared. During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation. During sonication proteins are released (lost from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity. High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. Conclusions The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells.

  2. Isolation of cell-free bacterial inclusion bodies

    Directory of Open Access Journals (Sweden)

    Rodríguez-Carmona Escarlata

    2010-09-01

    Full Text Available Abstract Background Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Results Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10-1 cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. Conclusions The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

  3. Micro-magnet arrays for specific single bacterial cell positioning

    Science.gov (United States)

    Pivetal, Jérémy; Royet, David; Ciuta, Georgeta; Frenea-Robin, Marie; Haddour, Naoufel; Dempsey, Nora M.; Dumas-Bouchiat, Frédéric; Simonet, Pascal

    2015-04-01

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications.

  4. Improved immunoadsorption procedure with anion-exchange bacterial cell columns.

    Science.gov (United States)

    McKinney, R M; Thacker, L; Wong, M C; Hebert, G A

    1978-01-01

    Bacterial cell columns for immunoadsorption were prepared with Streptococcus cells and triethylaminoethyl cellulose (Cellex-T) matrix material as a model system. Good column flow properties and satisfactory retention of the cells were obtained with ratios as high as 2 ml of packed cells/3 g dry weight of cellulose. Anion-exchange fractionation of whole serum by the Cellex-T was prevented by using 0.25 M NaCl in the developing buffer. Antibodies were adsorbed directly from whole serum and recovered in high yield by desorption at pH 2.3. Pre-exposing bacterial cells to formalin and washing them with acetone was necessary to ensure that they remained on the columns. One strain of Streptococcus salivarius (SS 908) was satisfactorily retained on a column only after cells were labeled with fluorescein isothiocyanate and washed with acetone. The means by which Cellex-T retains bacterial cells appears to be a combination of electronic attraction and physical entrapment.

  5. GTPases in bacterial cell polarity and signalling.

    Science.gov (United States)

    Bulyha, Iryna; Hot, Edina; Huntley, Stuart; Søgaard-Andersen, Lotte

    2011-12-01

    In bacteria, large G domain GTPases have well-established functions in translation, protein translocation, tRNA modification and ribosome assembly. In addition, bacteria also contain small Ras-like GTPases consisting of stand-alone G domains. Recent data have revealed that small Ras-like GTPases as well as large G domain GTPases in bacteria function in the regulation of cell polarity, signal transduction and possibly also in cell division. The small Ras-like GTPase MglA together with its cognate GAP MglB regulates cell polarity in Myxococcus xanthus, and the small Ras-like GTPase CvnD9 in Streptomyces coelicolor is involved in signal transduction. Similarly, the large GTPase FlhF together with the ATPase FlhG regulates the localization and number of flagella in polarly flagellated bacteria. Moreover, large dynamin-like GTPases in bacteria may function in cell division. Thus, the function of GTPases in bacteria may be as pervasive as in eukaryotes.

  6. A Bacterial Cell Shape-Determining Inhibitor.

    Science.gov (United States)

    Liu, Yanjie; Frirdich, Emilisa; Taylor, Jennifer A; Chan, Anson C K; Blair, Kris M; Vermeulen, Jenny; Ha, Reuben; Murphy, Michael E P; Salama, Nina R; Gaynor, Erin C; Tanner, Martin E

    2016-04-15

    Helicobacter pylori and Campylobacter jejuni are human pathogens and causative agents of gastric ulcers/cancer and gastroenteritis, respectively. Recent studies have uncovered a series of proteases that are responsible for maintaining the helical shape of these organisms. The H. pylori metalloprotease Csd4 and its C. jejuni homologue Pgp1 cleave the amide bond between meso-diaminopimelate and iso-d-glutamic acid in truncated peptidoglycan side chains. Deletion of either csd4 or pgp1 results in bacteria with a straight rod phenotype, a reduced ability to move in viscous media, and reduced pathogenicity. In this work, a phosphinic acid-based pseudodipeptide inhibitor was designed to act as a tetrahedral intermediate analog against the Csd4 enzyme. The phosphinic acid was shown to inhibit the cleavage of the alternate substrate, Ac-l-Ala-iso-d-Glu-meso-Dap, with a Ki value of 1.5 μM. Structural analysis of the Csd4-inhibitor complex shows that the phosphinic acid displaces the zinc-bound water and chelates the metal in a bidentate fashion. The phosphinate oxygens also interact with the key acid/base residue, Glu222, and the oxyanion-stabilizing residue, Arg86. The results are consistent with the "promoted-water pathway" mechanism for carboxypeptidase A catalysis. Studies on cultured bacteria showed that the inhibitor causes significant cell straightening when incubated with H. pylori at millimolar concentrations. A diminished, yet observable, effect on the morphology of C. jejuni was also apparent. Cell straightening was more pronounced with an acapsular C. jejuni mutant strain compared to the wild type, suggesting that the capsule impaired inhibitor accessibility. These studies demonstrate that a highly polar compound is capable of crossing the outer membrane and altering cell shape, presumably by inhibiting cell shape determinant proteases. Peptidoglycan proteases acting as cell shape determinants represent novel targets for the development of antimicrobials

  7. Bacterial cell surface structures in Yersinia enterocolitica.

    Science.gov (United States)

    Białas, Nataniel; Kasperkiewicz, Katarzyna; Radziejewska-Lebrecht, Joanna; Skurnik, Mikael

    2012-06-01

    Yersinia enterocolitica is a widespread member of the family of Enterobacteriaceae that contains both non-virulent and virulent isolates. Pathogenic Y. enterocolitica strains, especially belonging to serotypes O:3, O:5,27, O:8 and O:9 are etiologic agents of yersiniosis in animals and humans. Y. enterocolitica cell surface structures that play a significant role in virulence have been subject to many investigations. These include outer membrane (OM) glycolipids such as lipopolysaccharide (LPS) and enterobacterial common antigen (ECA) and several cell surface adhesion proteins present only in virulent Y. enterocolitica, i.e., Inv, YadA and Ail. While the yadA gene is located on the Yersinia virulence plasmid the Ail, Inv, LPS and ECA are chromosomally encoded. These structures ensure the correct architecture of the OM, provide adhesive properties as well as resistance to antimicrobial peptides and to host innate immune response mechanisms.

  8. Resistance to antibiotics targeted to the bacterial cell wall.

    Science.gov (United States)

    Nikolaidis, I; Favini-Stabile, S; Dessen, A

    2014-03-01

    Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units crosslinked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies toward the development of novel inhibitors that could overcome resistance are also discussed.

  9. Fluid dynamics and noise in bacterial cell-cell and cell-surface scattering

    CERN Document Server

    Drescher, Knut; Cisneros, Luis H; Ganguly, Sujoy; Goldstein, Raymond E; 10.1073/pnas.1019079108

    2011-01-01

    Bacterial processes ranging from gene expression to motility and biofilm formation are constantly challenged by internal and external noise. While the importance of stochastic fluctuations has been appreciated for chemotaxis, it is currently believed that deterministic long-range fluid dynamical effects govern cell-cell and cell-surface scattering - the elementary events that lead to swarming and collective swimming in active suspensions and to the formation of biofilms. Here, we report the first direct measurements of the bacterial flow field generated by individual swimming Escherichia coli both far from and near to a solid surface. These experiments allowed us to examine the relative importance of fluid dynamics and rotational diffusion for bacteria. For cell-cell interactions it is shown that thermal and intrinsic stochasticity drown the effects of long-range fluid dynamics, implying that physical interactions between bacteria are determined by steric collisions and near-field lubrication forces. This dom...

  10. Bounds on bacterial cell growth rates

    CERN Document Server

    Landy, Jonathan

    2013-01-01

    Recent experiments have shown that rod-like bacteria in nutrient-rich media grow in length at an exponential rate. Here, I point out that it is the elongated shape of these bacteria that allows for this behavior. Further, I show that when a bacterium's growth is limited by some nutrient -- taken in by the cell through a diffusion-to-capture process -- its growth is suppressed: In three-dimensional geometries, the length $L$ is bounded by $\\log L \\lesssim t^{1/2}$, while in two dimensions the length is bounded by a power-law form. Fits of experimental growth curves to these predicted, sub-exponential forms could allow for direct measures of quantities relating to cellular metabolic rates.

  11. Dental pulp response to bacterial cell wall material.

    Science.gov (United States)

    Warfvinge, J; Dahlén, G; Bergenholtz, G

    1985-08-01

    Lipopolysaccharides (LPS) from Bacteroides oralis and Veillonella parvula and cell wall material from Lactobacillus casei were studied for their capacity to induce leukocyte migration in the dental pulp and in an implanted wound chamber. Three adult monkeys were challenged using lyophilized material sealed into buccal Class V cavities prepared in dentin. Pulp tissue responses were observed histologically eight and 72 hours after initiation of the experiment. Subjacent to cut dentinal tubules, bacterial materials induced polymorphonuclear leukocyte (PMN's) infiltration in the pulp tissue of the majority of test teeth examined. Responses were similar for the three bacterial test materials at both time periods. Topical applications of bovine serum albumin (BSA), used as a control, induced significantly less accumulation of PMN's. Assessments of induced exudate volumes and leukocyte densities in chambers implanted in rats showed comparable rankings with pulpal experiment between test (i.e., bacterial) and control (BSA) materials. Analysis of the data indicates that high-molecular-weight complexes of bacterial cell walls may adversely affect pulpal tissue across freshly exposed dentin.

  12. Extracellular heme uptake and the challenges of bacterial cell membranes.

    Science.gov (United States)

    Smith, Aaron D; Wilks, Angela

    2012-01-01

    In bacteria, the fine balance of maintaining adequate iron levels while preventing the deleterious effects of excess iron has led to the evolution of sophisticated cellular mechanisms to obtain, store, and regulate iron. Iron uptake provides a significant challenge given its limited bioavailability and need to be transported across the bacterial cell wall and membranes. Pathogenic bacteria have circumvented the iron-availability issue by utilizing the hosts' heme-containing proteins as a source of iron. Once internalized, iron is liberated from the porphyrin enzymatically for cellular processes within the bacterial cell. Heme, a lipophilic and toxic molecule, poses a significant challenge in terms of transport given its chemical reactivity. As such, pathogenic bacteria have evolved sophisticated membrane transporters to coordinate, sequester, and transport heme. Recent advances in the biochemical and structural characterization of the membrane-bound heme transport proteins are discussed in the context of ligand coordination, protein-protein interaction, and heme transfer.

  13. The Role of Lipid Domains in Bacterial Cell Processes

    Directory of Open Access Journals (Sweden)

    Katarína Muchová

    2013-02-01

    Full Text Available Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function.

  14. Virus and Bacterial Cell Chemical Analysis by NanoSIMS

    Energy Technology Data Exchange (ETDEWEB)

    Weber, P; Holt, J

    2008-07-28

    In past work for the Department of Homeland Security, the LLNL NanoSIMS team has succeeded in extracting quantitative elemental composition at sub-micron resolution from bacterial spores using nanometer-scale secondary ion mass spectrometry (NanoSIMS). The purpose of this task is to test our NanoSIMS capabilities on viruses and bacterial cells. This initial work has proven successful. We imaged Tobacco Mosaic Virus (TMV) and Bacillus anthracis Sterne cells using scanning electron microscopy (SEM) and then analyzed those samples by NanoSIMS. We were able resolve individual viral particles ({approx}18 nm by 300 nm) in the SEM and extract correlated elemental composition in the NanoSIMS. The phosphorous/carbon ratio observed in TMV is comparable to that seen in bacterial spores (0.033), as was the chlorine/carbon ratio (0.11). TMV elemental composition is consistent from spot to spot, and TMV is readily distinguished from debris by NanoSIMS analysis. Bacterial cells were readily identified in the SEM and relocated in the NanoSIMS for elemental analysis. The Ba Sterne cells were observed to have a measurably lower phosphorous/carbon ratio (0.005), as compared to the spores produced in the same run (0.02). The chlorine/carbon ratio was approximately 2.5X larger in the cells (0.2) versus the spores (0.08), while the fluorine/carbon ratio was approximately 10X lower in the cells (0.008) than the spores (0.08). Silicon/carbon ratios for both cells and spores encompassed a comparable range. The initial data in this study suggest that high resolution analysis is useful because it allows the target agent to be analyzed separate from particulates and other debris. High resolution analysis would also be useful for trace sample analysis. The next step in this work is to determine the potential utility of elemental signatures in these kinds of samples. We recommend bulk analyses of media and agent samples to determine the range of media compositions in use, and to determine how

  15. Mechanical Genomics Identifies Diverse Modulators of Bacterial Cell Stiffness.

    Science.gov (United States)

    Auer, George K; Lee, Timothy K; Rajendram, Manohary; Cesar, Spencer; Miguel, Amanda; Huang, Kerwyn Casey; Weibel, Douglas B

    2016-06-22

    Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of ∼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.

  16. Bacterial actin and tubulin homologs in cell growth and division.

    Science.gov (United States)

    Busiek, Kimberly K; Margolin, William

    2015-03-16

    In contrast to the elaborate cytoskeletal machines harbored by eukaryotic cells, such as mitotic spindles, cytoskeletal structures detectable by typical negative stain electron microscopy are generally absent from bacterial cells. As a result, for decades it was thought that bacteria lacked cytoskeletal machines. Revolutions in genomics and fluorescence microscopy have confirmed the existence not only of smaller-scale cytoskeletal structures in bacteria, but also of widespread functional homologs of eukaryotic cytoskeletal proteins. The presence of actin, tubulin, and intermediate filament homologs in these relatively simple cells suggests that primitive cytoskeletons first arose in bacteria. In bacteria such as Escherichia coli, homologs of tubulin and actin directly interact with each other and are crucial for coordinating cell growth and division. The function and direct interactions between these proteins will be the focus of this review.

  17. Factors affecting daughter cells' arrangement during the early bacterial divisions.

    Directory of Open Access Journals (Sweden)

    Pin-Tzu Su

    Full Text Available On agar plates, daughter cells of Escherichia coli mutually slide and align side-by-side in parallel during the first round of binary fission. This phenomenon has been previously attributed to an elastic material that restricts apparently separated bacteria from being in string. We hypothesize that the interaction between bacteria and the underneath substratum may affect the arrangement of the daughter bacteria. To test this hypothesis, bacterial division on hyaluronic acid (HA gel, as an alternative substratum, was examined. Consistent with our proposition, the HA gel differs from agar by suppressing the typical side-by-side alignments to a rare population. Examination of bacterial surface molecules that may contribute to the daughter cells' arrangement yielded an observation that, with disrupted lpp, the E. coli daughter cells increasingly formed non-typical patterns, i.e. neither sliding side-by-side in parallel nor forming elongated strings. Therefore, our results suggest strongly that the early cell patterning is affected by multiple interaction factors. With oscillatory optical tweezers, we further demonstrated that the interaction force decreased in bacteria without Lpp, a result substantiating our notion that the side-by-side sliding phenomenon directly reflects the strength of in-situ interaction between bacteria and substratum.

  18. Tiny cells meet big questions: a closer look at bacterial cell biology.

    Science.gov (United States)

    Goley, Erin D

    2013-04-01

    While studying actin assembly as a graduate student with Matt Welch at the University of California at Berkeley, my interest was piqued by reports of surprising observations in bacteria: the identification of numerous cytoskeletal proteins, actin homologues fulfilling spindle-like functions, and even the presence of membrane-bound organelles. Curiosity about these phenomena drew me to Lucy Shapiro's lab at Stanford University for my postdoctoral research. In the Shapiro lab, and now in my lab at Johns Hopkins, I have focused on investigating the mechanisms of bacterial cytokinesis. Spending time as both a eukaryotic cell biologist and a bacterial cell biologist has convinced me that bacterial cells present the same questions as eukaryotic cells: How are chromosomes organized and accurately segregated? How is force generated for cytokinesis? How is polarity established? How are signals transduced within and between cells? These problems are conceptually similar between eukaryotes and bacteria, although their solutions can differ significantly in specifics. In this Perspective, I provide a broad view of cell biological phenomena in bacteria, the technical challenges facing those of us who peer into bacterial cells, and areas of common ground as research in eukaryotic and bacterial cell biology moves forward.

  19. High resolution imaging of surface patterns of single bacterial cells

    Energy Technology Data Exchange (ETDEWEB)

    Greif, Dominik; Wesner, Daniel [Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany); Regtmeier, Jan, E-mail: jan.regtmeier@physik.uni-bielefeld.de [Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany); Anselmetti, Dario [Experimental Biophysics and Applied Nanoscience, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld (Germany)

    2010-09-15

    We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.

  20. Dislocation-mediated growth of bacterial cell walls

    CERN Document Server

    Amir, Ariel

    2012-01-01

    Recent experiments have illuminated a remarkable growth mechanism of rod-shaped bacteria: proteins associated with cell wall extension move at constant velocity in circles oriented approximately along the cell circumference (Garner et al., Science (2011), Dominguez-Escobar et al. Science (2011), van Teeffelen et al. PNAS (2011). We view these as dislocations in the partially ordered peptidoglycan structure, activated by glycan strand extension machinery, and study theoretically the dynamics of these interacting defects on the surface of a cylinder. Generation and motion of these interacting defects lead to surprising effects arising from the cylindrical geometry, with important implications for growth. We also discuss how long range elastic interactions and turgor pressure affect the dynamics of the fraction of actively moving dislocations in the bacterial cell wall.

  1. Structure of a bacterial cell surface decaheme electron conduit.

    Science.gov (United States)

    Clarke, Thomas A; Edwards, Marcus J; Gates, Andrew J; Hall, Andrea; White, Gaye F; Bradley, Justin; Reardon, Catherine L; Shi, Liang; Beliaev, Alexander S; Marshall, Matthew J; Wang, Zheming; Watmough, Nicholas J; Fredrickson, James K; Zachara, John M; Butt, Julea N; Richardson, David J

    2011-06-07

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along "nanowire" appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

  2. Metabolic activity of bacterial cells enumerated by direct viable count

    Energy Technology Data Exchange (ETDEWEB)

    Roszak, D.B.; Colwell, R.R.

    1987-12-01

    The direct viable count (DVC) method was modified by incorporating radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included (methyl-tritium thymidine or (Uranium-Carbon 14) glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate.

  3. A cell cycle and nutritional checkpoint controlling bacterial surface adhesion.

    Directory of Open Access Journals (Sweden)

    Aretha Fiebig

    2014-01-01

    Full Text Available In natural environments, bacteria often adhere to surfaces where they form complex multicellular communities. Surface adherence is determined by the biochemical composition of the cell envelope. We describe a novel regulatory mechanism by which the bacterium, Caulobacter crescentus, integrates cell cycle and nutritional signals to control development of an adhesive envelope structure known as the holdfast. Specifically, we have discovered a 68-residue protein inhibitor of holdfast development (HfiA that directly targets a conserved glycolipid glycosyltransferase required for holdfast production (HfsJ. Multiple cell cycle regulators associate with the hfiA and hfsJ promoters and control their expression, temporally constraining holdfast development to the late stages of G1. HfiA further functions as part of a 'nutritional override' system that decouples holdfast development from the cell cycle in response to nutritional cues. This control mechanism can limit surface adhesion in nutritionally sub-optimal environments without affecting cell cycle progression. We conclude that post-translational regulation of cell envelope enzymes by small proteins like HfiA may provide a general means to modulate the surface properties of bacterial cells.

  4. Lung Dendritic Cells Facilitate Extrapulmonary Bacterial Dissemination during Pneumococcal Pneumonia

    Directory of Open Access Journals (Sweden)

    Alva eRosendahl

    2013-06-01

    Full Text Available Streptococcus pneumoniae is a leading cause of bacterial pneumonia worldwide. Given the critical role of dendritic cells (DCs in regulating and modulating the immune response to pathogens, we investigated here the role of DCs in S. pneumoniae lung infections. Using a well-established transgenic mouse line which allows the conditional transient depletion of DCs, we showed that ablation of DCs resulted in enhanced resistance to intranasal challenge with S. pneumoniae. DC-depleted mice exhibited delayed bacterial systemic dissemination, significantly reduced bacterial loads in the infected organs and lower levels of serum inflammatory mediators than non-depleted animals. The increased resistance of DC-depleted mice to S. pneumoniae was associated with a better capacity to restrict pneumococci extrapulmonary dissemination. Furthermore, we demonstrated that S. pneumoniae disseminated from the lungs into the regional lymph nodes in a cell-independent manner and that this direct way of dissemination was much more efficient in the presence of DCs. We also provide evidence that S. pneumoniae induces expression and activation of matrix metalloproteinase-9 (MMP-9 in cultured bone marrow-derived DCs. MMP-9 is a protease involved in the breakdown of extracellular matrix proteins and is critical for DC trafficking across extracellular matrix and basement membranes during the migration from the periphery to the lymph nodes. MMP-9 was also significantly up-regulated in the lungs of mice after intranasal infection with S. pneumoniae. Notably, the expression levels of MMP-9 in the infected lungs were significantly decreased after depletion of DCs suggesting the involvement of DCs in MMP-9 production during pneumococcal pneumonia. Thus, we propose that S. pneumoniae can exploit the DC-derived proteolysis to open tissue barriers thereby facilitating its own dissemination from the local site of infection.

  5. Segrosome complex formation during DNA trafficking in bacterial cell division

    Directory of Open Access Journals (Sweden)

    Maria A. Oliva

    2016-09-01

    Full Text Available Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialised partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

  6. Transmission electron microscopy and atomic force microscopy characterization of nickel deposition on bacterial cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Recently bacterial cells have become attractive biological templates for the fabrication of metal nano- structures or nanomaterials due to their inherent small size, various standard geometrical shapes and abundant source. In this paper, nickel-coated bacterial cells (gram-negative bacteria of Escherichia coli) were fabricated via electroless chemical plating. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) characterization results reveal evident morphological difference between bacterial cells before and after deposition with nickel. The bare cells with smooth surface presented transverse outspreading effect at mica surface. Great changes took place in surface roughness for those bacterial cells after metallization. A large number of nickel nanoparticles were observed to be equably distributed at bacterial surface after activation and subsequent metallization. Furthermore, ultra thin section analytic results validated the presence and uniformity of thin nickel coating at bacterial surface after metallization.

  7. (p)ppGpp and the bacterial cell cycle

    Indian Academy of Sciences (India)

    Aanisa Nazir; Rajendran Harinarayanan

    2016-06-01

    Genes of the Rel/Spo homolog (RSH) superfamily synthesize and/or hydrolyse the modified nucleotides pppGpp/ppGpp (collectively referred to as (p)ppGpp) and are prevalent across diverse bacteria and in plant chloroplasts. Bacteria accumulate (p)ppGpp in response to nutrient deprivation (generically called the stringent response) and elicit appropriate adaptive responses mainly through the regulation of transcription. Although at different concentrations (p)ppGpp affect the expression of distinct set of genes, the two well-characterized responses are reduction in expression of the protein synthesis machinery and increase in the expression of genes coding for amino acid biosynthesis. In Escherichia coli, the cellular (p)ppGpp level inversely correlates with the growth rate and increasing its concentration decreases the steady state growth rate in a defined growth medium. Since change in growth rate must be accompanied by changes in cell cycle parameters set through the activities of the DNA replication and cell division apparatus, (p)ppGpp could coordinate protein synthesis (cell mass increase) with these processes. Here we review the role of (p)ppGpp in bacterial cell cycle regulation.

  8. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  9. (p)ppGpp and the bacterial cell cycle.

    Science.gov (United States)

    Nazir, Aanisa; Harinarayanan, Rajendran

    2016-06-01

    Genes of the Rel/Spo homolog (RSH) superfamily synthesize and/or hydrolyse the modified nucleotides pppGpp/ ppGpp (collectively referred to as (p)ppGpp) and are prevalent across diverse bacteria and in plant chloroplasts. Bacteria accumulate (p)ppGpp in response to nutrient deprivation (generically called the stringent response) and elicit appropriate adaptive responses mainly through the regulation of transcription. Although at different concentrations (p)ppGpp affect the expression of distinct set of genes, the two well-characterized responses are reduction in expression of the protein synthesis machinery and increase in the expression of genes coding for amino acid biosynthesis. In Escherichia coli, the cellular (p)ppGpp level inversely correlates with the growth rate and increasing its concentration decreases the steady state growth rate in a defined growth medium. Since change in growth rate must be accompanied by changes in cell cycle parameters set through the activities of the DNA replication and cell division apparatus, (p)ppGpp could coordinate protein synthesis (cell mass increase) with these processes. Here we review the role of (p)ppGpp in bacterial cell cycle regulation.

  10. Bacterial protein toxins : tools to study mammalian molecular cell biology

    NARCIS (Netherlands)

    Wüthrich, I.W.

    2014-01-01

    Bacterial protein toxins are genetically encoded proteinaceous macromolecules that upon exposure causes perturbation of cellular metabolism in a susceptible host. A bacterial toxin can work at a distance from the site of infection, and has direct and quantifiable actions. Bacterial protein toxins ca

  11. Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis.

    Science.gov (United States)

    Liu, Xiaokun; Grabherr, Heini M; Willmann, Roland; Kolb, Dagmar; Brunner, Frédéric; Bertsche, Ute; Kühner, Daniel; Franz-Wachtel, Mirita; Amin, Bushra; Felix, Georg; Ongena, Marc; Nürnberger, Thorsten; Gust, Andrea A

    2014-06-23

    Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites.

  12. Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

    DEFF Research Database (Denmark)

    Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W;

    2007-01-01

    Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients...

  13. Regulation of bacterial cell polarity by small GTPases.

    Science.gov (United States)

    Keilberg, Daniela; Søgaard-Andersen, Lotte

    2014-04-01

    Bacteria are polarized with many proteins localizing dynamically to specific subcellular sites. Two GTPase families have important functions in the regulation of bacterial cell polarity, FlhF homologues and small GTPases of the Ras superfamily. The latter consist of only a G domain and are widespread in bacteria. The rod-shaped Myxococcus xanthus cells have two motility systems, one for gliding and one that depends on type IV pili. The function of both systems hinges on proteins that localize asymmetrically to the cell poles. During cellular reversals, these asymmetrically localized proteins are released from their respective poles and then bind to the opposite pole, resulting in an inversion of cell polarity. Here, we review genetic, cell biological, and biochemical analyses that identified two modules containing small Ras-like GTPases that regulate the dynamic polarity of motility proteins. The GTPase SofG interacts directly with the bactofilin cytoskeletal protein BacP to ensure polar localization of type IV pili proteins. In the second module, the small GTPase MglA, its cognate GTPase activating protein (GAP) MglB, and the response regulator RomR localize asymmetrically to the poles and sort dynamically localized motility proteins to the poles. During reversals, MglA, MglB, and RomR switch poles, in that way inducing the relocation of dynamically localized motility proteins. Structural analyses have demonstrated that MglB has a Roadblock/LC7 fold, the central β2 strand in MglA undergoes an unusual screw-type movement upon GTP binding, MglA contains an intrinsic Arg finger required for GTP hydrolysis, and MglA and MglB form an unusual G protein/GAP complex with a 1:2 stoichiometry.

  14. Characterization and use of crystalline bacterial cell surface layers

    Science.gov (United States)

    Sleytr, Uwe B.; Sára, Margit; Pum, Dietmar; Schuster, Bernhard

    2001-10-01

    Crystalline bacterial cell surface layers (S-layers) are one of the most common outermost cell envelope components of prokaryotic organisms (archaea and bacteria). S-layers are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. S-layers as the most abundant of prokaryotic cellular proteins are appealing model systems for studying the structure, synthesis, genetics, assembly and function of proteinaceous supramolecular structures. The wealth of information existing on the general principle of S-layers have revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for chemical modifications and binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from a variety of organisms are capable of recrystallizing as closed monolayers onto solid supports (e.g., metals, polymers, silicon wafers) at the air-water interface, on lipid films or onto the surface of liposomes; (iv) functional domains can be incorporated in S-layer proteins by genetic engineering. Thus, S-layer technologies particularly provide new approaches for biotechnology, biomimetics, molecular nanotechnology, nanopatterning of surfaces and formation of ordered arrays of metal clusters or nanoparticles as required for nanoelectronics.

  15. Probing living bacterial adhesion by single cell force spectroscopy using atomic force microscopy

    DEFF Research Database (Denmark)

    Zeng, Guanghong; Ogaki, Ryosuke; Regina, Viduthalai R.;

    of the mechanisms of bacterial attachment. An alternative way to study the adhesion of single bacterial cells is to measure the adhesion between immobilized bacterial cells and coated AFM cantilevers. This strategy was used to investigate the adhesive properties of novel high density poly(ethylene glycol) (PEG......) coatings on titanium. We investigate the ability of a high density poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) coating to resist bacterial adhesion and biofilm formation from three clinically relevant bacteria: Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermis. The high...

  16. Comparison of the cytotoxic effect of polystyrene latex nanoparticles on planktonic cells and bacterial biofilms

    Science.gov (United States)

    Nomura, Toshiyuki; Fujisawa, Eri; Itoh, Shikibu; Konishi, Yasuhiro

    2016-06-01

    The cytotoxic effect of positively charged polystyrene latex nanoparticles (PSL NPs) was compared between planktonic bacterial cells and bacterial biofilms using confocal laser scanning microscopy, atomic force microscopy, and a colony counting method. Pseudomonas fluorescens, which is commonly used in biofilm studies, was employed as the model bacteria. We found that the negatively charged bacterial surface of the planktonic cells was almost completely covered with positively charged PSL NPs, leading to cell death, as indicated by the NP concentration being greater than that required to achieve single layer coverage. In addition, the relationship between surface coverage and cell viability of P. fluorescens cells correlated well with the findings in other bacterial cells ( Escherichia coli and Lactococcus lactis). However, most of the bacterial cells that formed the biofilm were viable despite the positively charged PSL NPs being highly toxic to planktonic bacterial cells. This indicated that bacterial cells embedded in the biofilm were protected by self-produced extracellular polymeric substances (EPS) that provide resistance to antibacterial agents. In conclusion, mature biofilms covered with EPS exhibit resistance to NP toxicity as well as antibacterial agents.

  17. Guidelines for monitoring bulk tank milk somatic cell and bacterial counts.

    Science.gov (United States)

    Jayarao, B M; Pillai, S R; Sawant, A A; Wolfgang, D R; Hegde, N V

    2004-10-01

    This study was conducted to establish guidelines for monitoring bulk tank milk somatic cell count and bacterial counts, and to understand the relationship between different bacterial groups that occur in bulk tank milk. One hundred twenty-six dairy farms in 14 counties of Pennsylvania participated, each providing one bulk tank milk sample every 15 d for 2 mo. The 4 bulk tank milk samples from each farm were examined for bulk tank somatic cell count and bacterial counts including standard plate count, preliminary incubation count, laboratory pasteurization count, coagulase-negative staphylococcal count, environmental streptococcal count, coliform count, and gram-negative noncoliform count. The milk samples were also examined for presence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma. The bacterial counts of 4 bulk tank milk samples examined over an 8-wk period were averaged and expressed as mean bacterial count per milliliter. The study revealed that an increase in the frequency of isolation of Staphylococcus aureus and Streptococcus agalactiae was significantly associated with an increased bulk tank somatic cell count. Paired correlation analysis showed that there was low correlation between different bacterial counts. Bulk tank milk with low (standard plate count also had a significantly low level of mean bulk tank somatic cell count (count (count (counts (count (count was less likely to be associated with somatic cell or other bacterial counts. Herd size and farm management practices had considerable influence on somatic cell and bacterial counts in bulk tank milk. Dairy herds that used automatic milking detachers, sand as bedding material, dip cups for teat dipping instead of spraying, and practiced pre-and postdipping had significantly lower bulk tank somatic cell and/or bacterial counts. In conclusion, categorized bulk tank somatic cell and bacterial counts could serve as indicators and facilitate monitoring of herd udder health and milk

  18. Bacterial colonization of host cells in the absence of cholesterol.

    Directory of Open Access Journals (Sweden)

    Stacey D Gilk

    2013-01-01

    Full Text Available Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/- mouse embryonic fibroblasts (MEFs. DHCR24(-/- MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/- MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/- MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(Vβ(3 integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/- MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

  19. The bacterial cell cycle checkpoint protein Obg and its role in programmed cell death

    Directory of Open Access Journals (Sweden)

    Liselot Dewachter

    2016-03-01

    Full Text Available The phenomenon of programmed cell death (PCD, in which cells initiate their own demise, is not restricted to multicellular organisms. Unicellular organisms, both eukaryotes and prokaryotes, also possess pathways that mediate PCD. We recently identified a PCD mechanism in Escherichia coli that is triggered by a mutant isoform of the essential GTPase ObgE (Obg of E. coli. Importantly, the PCD pathway mediated by mutant Obg (Obg* differs fundamentally from other previously described bacterial PCD pathways and thus constitutes a new mode of PCD. ObgE was previously proposed to act as a cell cycle checkpoint protein able to halt cell division. The implication of ObgE in the regulation of PCD further increases the similarity between this protein and eukaryotic cell cycle regulators that are capable of doing both. Moreover, since Obg is conserved in eukaryotes, the elucidation of this cell death mechanism might contribute to the understanding of PCD in higher organisms. Additionally, if Obg*-mediated PCD is conserved among different bacterial species, it will be a prime target for the development of innovative antibacterials that artificially induce this pathway.

  20. EEVD motif of heat shock cognate protein 70 contributes to bacterial uptake by trophoblast giant cells

    Directory of Open Access Journals (Sweden)

    Kim Suk

    2009-12-01

    Full Text Available Abstract Background The uptake of abortion-inducing pathogens by trophoblast giant (TG cells is a key event in infectious abortion. However, little is known about phagocytic functions of TG cells against the pathogens. Here we show that heat shock cognate protein 70 (Hsc70 contributes to bacterial uptake by TG cells and the EEVD motif of Hsc70 plays an important role in this. Methods Brucella abortus and Listeria monocytogenes were used as the bacterial antigen in this study. Recombinant proteins containing tetratricopeptide repeat (TPR domains were constructed and confirmation of the binding capacity to Hsc70 was assessed by ELISA. The recombinant TPR proteins were used for investigation of the effect of TPR proteins on bacterial uptake by TG cells and on pregnancy in mice. Results The monoclonal antibody that inhibits bacterial uptake by TG cells reacted with the EEVD motif of Hsc70. Bacterial TPR proteins bound to the C-terminal of Hsc70 through its EEVD motif and this binding inhibited bacterial uptake by TG cells. Infectious abortion was also prevented by blocking the EEVD motif of Hsc70. Conclusions Our results demonstrate that surface located Hsc70 on TG cells mediates the uptake of pathogenic bacteria and proteins containing the TPR domain inhibit the function of Hsc70 by binding to its EEVD motif. These molecules may be useful in the development of methods for preventing infectious abortion.

  1. Phase Diagram of Collective Motion of Bacterial Cells in a Shallow Circular Pool

    CERN Document Server

    Wakita, Jun-ichi; Yamamoto, Ken; Katori, Makoto; Yamada, Yasuyuki

    2015-01-01

    The collective motion of bacterial cells in a shallow circular pool is systematically studied using the bacterial species $Bacillus$ $subtilis$. The ratio of cell length to pool diameter (i.e., the reduced cell length) ranges from 0.06 to 0.43 in our experiments. Bacterial cells in a circular pool show various types of collective motion depending on the cell density in the pool and the reduced cell length. The motion is classified into six types, which we call random motion, turbulent motion, one-way rotational motion, two-way rotational motion, random oscillatory motion, and ordered oscillatory motion. Two critical values of reduced cell lengths are evaluated, at which drastic changes in collective motion are induced. A phase diagram is proposed in which the six phases are arranged.

  2. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    Energy Technology Data Exchange (ETDEWEB)

    Yu, L.D., E-mail: yuld@thep-center.org [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Sangwijit, K. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Prakrajang, K. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Faculty of Science, Maejo University, Chiang Mai 50290 (Thailand); Phanchaisri, B. [Institute of Science and Technology Research, Chiang Mai University, Chiang Mai 50200 (Thailand); Thongkumkoon, P. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thopan, P. [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Singkarat, S. [Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Anuntalabhochai, S. [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2014-05-01

    Highlights: • Ion bombardment could induce DNA transfer into E. coli cells. • The DNA transfer induction depended on ion energy and fluence. • The mechanism was associated with the bacterial cell envelope structure. • A mechanism phase diagram was proposed to summarize the mechanism. - Abstract: As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10–20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

  3. Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations

    Directory of Open Access Journals (Sweden)

    Carolina Tropini

    2014-11-01

    Full Text Available Although bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

  4. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S

    2001-01-01

    Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  5. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

    DEFF Research Database (Denmark)

    Schneider, Tanja; Kruse, Thomas; Wimmer, Reinhard

    2010-01-01

    that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular...

  6. Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

    Directory of Open Access Journals (Sweden)

    Schuren Frank H

    2008-12-01

    Full Text Available Abstract Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability.

  7. Multiscale modeling of bacterial colonies: how pili mediate the dynamics of single cells and cellular aggregates

    Science.gov (United States)

    Pönisch, Wolfram; Weber, Christoph A.; Juckeland, Guido; Biais, Nicolas; Zaburdaev, Vasily

    2017-01-01

    Neisseria gonorrhoeae is the causative agent of one of the most common sexually transmitted diseases, gonorrhea. Over the past two decades there has been an alarming increase of reported gonorrhea cases where the bacteria were resistant to the most commonly used antibiotics thus prompting for alternative antimicrobial treatment strategies. The crucial step in this and many other bacterial infections is the formation of microcolonies, agglomerates consisting of up to several thousands of cells. The attachment and motility of cells on solid substrates as well as the cell-cell interactions are primarily mediated by type IV pili, long polymeric filaments protruding from the surface of cells. While the crucial role of pili in the assembly of microcolonies has been well recognized, the exact mechanisms of how they govern the formation and dynamics of microcolonies are still poorly understood. Here, we present a computational model of individual cells with explicit pili dynamics, force generation and pili-pili interactions. We employ the model to study a wide range of biological processes, such as the motility of individual cells on a surface, the heterogeneous cell motility within the large cell aggregates, and the merging dynamics and the self-assembly of microcolonies. The results of numerical simulations highlight the central role of pili generated forces in the formation of bacterial colonies and are in agreement with the available experimental observations. The model can quantify the behavior of multicellular bacterial colonies on biologically relevant temporal and spatial scales and can be easily adjusted to include the geometry and pili characteristics of various bacterial species. Ultimately, the combination of the microbiological experimental approach with the in silico model of bacterial colonies might provide new qualitative and quantitative insights on the development of bacterial infections and thus pave the way to new antimicrobial treatments.

  8. Behind the lines–actions of bacterial type III effector proteins in plant cells

    OpenAIRE

    Büttner, Daniela

    2016-01-01

    Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Ty...

  9. New method for estimating bacterial cell abundances in natural samples by use of sublimation

    Science.gov (United States)

    Glavin, Daniel P.; Cleaves, H. James; Schubert, Michael; Aubrey, Andrew; Bada, Jeffrey L.

    2004-01-01

    We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500 degrees C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approximately 10(5) to 10(9) E. coli cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.

  10. [Influence of human gastrointestinal tract bacterial pathogens on host cell apoptosis].

    Science.gov (United States)

    Wronowska, Weronika; Godlewska, Renata; Jagusztyn-Krynicka, Elzbieta Katarzyna

    2005-01-01

    Several pathogenic bacteria are able to trigger apoptosis in the host cell, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Induction and/or blockage of programmed cell death upon infection is a result of complex interaction of bacterial proteins with cellular proteins involved in signal transduction and apoptosis. In this review we focus on pro/anti-apoptotic activities exhibited by two enteric pathogens Salmonella enterica, Yersinia spp. and gastric pathogen Helicobacter pylori. We present current knowledge on how interaction between mammalian and bacterial cell relates to the molecular pathways of apoptosis, and what is the role of apoptosis in pathogenesis.

  11. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.;

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...

  12. The innate immune protein Nod2 binds directly to MDP, a bacterial cell wall fragment.

    Science.gov (United States)

    Grimes, Catherine Leimkuhler; Ariyananda, Lushanti De Zoysa; Melnyk, James E; O'Shea, Erin K

    2012-08-22

    Mammalian Nod2 is an intracellular protein that is implicated in the innate immune response to the bacterial cell wall and is associated with the development of Crohn's disease, Blau syndrome, and gastrointestinal cancers. Nod2 is required for an immune response to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial cell wall, but it is not known whether MDP binds directly to Nod2. We report the expression and purification of human Nod2 from insect cells. Using novel MDP self-assembled monolayers (SAMs), we provide the first biochemical evidence for a direct, high-affinity interaction between Nod2 and MDP.

  13. Selective Removal of DNA from Dead Cells of Mixed Bacterial Communities by Use of Ethidium Monoazide

    OpenAIRE

    Nocker, Andreas; Anne K. Camper

    2006-01-01

    The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A prom...

  14. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Science.gov (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  15. Bacterial delivery of large intact genomic-DNA-containing BACs into mammalian cells.

    Science.gov (United States)

    Cheung, Wing; Kotzamanis, George; Abdulrazzak, Hassan; Goussard, Sylvie; Kaname, Tadashi; Kotsinas, Athanassios; Gorgoulis, Vassilis G; Grillot-Courvalin, Catherine; Huxley, Clare

    2012-01-01

    Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.

  16. Depletion of dendritic cells enhances innate anti-bacterial host defense through modulation of phagocyte homeostasis.

    Directory of Open Access Journals (Sweden)

    Stella E Autenrieth

    2012-02-01

    Full Text Available Dendritic cells (DCs as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye. We used CD11c-diphtheria toxin (DT mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.

  17. Evaluation of the sensitivity of bacterial and yeast cells to cold atmospheric plasma jet treatments.

    Science.gov (United States)

    Sharkey, Michael A; Chebbi, Ahmed; McDonnell, Kevin A; Staunton, Claire; Dowling, Denis P

    2015-06-07

    The focus of this research was first to determine the influence of the atmospheric plasma drive frequency on the generation of atomic oxygen species and its correlation with the reduction of bacterial load after treatment in vitro. The treatments were carried out using a helium-plasma jet source called PlasmaStream™. The susceptibility of multiple microbial cell lines was investigated in order to compare the response of gram-positive and gram-negative bacteria, as well as a yeast cell line to the atmospheric plasma treatment. It was observed for the source evaluated that at a frequency of 160 kHz, increased levels of oxygen-laden active species (i.e., OH, NO) were generated. At this frequency, the maximum level of bacterial inactivation in vitro was also achieved. Ex vivo studies (using freshly excised porcine skin as a human analog) were also carried out to verify the antibacterial effect of the plasma jet treatment at this optimal operational frequency and to investigate the effect of treatment duration on the reduction of bacterial load. The plasma jet treatment was found to yield a 4 log reduction in bacterial load after 6 min of treatment, with no observable adverse effects on the treatment surface. The gram-negative bacterial cell lines were found to be far more susceptible to the atmospheric plasma treatments than the gram-positive bacteria. Flow cytometric analysis of plasma treated bacterial cells (Escherichia coli) was conducted in order to attain a fundamental understanding of the mode of action of the treatment on bacteria at a cellular level. This study showed that after treatment with the plasma jet, E. coli cells progressed through the following steps of cell death; the inactivation of transport systems, followed by depolarization of the cytoplasmic membrane, and finally permeabilization of the cell wall.

  18. Development of method for evaluating cell hardness and correlation between bacterial spore hardness and durability

    Directory of Open Access Journals (Sweden)

    Nakanishi Koichi

    2012-06-01

    Full Text Available Abstract Background Despite the availability of conventional devices for making single-cell manipulations, determining the hardness of a single cell remains difficult. Here, we consider the cell to be a linear elastic body and apply Young’s modulus (modulus of elasticity, which is defined as the ratio of the repulsive force (stress in response to the applied strain. In this new method, a scanning probe microscope (SPM is operated with a cantilever in the “contact-and-push” mode, and the cantilever is applied to the cell surface over a set distance (applied strain. Results We determined the hardness of the following bacterial cells: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and five Bacillus spp. In log phase, these strains had a similar Young’s modulus, but Bacillus spp. spores were significantly harder than the corresponding vegetative cells. There was a positive, linear correlation between the hardness of bacterial spores and heat or ultraviolet (UV resistance. Conclusions Using this technique, the hardness of a single vegetative bacterial cell or spore could be determined based on Young’s modulus. As an application of this technique, we demonstrated that the hardness of individual bacterial spores was directly proportional to heat and UV resistance, which are the conventional measures of physical durability. This technique allows the rapid and direct determination of spore durability and provides a valuable and innovative method for the evaluation of physical properties in the field of microbiology.

  19. Dynamics of phenotypic reversibility of bacterial cells with oscillating hydrostatic pressure

    Science.gov (United States)

    Nepal, Sudip; Kumar, Pradeep

    Bacterial cells encounter and respond to physiochemical fluctuations. The response depends on the extent and type of the stresses applied. The response of bacterial cells to the fluctuating stress is relatively unknown. Here, we have studied the response of wild type Escherichia coli (E. coli) under fluctuating hydrostatic pressures ranging from 1 atm to 500 atm. High pressure acts as a stress to E. coli since these bacteria are adapted to grow optimally at atmospheric pressure. Cell division of E. coli is inhibited at high pressures resulting in increase in the length of the cells. Cell-length is reversible in nature and bacterial cells revert back to normal size on a time scale that is proportional to the strength and time of continuous pressure applied upon relaxing the high pressure condition. We have studied the dynamics of cellular reversibility of E. coli under the conditions in which continuous pressure is applied and subsequently relaxed over different time scales. We have quantified the dynamics of cellular reversibility with different relaxation times. Furthermore, we propose a model to describe the reversibility of the bacterial cell with the relaxation time. Our theoretical model fits well to the experimental data. We further

  20. Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level.

    Science.gov (United States)

    Cinquin, Bertrand; Maigre, Laure; Pinet, Elizabeth; Chevalier, Jacqueline; Stavenger, Robert A; Mills, Scott; Réfrégiers, Matthieu; Pagès, Jean-Marie

    2015-12-11

    Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake.

  1. The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes

    DEFF Research Database (Denmark)

    Chindera, Kantaraja; Mahato, Manohar; Sharma, Ashwani Kumar

    2016-01-01

    To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption...... by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when...... to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance....

  2. Mechanisms of bacterial morphogenesis: evolutionary cell biology approaches provide new insights.

    Science.gov (United States)

    Jiang, Chao; Caccamo, Paul D; Brun, Yves V

    2015-04-01

    How Darwin's "endless forms most beautiful" have evolved remains one of the most exciting questions in biology. The significant variety of bacterial shapes is most likely due to the specific advantages they confer with respect to the diverse environments they occupy. While our understanding of the mechanisms generating relatively simple shapes has improved tremendously in the last few years, the molecular mechanisms underlying the generation of complex shapes and the evolution of shape diversity are largely unknown. The emerging field of bacterial evolutionary cell biology provides a novel strategy to answer this question in a comparative phylogenetic framework. This relatively novel approach provides hypotheses and insights into cell biological mechanisms, such as morphogenesis, and their evolution that would have been difficult to obtain by studying only model organisms. We discuss the necessary steps, challenges, and impact of integrating "evolutionary thinking" into bacterial cell biology in the genomic era.

  3. Genetic Screening for Bacterial Mutants in Liquid Growth Media By Fluorescence-Activated Cell Sorting

    Science.gov (United States)

    Abuaita, Basel H.; Withey, Jeffrey H.

    2010-01-01

    Many bacterial pathogens have defined in vitro virulence inducing conditions in liquid media which lead to production of virulence factors important during an infection. Identifying mutants that no longer respond to virulence inducing conditions will increase our understanding of bacterial pathogenesis. However, traditional genetic screens require growth on solid media. Bacteria in a single colony are in every phase of the growth curve, which complicates the analysis and make screens for growth phase-specific mutants problematic. Here, we utilize fluorescence-activated cell sorting in conjunction with random transposon mutagenesis to isolate bacteria grown in liquid media that are defective in virulence activation. This method permits analysis of an entire bacterial population in real time and selection of individual bacterial mutants with the desired gene expression profile at any time point after induction. We have used this method to identify Vibrio cholerae mutants defective in virulence induction. PMID:21094189

  4. Disturbance of the bacterial cell wall specifically interferes with biofilm formation.

    Science.gov (United States)

    Bucher, Tabitha; Oppenheimer-Shaanan, Yaara; Savidor, Alon; Bloom-Ackermann, Zohar; Kolodkin-Gal, Ilana

    2015-12-01

    In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment.

  5. Responses of synovial fluid and peripheral blood mononuclear cells to bacterial antigens and autologous antigen presenting cells.

    Science.gov (United States)

    Klasen, I S; Melief, M J; Swaak, T J; Severijnen, A J; Hazenberg, M P

    1993-01-01

    The specificity of T cells in the inflamed joints of patients with rheumatoid arthritis (RA) has been the subject of much study. Bacterial antigens are suspect in the aetiology of rheumatic diseases. The responsiveness of the mononuclear cell fraction of peripheral blood and synovial fluid of patients with RA and of patients with rheumatic diseases other than RA to bacterial antigens such as cell wall fragments of the anaerobic intestinal flora, cell wall fragments of Streptococcus pyogenes, intestinal flora derived peptidoglycan polysaccharide complexes, the 65 kilodalton protein of Mycobacterium tuberculosis, and muramyldipeptide was investigated. No significant difference in response was found to all these bacterial antigens in the synovial fluid of patients with RA compared with the responses in patients with other rheumatic diseases. The highest responsiveness in the synovial fluid of the patients with RA was to the streptococcal cell wall fragments and to the 65 kilodalton protein. Higher responses to several bacterial antigens in the synovial fluid of patients with RA were found compared with peripheral blood from the same patient group. The antigen presenting cell population of the synovial fluid in patients with RA and the patients with other rheumatic diseases was found to be stimulatory for autologous peripheral blood T cells even in the absence of antigen. This suggests an important role for the synovial antigen presenting cell in the aetiology of inflammatory joint diseases. PMID:8447692

  6. Bacterial cell-cell communication in the host via RRNPP peptide-binding regulators

    Directory of Open Access Journals (Sweden)

    David ePerez-Pascual

    2016-05-01

    Full Text Available Human microbiomes are composed of complex and dense bacterial consortia. In these environments, bacteria are able to react quickly to change by coordinating their gene expression at the population level via small signaling molecules. In Gram-positive bacteria, cell-cell communication is mostly mediated by peptides that are released into the extracellular environment. Cell-cell communication based on these peptides is especially widespread in the group Firmicutes, in which they regulate a wide array of biological processes, including functions related to host-microbe interactions. Among the different agents of communication, the RRNPP family of cytoplasmic transcriptional regulators, together with their cognate re-internalized signaling peptides, represents a group of emerging importance. RRNPP members that have been studied so far are found mainly in species of bacilli, streptococci, and enterococci. These bacteria are characterized as both human commensal and pathogenic, and share different niches in the human body with other microorganisms. The goal of this mini-review is to present the current state of research on the biological relevance of RRNPP mechanisms in the context of the host, highlighting their specific roles in commensalism or virulence.

  7. Bacterial swarmer cells in confinement: A mesoscale hydrodynamic simulation study

    CERN Document Server

    Eisenstecken, Thomas; Winkler, Roland G

    2016-01-01

    A wide spectrum of Peritrichous bacteria undergo considerable physiological changes when they are inoculated onto nutrition-rich surfaces and exhibit a rapid and collective migration denoted as swarming. Thereby, the length of such swarmer cells and their number of flagella increases substantially. In this article, we investigated the properties of individual E. coli-type swarmer cells confined between two parallel walls via mesoscale hydrodynamic simulations, combining molecular dynamics simulations of the swarmer cell with the multiparticle particle collision dynamics approach for the embedding fluid. E. coli-type swarmer cells are three-times longer than their planktonic counter parts, but their flagella density is comparable. By varying the wall separation, we analyze the confinement effect on the flagella arrangement, on the distribution of cells in the gap between the walls, and on the cell dynamics. We find only a weak dependence of confinement on the bundle structure and dynamics. The distribution of ...

  8. On the chronology and topography of bacterial cell division.

    Science.gov (United States)

    Vicente, M; Palacios, P; Dopazo, A; Garrido, T; Pla, J; Aldea, M

    1991-01-01

    Gene products that play a role in the formation of cell septum should be expected to be endowed with a set of specific properties. In principle, septal proteins should be located at the cell envelope. The expression of division genes should ensure the synthesis of septal proteins at levels commensurate with the needs of cell division at different rates of cell duplication. We have results indicating that some fts genes located within the 2.5-min cluster in the Escherichia coli chromosome conform to these predictions.

  9. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring.

    Science.gov (United States)

    Van Nevel, S; Koetzsch, S; Proctor, C R; Besmer, M D; Prest, E I; Vrouwenvelder, J S; Knezev, A; Boon, N; Hammes, F

    2017-04-15

    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water.

  10. Effect of cell physicochemical characteristics and motility on bacterial transport in groundwater

    Science.gov (United States)

    Becker, M.W.; Collins, S.A.; Metge, D.W.; Harvey, R.W.; Shapiro, A.M.

    2004-01-01

    The influence of physicochemical characteristics and motility on bacterial transport in groundwater were examined in flow-through columns. Four strains of bacteria isolated from a crystalline rock groundwater system were investigated, with carboxylate-modified and amidine-modified latex microspheres and bromide as reference tracers. The bacterial isolates included a gram-positive rod (ML1), a gram-negative motile rod (ML2), a nonmotile mutant of ML2 (ML2m), and a gram-positive coccoid (ML3). Experiments were repeated at two flow velocities, in a glass column packed with glass beads, and in another packed with iron-oxyhydroxide coated glass beads. Bacteria breakthrough curves were interpreted using a transport equation that incorporates a sorption model from microscopic observation of bacterial deposition in flow-cell experiments. The model predicts that bacterial desorption rate will decrease exponentially with the amount of time the cell is attached to the solid surface. Desorption kinetics appeared to influence transport at the lower flow rate, but were not discernable at the higher flow rate. Iron-oxyhydroxide coatings had a lower-than-expected effect on bacterial breakthrough and no effect on the microsphere recovery in the column experiments. Cell wall type and shape also had minor effects on breakthrough. Motility tended to increase the adsorption rate, and decrease the desorption rate. The transport model predicts that at field scale, desorption rate kinetics may be important to the prediction of bacteria transport rates. ?? 2003 Elsevier B.V. All rights reserved.

  11. Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation.

    Science.gov (United States)

    Boix-Amorós, Alba; Collado, Maria C; Mira, Alex

    2016-01-01

    Human breast milk is considered the optimal nutrition for infants, providing essential nutrients and a broad range of bioactive compounds, as well as its own microbiota. However, the interaction among those components and the biological role of milk microorganisms is still uncovered. Thus, our aim was to identify the relationships between milk microbiota composition, bacterial load, macronutrients, and human cells during lactation. Bacterial load was estimated in milk samples from a total of 21 healthy mothers through lactation time by bacteria-specific qPCR targeted to the single-copy gene fusA. Milk microbiome composition and diversity was estimated by 16S-pyrosequencing and the structure of these bacteria in the fluid was studied by flow cytometry, qPCR, and microscopy. Fat, protein, lactose, and dry extract of milk as well as the number of somatic cells were also analyzed. We observed that milk bacterial communities were generally complex, and showed individual-specific profiles. Milk microbiota was dominated by Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter. Staphylococcus aureus was not detected in any of these samples from healthy mothers. There was high variability in composition and number of bacteria per milliliter among mothers and in some cases even within mothers at different time points. The median bacterial load was 10(6) bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods. Furthermore, milk bacteria were present in a free-living, "planktonic" state, but also in equal proportion associated to human immune cells. There was no correlation between bacterial load and the amount of immune cells in milk, strengthening the idea that milk bacteria are not sensed as an infection by the immune system.

  12. A mechanistic stochastic framework for regulating bacterial cell division.

    Science.gov (United States)

    Ghusinga, Khem Raj; Vargas-Garcia, Cesar A; Singh, Abhyudai

    2016-07-26

    How exponentially growing cells maintain size homeostasis is an important fundamental problem. Recent single-cell studies in prokaryotes have uncovered the adder principle, where cells add a fixed size (volume) from birth to division, irrespective of their size at birth. To mechanistically explain the adder principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. Intriguingly, our results show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This was dramatically seen in experimental studies, where histograms of the added volume corresponding to different newborn sizes collapsed on top of each other. The model provides further insights consistent with experimental observations: the distribution of the added volume when scaled by its mean becomes invariant of the growth rate. In summary, our simple yet elegant model explains key experimental findings and suggests a mechanism for regulating both the mean and fluctuations in cell-division timing for controlling size.

  13. SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells.

    Science.gov (United States)

    Stylianidou, Stella; Brennan, Connor; Nissen, Silas B; Kuwada, Nathan J; Wiggins, Paul A

    2016-11-01

    Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame-to-frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB-based image processing package well-suited to quantitative analysis of high-throughput live-cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine-learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame-to-frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell-cycle dynamics in bacteria as well as cell-contact mediated phenomena. This package has a range of built-in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution.

  14. Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen.

    Directory of Open Access Journals (Sweden)

    Andrew S Brown

    2016-06-01

    Full Text Available Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC, which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.

  15. Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen.

    Science.gov (United States)

    Brown, Andrew S; Yang, Chao; Fung, Ka Yee; Bachem, Annabell; Bourges, Dorothée; Bedoui, Sammy; Hartland, Elizabeth L; van Driel, Ian R

    2016-06-01

    Legionella pneumophila is the causative agent of Legionnaires' disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity.

  16. Water Diffusion from a Bacterial Cell in Low-Moisture Foods.

    Science.gov (United States)

    Syamaladevi, Roopesh M; Tang, Juming; Zhong, QingPing

    2016-09-01

    We used a Fick's unsteady state diffusion equation to estimate the time required for a single spherical shaped bacterium (assuming Enterococcus faecium as the target microorganism) in low-moisture foods to equilibrate with the environment. We generated water sorption isotherms of freeze-dried E. faecium. The water activity of bacterial cells at given water content increased considerably as temperature increased from 20 to 80 °C, as observed in the sorption isotherms of bacterial cells. When the water vapor diffusion coefficient was assumed as between 10(-12) and 10(-10) m(2) /s for bacterial cells, the predicted equilibration times (teq ) ranged from 8.24×10(-4) to 8.24×10(-2) s. Considering a cell membrane barrier with a lower water diffusion coefficient (10(-15) m(2) /s) around the bacterial cell with a water diffusion coefficient of 10(-12) m(2) /s, the teq predicted using COMSOL Multiphysics program was 3.8×10(-1) s. This result suggests that a single bacterium equilibrates rapidly (within seconds) with change in environmental humidity and temperature.

  17. In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells.

    Science.gov (United States)

    Chen, H; Ouyang, W; Jones, M; Haque, T; Lawuyi, B; Prakash, S

    2005-08-01

    Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

  18. Nanoscale Electric Permittivity of Single Bacterial Cells at Gigahertz Frequencies by Scanning Microwave Microscopy.

    Science.gov (United States)

    Biagi, Maria Chiara; Fabregas, Rene; Gramse, Georg; Van Der Hofstadt, Marc; Juárez, Antonio; Kienberger, Ferry; Fumagalli, Laura; Gomila, Gabriel

    2016-01-26

    We quantified the electric permittivity of single bacterial cells at microwave frequencies and nanoscale spatial resolution by means of near-field scanning microwave microscopy. To this end, calibrated complex admittance images have been obtained at ∼19 GHz and analyzed with a methodology that removes the nonlocal topographic cross-talk contributions and thus provides quantifiable intrinsic dielectric images of the bacterial cells. Results for single Escherichia coli cells provide a relative electric permittivity of ∼4 in dry conditions and ∼20 in humid conditions, with no significant loss contributions. Present findings, together with the ability of microwaves to penetrate the cell membrane, open an important avenue in the microwave label-free imaging of single cells with nanoscale spatial resolution.

  19. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    Science.gov (United States)

    Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

    2016-09-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

  20. Vehicles, Replicators, and Intercellular Movement of Genetic Information: Evolutionary Dissection of a Bacterial Cell

    Directory of Open Access Journals (Sweden)

    Matti Jalasvuori

    2012-01-01

    Full Text Available Prokaryotic biosphere is vastly diverse in many respects. Any given bacterial cell may harbor in different combinations viruses, plasmids, transposons, and other genetic elements along with their chromosome(s. These agents interact in complex environments in various ways causing multitude of phenotypic effects on their hosting cells. In this discussion I perform a dissection for a bacterial cell in order to simplify the diversity into components that may help approach the ocean of details in evolving microbial worlds. The cell itself is separated from all the genetic replicators that use the cell vehicle for preservation and propagation. I introduce a classification that groups different replicators according to their horizontal movement potential between cells and according to their effects on the fitness of their present host cells. The classification is used to discuss and improve the means by which we approach general evolutionary tendencies in microbial communities. Moreover, the classification is utilized as a tool to help formulating evolutionary hypotheses and to discuss emerging bacterial pathogens as well as to promote understanding on the average phenotypes of different replicators in general. It is also discussed that any given biosphere comprising prokaryotic cell vehicles and genetic replicators may naturally evolve to have horizontally moving replicators of various types.

  1. Heterotrophic free-living and particle-bound bacterial cell size in the river Cauvery and its downstream tributaries

    Indian Academy of Sciences (India)

    T S Harsha; Sadanand M Yamakanamardi; M Mahadevaswamy

    2007-03-01

    This is the first comprehensive study on planktonic heterotrophic bacterial cell size in the river Cauvery and its important tributaries in Karnataka State, India. The initial hypothesis that the mean cell size of planktonic heterotrophic bacteria in the four tributaries are markedly different from each other and also from that in the main river Cauvery was rejected, because all five watercourses showed similar planktonic heterotrophic bacterial cell size. Examination of the correlation between mean heterotrophic bacterial cell size and environmental variables showed four correlations in the river Arkavathy and two in the river Shimsha. Regression analysis revealed that 18% of the variation in mean heterotrophic free-living bacterial cell size was due to biological oxygen demand (BOD) in the river Arkavathy, 11% due to surface water velocity (SWV) in the river Cauvery and 11% due to temperature in the river Kapila. Heterotrophic particle-bound bacterial cell size variation was 28% due to chloride and BOD in the river Arkavathy, 11% due to conductivity in the river Kapila and 8% due to calcium in the river Cauvery. This type of relationship between heterotrophic bacterial cell size and environmental variables suggests that, though the mean heterotrophic bacterial cell size was similar in all the five water courses, different sets of environmental variables apparently control the heterotrophic bacterial cell size in the various water bodies studied in this investigation. The possible cause for this environmental (bottom–up) control is discussed.

  2. Synchronization of Caulobacter crescentus for investigation of the bacterial cell cycle.

    Science.gov (United States)

    Schrader, Jared M; Shapiro, Lucy

    2015-04-08

    The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

  3. Lactic acid bacterial cell factories for gamma-aminobutyric acid.

    Science.gov (United States)

    Li, Haixing; Cao, Yusheng

    2010-11-01

    Gamma-aminobutyric acid is a non-protein amino acid that is widely present in organisms. Several important physiological functions of gamma-aminobutyric acid have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, and tranquilizer effects. Many microorganisms can produce gamma-aminobutyric acid including bacteria, fungi and yeasts. Among them, gamma-aminobutyric acid-producing lactic acid bacteria have been a focus of research in recent years, because lactic acid bacteria possess special physiological activities and are generally regarded as safe. They have been extensively used in food industry. The production of lactic acid bacterial gamma-aminobutyric acid is safe and eco-friendly, and this provides the possibility of production of new naturally fermented health-oriented products enriched in gamma-aminobutyric acid. The gamma-aminobutyric acid-producing species of lactic acid bacteria and their isolation sources, the methods for screening of the strains and increasing their production, the enzymatic properties of glutamate decarboxylases and the relative fundamental research are reviewed in this article. And the potential applications of gamma-aminobutyric acid-producing lactic acid bacteria were also referred to.

  4. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

    2007-04-05

    The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

  5. Homeostatic interplay between bacterial cell-cell signaling and iron in virulence.

    Directory of Open Access Journals (Sweden)

    Ronen Hazan

    2010-03-01

    Full Text Available Pathogenic bacteria use interconnected multi-layered regulatory networks, such as quorum sensing (QS networks to sense and respond to environmental cues and external and internal bacterial cell signals, and thereby adapt to and exploit target hosts. Despite the many advances that have been made in understanding QS regulation, little is known regarding how these inputs are integrated and processed in the context of multi-layered QS regulatory networks. Here we report the examination of the Pseudomonas aeruginosa QS 4-hydroxy-2-alkylquinolines (HAQs MvfR regulatory network and determination of its interaction with the QS acyl-homoserine-lactone (AHL RhlR network. The aim of this work was to elucidate paradigmatically the complex relationships between multi-layered regulatory QS circuitries, their signaling molecules, and the environmental cues to which they respond. Our findings revealed positive and negative homeostatic regulatory loops that fine-tune the MvfR regulon via a multi-layered dependent homeostatic regulation of the cell-cell signaling molecules PQS and HHQ, and interplay between these molecules and iron. We discovered that the MvfR regulon component PqsE is a key mediator in orchestrating this homeostatic regulation, and in establishing a connection to the QS rhlR system in cooperation with RhlR. Our results show that P. aeruginosa modulates the intensity of its virulence response, at least in part, through this multi-layered interplay. Our findings underscore the importance of the homeostatic interplay that balances competition within and between QS systems via cell-cell signaling molecules and environmental cues in the control of virulence gene expression. Elucidation of the fine-tuning of this complex relationship offers novel insights into the regulation of these systems and may inform strategies designed to limit infections caused by P. aeruginosa and related human pathogens.

  6. Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

    Science.gov (United States)

    Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

    2016-12-01

    We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process.

  7. Effects of bacterial cells and two types of extracellular polymers on bioclogging of sand columns

    Science.gov (United States)

    Xia, Lu; Zheng, Xilai; Shao, Haibing; Xin, Jia; Sun, Zhaoyue; Wang, Leyun

    2016-04-01

    Microbially induced reductions in the saturated hydraulic conductivity, Ks, of natural porous media, conventionally called bioclogging, occurs frequently in natural and engineered subsurface systems. Bioclogging can affect artificial groundwater recharge, in situ bioremediation of contaminated aquifers, or permeable reactive barriers. In this study, we designed a series of percolation experiments to simulate the growth and metabolism of bacteria in sand columns. The experimental results showed that the bacterial cell amount gradually increased to a maximum of 8.91 log10 CFU/g sand at 144 h during the bioclogging process, followed by a decrease to 7.89 log10 CFU/g sand until 336 h. The same variation pattern was found for the concentration of tightly bound extracellular polymeric substances (TB-EPS), which had a peak value of 220.76 μg/g sand at 144 h. In the same experiments, the concentration of loosely bound extracellular polymeric substances (LB-EPS) increased sharply from 54.45 to 575.57 μg/g sand in 192 h, followed by a slight decline to 505.04 μg/g sand. The increase of the bacterial cell amount along with the other two concentrations could reduce the Ks of porous media, but their relative contributions varied to a large degree during different percolation stages. At the beginning of the tests (e.g., 48 h before), bacterial cells were likely responsible for the Ks reduction of porous media because no increase was found for the other two concentrations. With the accumulation of cells and EPS production from 48 to 144 h, both were important for the reduction of Ks. However, in the late period of percolation tests from 144 to 192 h, LB-EPS was probably responsible for the further reduction of Ks, as the bacterial cell amount and TB-EPS concentration decreased. Quantitative contributions of bacterial cell amount and the two types of extracellular polymers to Ks reductions were also evaluated.

  8. Interspecies communication between pathogens and immune cells via bacterial membrane vesicles

    Directory of Open Access Journals (Sweden)

    Katerina S Jurkoshek

    2016-11-01

    Full Text Available The production of extracellular vesicles is a universal mechanism for intercellular communication that is conserved across kingdoms. Prokaryotes secrete 50–250 nm membrane vesicles (MVs in a manner that is regulated by environmental stress and is thought to promote survival. Since many types of host-derived stress are encountered during infection, this implies an important role for MV secretion in bacterial pathogenesis. Accordingly, MVs produced by gram-positive and gram-negative pathogens contain toxins, virulence factors, and other molecules that promote survival in the host. However, recent studies have also shown that bacterial MVs are enriched for molecules that stimulate innate and adaptive immune responses. As an example, MVs may serve multiple, important roles in regulating the host response to Mycobacterium tuberculosis (Mtb, an intracellular pathogen that infects lung macrophages and resides within modified phagosomes. Previously, we demonstrated that Mtb secretes MVs during infection that may regulate infected and uninfected immune cells. Our present data demonstrates that Mtb MVs inhibit the functions of macrophages and T cells, but promote MHC-II antigen presentation by dendritic cells. We conclude that bacterial MVs serve dual and opposing roles in the activation of and defense against host immune responses to Mtb and other bacterial pathogens. We also propose that MV secretion is a central mechanism for interspecies communication between bacteria and host cells during infection.

  9. Bacterial vaginosis (clue cell-positive discharge) : diagnostic, ultra-structural and therapeutic aspects

    NARCIS (Netherlands)

    W.I. van der Meijden (Willem)

    1987-01-01

    textabstractThis thesis deals with several aspects of (abnormal) vaginal discharge, focusing especially on clue cell-positive discharge (bacterial vaginosis, nonspecific vaginitis). It reports data on epidemiology and clinical features, pathogenesis, and treatment of this vaginal disease entity, as

  10. Influence of setup and carbon source on the bacterial community of biocathodes in microbial electrolysis cells

    NARCIS (Netherlands)

    Croesea, E.; Jeremiasse, A.W.; Marshall, I.P.G.; Spormann, A.M.; Euverink, G.J.W.; Geelhoed, J.S.; Stams, A.J.M.; Plugge, C.M.

    2014-01-01

    The microbial electrolysis cell (MEC) biocathode has shown great potential as alternative for expensive metals as catalyst for H2 synthesis. Here, the bacterial communities at the biocathode of five hydrogen producing MECs using molecular techniques were characterized. The setups differed in design

  11. Influence of setup and carbon source on the bacterial community of biocathodes in microbial electrolysis cells

    NARCIS (Netherlands)

    Croese, Elsemiek; Jeremiasse, Adriaan W.; Marshall, Ian P.G.; Spormann, Alfred M.; Euverink, Gert-Jan W.; Geelhoed, Jeanine S.; Stams, Alfons J.M.; Plugge, Caroline M.

    2014-01-01

    The microbial electrolysis cell (MEC) biocathode has shown great potential as alternative for expensive metals as catalyst for H2synthesis. Here, the bacterial communities at the biocathode of five hydrogen producing MECs using molecular techniques were characterized. The setups differed in design (

  12. Quantification of bioavailable chlortetracycline in pig feces using a bacterial whole-cell biosensor

    DEFF Research Database (Denmark)

    Hansen, L. H.; Aarestrup, Frank Møller; Sørensen, S. J.

    2002-01-01

    Bacterial whole-cell biosensors were used to measure the concentration of chlortetracycline (CTC) in the feces of pigs. In this study, the Escherichia coli biosensor used has a detection limit of 0.03 mg/kg CTC in pig feces. The tetracycline concentration was correlated with the appearance...

  13. Increased electrical output when a bacterial ABTS oxidizer is used in a microbial fuel cell

    Science.gov (United States)

    Microbial fuel cells (MFCs) are a technology that provides electrical energy from the microbial oxidation of organic compounds. Most MFCs use oxygen as the oxidant in the cathode chamber. The present study examined the formation in culture of an unidentified bacterial oxidant and investigated the ...

  14. The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes.

    Science.gov (United States)

    Chindera, Kantaraja; Mahato, Manohar; Sharma, Ashwani Kumar; Horsley, Harry; Kloc-Muniak, Klaudia; Kamaruzzaman, Nor Fadhilah; Kumar, Satish; McFarlane, Alexander; Stach, Jem; Bentin, Thomas; Good, Liam

    2016-03-21

    To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

  15. Quantification of bioavailable chlortetracycline in pig feces using a bacterial whole-cell biosensor

    DEFF Research Database (Denmark)

    Hansen, Lars Hestbjerg; Aarestrup, Frank; Sørensen, Søren Johannes

    2002-01-01

    Bacterial whole-cell biosensors were used to measure the concentration of chlortetracycline (CTC) in the feces of pigs. In this study, the Escherichia coli biosensor used has a detection limit of 0.03 mg/kg CTC in pig feces. The tetracycline concentration was correlated with the appearance...

  16. Soluble triggering receptor expressed on myeloid cells 1: a biomarker for bacterial meningitis

    NARCIS (Netherlands)

    R.M. Determann; M. Weisfelt; J. de Gans; A. van der Ende; M.J. Schultz; D. van de Beek

    2006-01-01

    Objective: To evaluate whether soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) in CSF can serve as a biomarker for the presence of bacterial meningitis and outcome in patients with this disease. Design: Retrospective study of diagnostic accuracy. Setting and patients: CSF was coll

  17. Bacterial cell wall preservation during organic matter diagenesis in sediments off Peru

    DEFF Research Database (Denmark)

    Lomstein, Bente Aagaard; Niggemann, Jutta; Jørgensen, Bo Barker;

    BACTERIAL CELL WALL PRESERVATION DURING ORGANIC MATTER DIAGENESIS IN SEDIMENTS OFF PERU The spatial distribution of total hydrolysable amino acids, total hydrolysable amino sugars and amino acid enantiomers (D- and L-forms) were investigated in surface sediments at 20 stations in the Peru margin: 9...

  18. Arthrobacter Species as a Prey Cell Reservoir for Nonobligate Bacterial Predators in Soil

    Science.gov (United States)

    1989-01-01

    species as a prey cell reservoir for nonobligate bacterial predators in soil. Can. J. Microbiol. 35 : 559--564, tine investigation at etc entreprise sur...8217 .Stieprom tice ~s species and Bad//uhs instead of’ A. glohifrbrmis. to provide 1 .0 mng/g soil did not nivoie from the soil did not interfecre because

  19. Bacterial toxins fuel disease progression in cutaneous T-cell lymphoma

    DEFF Research Database (Denmark)

    Willerslev-Olsen, Andreas; Krejsgaard, Thorbjørn; Lindahl, Lise M

    2013-01-01

    In patients with cutaneous T-cell lymphoma (CTCL) bacterial infections constitute a major clinical problem caused by compromised skin barrier and a progressive immunodeficiency. Indeed, the majority of patients with advanced disease die from infections with bacteria, e.g., Staphylococcus aureus...

  20. Disinfection byproduct formation from chlorination of pure bacterial cells and pipeline biofilms.

    Science.gov (United States)

    Wang, Jun-Jian; Liu, Xin; Ng, Tsz Wai; Xiao, Jie-Wen; Chow, Alex T; Wong, Po Keung

    2013-05-15

    Disinfection byproduct (DBP) formation is commonly attributed to the reaction between natural organic matters and disinfectants, yet few have considered the contribution from disinfecting bacterial materials - the essential process of water disinfection. Here, we explored the DBP formation from chlorination and chloramination of Escherichia coli and found that most selected DBPs were detectable, including trihalomethanes, haloacetonitriles, chloral hydrate, chloropicrin, and 1,1,1-trichloro-2-propanone. A positive correlation (P = 0.08-0.09) between DBP formation and the log reduction of E. coli implied that breaking down of bacterial cells released precursors for DBP formation. As Pseudomonas aeruginosa is a dominant bacterial species in pipeline biofilms, the DBP formation potentials (DBPFPs) from its planktonic cells and biofilms were characterized. Planktonic cells formed 7-11 times greater trihalomethanes per carbon of those from biofilms but significantly lower (P disinfection of bacterial planktonic cells in source water and ex situ reaction between biofilms and residual chlorine in pipeline networks as hitherto unknown DBP sources in drinking water.

  1. Bacterial Cell Surface Adsorption of Rare Earth Elements

    Science.gov (United States)

    Jiao, Y.; Park, D.; Reed, D.; Fujita, Y.; Yung, M.; Anderko, A.; Eslamimanesh, A.

    2015-12-01

    Rare earth elements (REE) play a critical role in many emerging clean energy technologies, including high-power magnets, wind turbines, solar panels, hybrid/electric vehicle batteries and lamp phosphors. In order to sustain demand for such technologies given current domestic REE shortages, there is a need to develop new approaches for ore processing/refining and recycling of REE-containing materials. To this end, we have developed a microbially-mediated bioadsorption strategy with application towards enrichment of REE from complex mixtures. Specifically, the bacterium Caulobacter crescentus was genetically engineered to display lanthanide binding tags (LBTs), short peptides that possess high affinity and specificity for rare earth elements, on its cell surface S-layer protein. Under optimal conditions, LBT-displayed cells adsorbed greater than 5-fold more REE than control cells lacking LBTs. Competition binding experiments with a selection of REEs demonstrated that our engineered cells could facilitate separation of light- from heavy- REE. Importantly, binding of REE onto our engineered strains was much more favorable compared to non-REE metals. Finally, REE bound to the cell surface could be stripped off using citrate, providing an effective and non-toxic REE recovery method. Together, this data highlights the potential of our approach for selective REE enrichment from REE containing mixtures.

  2. Phase resetting reveals network dynamics underlying a bacterial cell cycle.

    Directory of Open Access Journals (Sweden)

    Yihan Lin

    Full Text Available Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS.

  3. Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture.

    Science.gov (United States)

    Little, Lauren E; Dane, Karen Y; Daugherty, Patrick S; Healy, Kevin E; Schaffer, David V

    2011-02-01

    Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.

  4. In situ probing the interior of single bacterial cells at nanometer scale

    Science.gov (United States)

    Liu, Boyin; Hemayet Uddin, Md; Ng, Tuck Wah; Paterson, David L.; Velkov, Tony; Li, Jian; Fu, Jing

    2014-10-01

    We report a novel approach to probe the interior of single bacterial cells at nanometre resolution by combining focused ion beam (FIB) and atomic force microscopy (AFM). After removing layers of pre-defined thickness in the order of 100 nm on the target bacterial cells with FIB milling, AFM of different modes can be employed to probe the cellular interior under both ambient and aqueous environments. Our initial investigations focused on the surface topology induced by FIB milling and the hydration effects on AFM measurements, followed by assessment of the sample protocols. With fine-tuning of the process parameters, in situ AFM probing beneath the bacterial cell wall was achieved for the first time. We further demonstrate the proposed method by performing a spatial mapping of intracellular elasticity and chemistry of the multi-drug resistant strain Klebsiella pneumoniae cells prior to and after it was exposed to the ‘last-line’ antibiotic polymyxin B. Our results revealed increased stiffness occurring in both surface and interior regions of the treated cells, suggesting loss of integrity of the outer membrane from polymyxin treatments. In addition, the hydrophobicity measurement using a functionalized AFM tip was able to highlight the evident hydrophobic portion of the cell such as the regions containing cell membrane. We expect that the proposed FIB-AFM platform will help in gaining deeper insights of bacteria-drug interactions to develop potential strategies for combating multi-drug resistance.

  5. Plasmonic imaging of protein interactions with single bacterial cells.

    Science.gov (United States)

    Syal, Karan; Wang, Wei; Shan, Xiaonan; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

    2015-01-15

    Quantifying the interactions of bacteria with external ligands is fundamental to the understanding of pathogenesis, antibiotic resistance, immune evasion, and mechanism of antimicrobial action. Due to inherent cell-to-cell heterogeneity in a microbial population, each bacterium interacts differently with its environment. This large variability is washed out in bulk assays, and there is a need of techniques that can quantify interactions of bacteria with ligands at the single bacterium level. In this work, we present a label-free and real-time plasmonic imaging technique to measure the binding kinetics of ligand interactions with single bacteria, and perform statistical analysis of the heterogeneity. Using the technique, we have studied interactions of antibodies with single Escherichia coli O157:H7 cells and demonstrated a capability of determining the binding kinetic constants of single live bacteria with ligands, and quantify heterogeneity in a microbial population.

  6. Human dental pulp stem cell behavior using natural nanotolith/bacterial cellulose scaffolds for regenerative medicine.

    Science.gov (United States)

    Olyveira, Gabriel Molina; Acasigua, Gerson Arisoly Xavier; Costa, Ligia Maria Manzine; Scher, Cristiane Regina; Xavier Filho, Lauro; Pranke, Patricia Helena Lucas; Basmaji, Pierre

    2013-08-01

    Adhesion and Viability study with human dental pulp stem cell using natural nanotolith/bacterial cellulose scaffolds for regenerative medicine are presented at first time in this work. Nanotolith, are osteoinductors, i.e., they stimulate bone regeneration, enabling higher cells migration for bone tissue regeneration formation. This is mainly because nanotoliths are rich minerals present in the internal ear of bony fish. In addition, are part of a system which acts as a depth sensor and balance, acting as a sound vibrations detector and considered essential for the bone mineralization process, as in hydroxiapatites. Nanotoliths influence in bacterial cellulose was analyzed using transmission infrared spectroscopy (FTIR). Results shows that fermentation process and nanotoliths agglomeration decrease initial human dental pulp stem cell adhesion however tested bionanocomposite behavior has cell viability increase over time.

  7. Bacterial conjugation in the cytoplasm of mouse cells.

    NARCIS (Netherlands)

    Lim, Y.M.; Groof, A.J.C. de; Bhattacharjee, M.K.; Figurski, D.H.; Schon, E.A.

    2008-01-01

    Intracellular pathogenic organisms such as salmonellae and shigellae are able to evade the effects of many antibiotics because the drugs are not able to penetrate the plasma membrane. In addition, these bacteria may be able to transfer genes within cells while protected from the action of drugs. The

  8. Pseudomonas aeruginosa serA Gene Is Required for Bacterial Translocation through Caco-2 Cell Monolayers

    Science.gov (United States)

    Yasuda, Masashi; Nagata, Syouya; Yamane, Satoshi; Kunikata, Chinami; Kida, Yutaka; Kuwano, Koichi; Suezawa, Chigusa; Okuda, Jun

    2017-01-01

    To specify critical factors responsible for Pseudomonas aeruginosa penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to P. aeruginosa PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the serA mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the serA gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type P. aeruginosa PAO1 strain. Together, these results suggest that serA is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Oral administration of L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by P. aeruginosa through inhibition of serA function. PMID:28046014

  9. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    Directory of Open Access Journals (Sweden)

    Anne-lie Ståhl

    2015-02-01

    Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  10. Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells.

    Science.gov (United States)

    Pan, Jian-yi; Zhang, Yue-ling; Wang, San-ying; Peng, Xuan-xian

    2008-01-01

    Hemocyanins are multi-functional proteins, although they are well known to be respiratory proteins of invertebrate to date. In the present study, the agglutination ability of two oligomers of hemocyanin, hexamer and dodecamer, with pathogenic bacteria and red blood cells (RBCs) is investigated in pacific white shrimp, Litopenaeus vannamei. Hexameric hemocyanin exhibits an extremely high stability even in the absence of Ca(2+) and in alkaline pH. Dodecamer (di-hexamer) is easily dissociated into hexamers in unphysiological conditions. Hexamer and dodecamer are interchanged reciprocally with environmental conditions. Both oligomers can bind to bacteria and RBCs, but agglutination is observed only using dodecamer but not using hexamer in agglutination assay. However, the agglutination is detected when hexamer is utilized in the presence of antiserum against hemocyanin. These results indicate that dodecamer of hemocyanin is required for agglutination with bacteria and RBCs. It can be logically inferred that there is only one carbohydrate-binding site to bacterial cells and RBCs in the hexamer, while at least two sites in the dodecamer. Our finding has provided new insights into structural-functional relationship of hemocyanin.

  11. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    Directory of Open Access Journals (Sweden)

    Chew Chieng Yeo

    2016-02-01

    Full Text Available Toxin-antitoxin (TA systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  12. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    Science.gov (United States)

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-19

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies.

  13. Mutagenic effect of accelerated heavy ions on bacterial cells

    Science.gov (United States)

    Boreyko, A. V.; Krasavin, E. A.

    2011-11-01

    The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac-, ton B-, col B) mutations for Esherichia coli cells and reverse his- → His+ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear "dose-effect" dependences are typical. The quadratic character of mutagenesis dose curves is determined by the "interaction" of two independent "hitting" events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific

  14. Chemical communication of antibiotic resistance by a highly resistant subpopulation of bacterial cells.

    Directory of Open Access Journals (Sweden)

    Omar M El-Halfawy

    Full Text Available The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.

  15. 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.

  16. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    Science.gov (United States)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  17. Cell fate regulation governed by a repurposed bacterial histidine kinase.

    Directory of Open Access Journals (Sweden)

    W Seth Childers

    2014-10-01

    Full Text Available One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  18. Analysis of gene expression levels in individual bacterial cells without image segmentation

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, In Hae; Son, Minjun [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States); Hagen, Stephen J., E-mail: sjhagen@ufl.edu [Physics Department, University of Florida, P.O. Box 118440, Gainesville, FL 32611-8440 (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  19. Bacterial ‘Cell’ Phones: Do cell phones carry potential pathogens?

    Directory of Open Access Journals (Sweden)

    Kiran Chawla

    2009-05-01

    Full Text Available Cell phones are important companions for professionals especially health care workers (HCWs for better communication in hospital. The present study compared the nature of the growth of potentially pathogenic bacterial flora on cell phones in hospital and community. 75% cell phones from both the categories grew at least one potentially pathogenic organism. Cell phones from HCWs grew significantly more potential pathogens like MRSA (20%, Acinetobacter species (5%, Pseudomonas species (2.5% as compared to the non HCWs. 97.5% HCWs use their cell phone in the hospital, 57.5% never cleaned their cell phone and 20% admitted that they did not wash their hands before or after attending patients, although majority (77.5% knows that cell phones can have harmful colonization and act as vector for nosocomial infections. It is recommended, therefore, that cell phones in the hospital should be regularly decontaminated. Moreover, utmost emphasis needs to be paid to hand washing practices among HCWs.

  20. Bacterial glycosidases for the production of universal red blood cells

    DEFF Research Database (Denmark)

    Liu, Qiyong P; Sulzenbacher, Gerlind; Yuan, Huaiping;

    2007-01-01

    Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating...... of the alpha-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions....

  1. Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count methods.

    Science.gov (United States)

    Le Blay, Gwenaëlle; Fliss, Ismaïl; Lacroix, Christophe

    2004-11-01

    Different methods are used to study bacterial adhesion to intestinal epithelial cells, which is an important step in pathogenic infection as well as in probiotic colonization of the intestinal tract. The aim of this study was to compare the ELISA-based method with more conventional plate count and radiolabeling methods for bacterial adhesion detection. An ELISA-based assay was optimized for the detection of Bifidobacterium longum and Escherichia coli O157:H7, which are low and highly adherent bacteria, respectively. In agreement with previous investigations, a percentage of adhesion below 1% was obtained for B. longum with ELISA. However, high nonspecific background and low positive signals were measured due to the use of polyclonal antibodies and the low adhesion capacity with this strain. In contrast, the ELISA-based method developed for E. coli adhesion detected a high adhesion percentage (15%). For this bacterium the three methods tested gave similar results for the highest bacterial concentrations (6.8 Log CFU added bacteria/well). However, differences among methods increased with the addition of decreased bacterial concentration due to different detection thresholds (5.9, 5.6 and 2.9 Log CFU adherent bacteria/well for radioactivity, ELISA and plate count methods, respectively). The ELISA-based method was shown to be a good predictor for bacterial adhesion compared to the radiolabeling method when good quality specific antibodies were used. This technique is convenient and allows handling of numerous samples.

  2. Predominance of single bacterial cells in composting bioaerosols

    Science.gov (United States)

    Galès, Amandine; Bru-Adan, Valérie; Godon, Jean-Jacques; Delabre, Karine; Catala, Philippe; Ponthieux, Arnaud; Chevallier, Michel; Birot, Emmanuel; Steyer, Jean-Philippe; Wéry, Nathalie

    2015-04-01

    Bioaerosols emitted from composting plants have become an issue because of their potential harmful impact on public or workers' health. Accurate knowledge of the particle-size distribution in bioaerosols emitted from open-air composting facilities during operational activity is a requirement for improved modeling of air dispersal. In order to investigate the aerodynamic diameter of bacteria in composting bioaerosols this study used an Electrical Low Pressure Impactor for sampling and quantitative real-time PCR for quantification. Quantitative PCR results show that the size of bacteria peaked between 0.95 μm and 2.4 μm and that the geometric mean diameter of the bacteria was 1.3 μm. In addition, total microbial cells were counted by flow cytometry and revealed that these qPCR results corresponded to single whole bacteria. Finally, the enumeration of cultivable thermophilic microorganisms allowed us to set the upper size limit for fragments at an aerodynamic diameter of ∼0.3 μm. Particle-size distributions of microbial groups previously used to monitor composting bioaerosols were also investigated. In collected the bioaerosols, the aerodynamic diameter of the actinomycetes Saccharopolyspora rectivirgula-and-relatives and also of the fungus Aspergillus fumigatus, appeared to be consistent with a majority of individual cells. Together, this study provides the first culture-independent data on particle-size distribution of composting bioaerosols and reveals that airborne single bacteria were emitted predominantly from open-air composting facilities.

  3. A central role for carbon-overflow pathways in the modulation of bacterial cell death.

    Directory of Open Access Journals (Sweden)

    Vinai Chittezham Thomas

    2014-06-01

    Full Text Available Similar to developmental programs in eukaryotes, the death of a subpopulation of cells is thought to benefit bacterial biofilm development. However mechanisms that mediate a tight control over cell death are not clearly understood at the population level. Here we reveal that CidR dependent pyruvate oxidase (CidC and α-acetolactate synthase/decarboxylase (AlsSD overflow metabolic pathways, which are active during staphylococcal biofilm development, modulate cell death to achieve optimal biofilm biomass. Whereas acetate derived from CidC activity potentiates cell death in cells by a mechanism dependent on intracellular acidification and respiratory inhibition, AlsSD activity effectively counters CidC action by diverting carbon flux towards neutral rather than acidic byproducts and consuming intracellular protons in the process. Furthermore, the physiological features that accompany metabolic activation of cell death bears remarkable similarities to hallmarks of eukaryotic programmed cell death, including the generation of reactive oxygen species and DNA damage. Finally, we demonstrate that the metabolic modulation of cell death not only affects biofilm development but also biofilm-dependent disease outcomes. Given the ubiquity of such carbon overflow pathways in diverse bacterial species, we propose that the metabolic control of cell death may be a fundamental feature of prokaryotic development.

  4. GROWTH AND METABOLISM OF INDIVIDUAL BACTERIAL CELLS UTILIZING NANOSIMS

    Energy Technology Data Exchange (ETDEWEB)

    NEALSON, H. K.

    2007-08-03

    This work involved the use of the Nano-SIMS Instrument at Lawrence Livermore Laboratory, in an effort to utilize this unique tool for experiments in Biology. The work consisted primarily of experiments to measure in real time, C and N fixation in cyanobacteria. The work revealed a number of the difficulties in using the nano-SIMS approach with biological material, but with collaboration from a number of individuals at USC and LLNL, major progress was made. The collaborators from LLNL were from the Chemistry Group (Dr. Peter Weber), and the Biology Group (Dr. Jennifer Pett-Ridge). In addition, there were a number of other scientists involved from LLNL. The USC group consisted of Dr. K.H. Nealson, the PI on the grant, Dr. R. Popa, a postdoctoral fellow and research associate at USC, Professor Douglas Capone, and Juliet Finze, a graduate student in biology. Two major experiments were done, both of which yielded new and exciting data. (1) We studied nitrogen and carbon fixation in Anabaena, demonstrating that fixation ofN occurred rapidly in the heterocysts, and that the fixed N was transported rapidly and completely to the vegetative cells. C fixation occurred in the vegetative cells, with labeled C remaining in these cells in support of their growth and metabolism. This work was accepted in the ISME Journal (Nature Publication), and published last month. (2) We studied nitrogen and carbon fixation in Trichodesmium, a non-heterocystous cyanobacterium that also fixes nitrogen. Interestingly, the nitrogen fixation was confined to regions within the filaments that seem to be identical to the so-called cyanophycaen granules. The fixed N is then transported to other parts of the cyanobacterium, as judged by movement of the heavy N throughout the filaments. On the basis of these very exciting results, we have applied for funding from the NSF to continue the collaboration with LLNL. The results of both studies were presented in the summer of 2007 at the Gordon Research

  5. Bacterial xylanase expression in mammalian cells and transgenic mice.

    Science.gov (United States)

    Fontes, C M; Ali, S; Gilbert, H J; Hazlewood, G P; Hirst, B H; Hall, J

    1999-06-11

    The energy which simple-stomached livestock can derive from dietary plant material is limited by the lack of plant polysaccharide degrading enzymes in their gastro-intestinal (GI) tract and the inefficient microbial fermentation of such material in their hind-gut. In poultry the non-starch polysaccharides found in cereal grains can also impair normal digestive function as they form viscous gels in the GI tract inhibiting the breakdown and absorption of nutrients. The nutrition of such livestock could, therefore, be improved by the introduction of enzymes able to degrade plant polysaccharides in the small intestine. We describe the expression of a xylanase, XYLY', from the bacterium Clostridium thermocellum in mammalian cells and the exocrine pancreas of transgenic mice. The enzyme is synthesised, secreted and functionally active in the eukaryote system. This work demonstrates the feasibility of generating animals with the endogenous capacity to depolymerise the xylan component of hemi-cellulose.

  6. Some ultrastructural information on intact, living bacterial cells and related cell-wall fragments as given by FTIR

    Science.gov (United States)

    Naumann, D.

    1984-05-01

    Living bacterial cells of Staphylococcus aureus have been measured from aqueous suspensions taking advantage of the solvent subtraction capabilities of FTIR. All spectral features, between 1800-800 cm -1, of the intact cells could be measured with a reproducibility of better than ±5% when applying strict metabolic control of cell growth and a highly standardized experimental procedure prior to IR measurements. IR bands near 1745, 1656, 1547, 1240 and 1200-1000 cm -1were tentatively assigned to: CO stretching of ester groups, amide I and amide II bands of the various peptides and proteins, asymmetric stretching of phosphate groups and complex vibrational modes resulting from polysaccharidal compounds, respectively. Absorbance subtraction of IR spectra of different intact baterial cells and cell-wall preparations yielded reasonable results on structural variations accompanying: (i) cell growth, (ii) use of different growth media, (iii) chemical treatment of cells and (iv) biochemical isolation processes of cell walls from the intact cells.

  7. Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.

    Directory of Open Access Journals (Sweden)

    Stephanie N Bartley

    Full Text Available We compared exemplar strains from two hypervirulent clonal complexes, strain NMB-CDC from ST-8/11 cc and strain MC58 from ST-32/269 cc, in host cell attachment and invasion. Strain NMB-CDC attached to and invaded host cells at a significantly greater frequency than strain MC58. Type IV pili retained the primary role for initial attachment to host cells for both isolates regardless of pilin class and glycosylation pattern. In strain MC58, the serogroup B capsule was the major inhibitory determinant affecting both bacterial attachment to and invasion of host cells. Removal of terminal sialylation of lipooligosaccharide (LOS in the presence of capsule did not influence rates of attachment or invasion for strain MC58. However, removal of either serogroup B capsule or LOS sialylation in strain NMB-CDC increased bacterial attachment to host cells to the same extent. Although the level of inhibition of attachment by capsule was different between these strains, the regulation of the capsule synthesis locus by the two-component response regulator MisR, and the level of surface capsule determined by flow cytometry were not significantly different. However, the diplococci of strain NMB-CDC were shown to have a 1.89-fold greater surface area than strain MC58 by flow cytometry. It was proposed that the increase in surface area without changing the amount of anchored glycolipid capsule in the outer membrane would result in a sparser capsule and increase surface hydrophobicity. Strain NMB-CDC was shown to be more hydrophobic than strain MC58 using hydrophobicity interaction chromatography and microbial adhesion-to-solvents assays. In conclusion, improved levels of adherence of strain NMB-CDC to cell lines was associated with increased bacterial cell surface and surface hydrophobicity. This study shows that there is diversity in bacterial cell surface area and surface hydrophobicity within N. meningitidis which influence steps in meningococcal pathogenesis.

  8. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring

    KAUST Repository

    Van Nevel, S.

    2017-02-08

    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically < 1% of all bacteria). FCM measurements are reproducible with relative standard deviations below 3% and can be available within 15 min of samples arriving in the laboratory. High throughput sample processing and complete automation are feasible and FCM analysis is arguably less expensive than HPC when measuring more than 15 water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water.

  9. Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

    Directory of Open Access Journals (Sweden)

    Marc Daigneault

    Full Text Available Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+ T-cells in PBMC cultures required 'classical' CD14(+ monocytes, which enhanced T-cell activation. CD3(+ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.

  10. Codon optimization of bacterial luciferase (lux) for expression in mammalian cells.

    Science.gov (United States)

    Patterson, Stacey S; Dionisi, Hebe M; Gupta, Rakesh K; Sayler, Gary S

    2005-03-01

    Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5x10(5) vs 2.9x10(6) relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7x10(7) RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells.

  11. Convergent development of anodic bacterial communities in microbial fuel cells.

    KAUST Repository

    Yates, Matthew D

    2012-05-10

    Microbial fuel cells (MFCs) are often inoculated from a single wastewater source. The extent that the inoculum affects community development or power production is unknown. The stable anodic microbial communities in MFCs were examined using three inocula: a wastewater treatment plant sample known to produce consistent power densities, a second wastewater treatment plant sample, and an anaerobic bog sediment. The bog-inoculated MFCs initially produced higher power densities than the wastewater-inoculated MFCs, but after 20 cycles all MFCs on average converged to similar voltages (470±20 mV) and maximum power densities (590±170 mW m(-2)). The power output from replicate bog-inoculated MFCs was not significantly different, but one wastewater-inoculated MFC (UAJA3 (UAJA, University Area Joint Authority Wastewater Treatment Plant)) produced substantially less power. Denaturing gradient gel electrophoresis profiling showed a stable exoelectrogenic biofilm community in all samples after 11 cycles. After 16 cycles the predominance of Geobacter spp. in anode communities was identified using 16S rRNA gene clone libraries (58±10%), fluorescent in-situ hybridization (FISH) (63±6%) and pyrosequencing (81±4%). While the clone library analysis for the underperforming UAJA3 had a significantly lower percentage of Geobacter spp. sequences (36%), suggesting that a predominance of this microbe was needed for convergent power densities, the lower percentage of this species was not verified by FISH or pyrosequencing analyses. These results show that the predominance of Geobacter spp. in acetate-fed systems was consistent with good MFC performance and independent of the inoculum source.

  12. The effect of natural organic matter on the adsorption of mercury to bacterial cells

    Science.gov (United States)

    Dunham-Cheatham, Sarrah; Mishra, Bhoopesh; Myneni, Satish; Fein, Jeremy B.

    2015-02-01

    We investigated the ability of non-metabolizing Bacillus subtilis, Shewanella oneidensis MR-1, and Geobacter sulfurreducens bacterial species to adsorb mercury in the absence and presence of Suwanee River fulvic acid (FA). Bulk adsorption and X-ray absorption spectroscopy (XAS) experiments were conducted at three pH conditions, and the results indicate that the presence of FA decreases the extent of Hg adsorption to biomass under all of the pH conditions studied. Hg XAS results show that the presence of FA does not alter the binding environment of Hg adsorbed onto the biomass regardless of pH or FA concentration, indicating that ternary bacteria-Hg-FA complexes do not form to an appreciable extent under the experimental conditions, and that Hg binding on the bacteria is dominated by sulfhydryl binding. We used the experimental results to calculate apparent partition coefficients, Kd, for Hg under each experimental condition. The calculations yield similar coefficients for Hg onto each of the bacterial species studies, suggesting there is no significant difference in Hg partitioning between the three bacterial species. The calculations also indicate similar coefficients for Hg-bacteria and Hg-FA complexes. S XAS measurements confirm the presence of sulfhydryl sites on both the FA and bacterial cells, and demonstrate the presence of a wide range of S moieties on the FA in contrast to the bacterial biomass, whose S sites are dominated by thiols. Our results suggest that although FA can compete with bacterial binding sites for aqueous Hg, because of the relatively similar partition coefficients for the types of sorbents, the competition is not dominated by either bacteria or FA unless the concentration of one type of site greatly exceeds that of the other.

  13. B cells moderate inflammatory progression and enhance bacterial containment upon pulmonary challenge with Mycobacterium tuberculosis.

    Science.gov (United States)

    Maglione, Paul J; Xu, Jiayong; Chan, John

    2007-06-01

    Though much is known about the function of T lymphocytes in the adaptive immune response against Mycobacterium tuberculosis, comparably little is understood regarding the corresponding role of B lymphocytes. Indicating B cells as components of lymphoid neogenesis during pulmonary tuberculosis, we have identified ectopic germinal centers (GCs) in the lungs of infected mice. B cells in these pulmonary lymphoid aggregates express peanut agglutinin and GL7, two markers of GC B cells, as well as CXCR5, and migrate in response to the lymphoid-associated chemokine CXCL13 ex vivo. CXCL13 is negatively regulated by the presence of B cells, as its production is elevated in lungs of B cell-deficient (B cell(-/-)) mice. Upon aerosol with 100 CFU of M. tuberculosis Erdman, B cell(-/-) mice have exacerbated immunopathology corresponding with elevated pulmonary recruitment of neutrophils. Infected B cell(-/-) mice show increased production of IL-10 in the lungs, whereas IFN-gamma, TNF-alpha, and IL-10R remain unchanged from wild type. B cell(-/-) mice have enhanced susceptibility to infection when aerogenically challenged with 300 CFU of M. tuberculosis corresponding with elevated bacterial burden in the lungs but not in the spleen or liver. Adoptive transfer of B cells complements the phenotypes of B cell(-/-) mice, confirming a role for B cells in both modulation of the host response and optimal containment of the tubercle bacillus. As components of ectopic GCs, moderators of inflammatory progression, and enhancers of local immunity against bacterial challenge, B cells may have a greater role in the host defense against M. tuberculosis than previously thought.

  14. DBIO Best Thesis Award: Mechanics, Dynamics, and Organization of the Bacterial Cytoskeleton and Cell Wall

    Science.gov (United States)

    Wang, Siyuan

    2012-02-01

    Bacteria come in a variety of shapes. While the peptidoglycan (PG) cell wall serves as an exoskeleton that defines the static cell shape, the internal bacterial cytoskeleton mediates cell shape by recruiting PG synthesis machinery and thus defining the pattern of cell-wall synthesis. While much is known about the chemistry and biology of the cytoskeleton and cell wall, much of their biophysics, including essential aspects of the functionality, dynamics, and organization, remain unknown. This dissertation aims to elucidate the detailed biophysical mechanisms of cytoskeleton guided wall synthesis. First, I find that the bacterial cytoskeleton MreB contributes nearly as much to the rigidity of an Escherichia coli cell as the cell wall. This conclusion implies that the cytoskeletal polymer MreB applies meaningful force to the cell wall, an idea favored by theoretical modeling of wall growth, and suggests an evolutionary origin of cytoskeleton-governed cell rigidity. Second, I observe that MreB rotates around the long axis of E. coli, and the motion depends on wall synthesis. This is the first discovery of a cell-wall assembly driven molecular motor in bacteria. Third, I prove that both cell-wall synthesis and the PG network have chiral ordering, which is established by the spatial pattern of MreB. This work links the molecular structure of the cytoskeleton and of the cell wall with organismal-scale behavior. Finally, I develop a mathematical model of cytoskeleton-cell membrane interactions, which explains the preferential orientation of different cytoskeleton components in bacteria.

  15. Mucosal-associated invariant T (MAIT cells: new players in anti-bacterial immunity

    Directory of Open Access Journals (Sweden)

    James E Ussher

    2014-10-01

    Full Text Available Mucosal-associated invariant T (MAIT cells are an innate-like T cell population involved in antibacterial immunity. In humans, MAIT cells are abundant, comprising ~ 10% of the CD8+ T cell compartment in blood. They are enriched at mucosal sites and are particularly prevalent within the liver. MAIT cells are defined by the expression of a semi-invariant T cell receptor (Vα7.2-Jα33/12/20 and are restricted by the non-polymorphic, highly evolutionarily conserved MHC class Ib molecule, MR1. MR1 has recently been shown to present an unstable pyrimidine intermediate derived from a biosynthetic precursor of riboflavin; riboflavin biosynthesis occurs in many bacteria but not in humans. Consistent with this, MAIT cells are responsive to riboflavin-metabolizing bacteria, including Salmonella. In mouse models, MAIT cells have been shown to play a non-redundant role in antibacterial immunity, including against E. coli, Klebsiella pneumoniae, and Mycobacterium bovis BCG. In humans, MAIT cells are decreased in frequency in the blood of patients with tuberculosis or pneumonia, and their frequency has been inversely correlated with the risk of subsequent systemic bacterial infection in patients in intensive care. Intriguingly, MAIT cells are also depleted from the blood early in HIV infection and fail to recover with antiretroviral therapy, which may contribute to the susceptibility of patients infected with HIV to certain bacterial infections, including with non-typhoidal Salmonella. In this review we will discuss what is currently known about MAIT cells, the role that Salmonella has played in elucidating MAIT cell restriction and function, and the role MAIT cells might play in the control of Salmonella infection.

  16. Multivalent nanofibers of a controlled length: regulation of bacterial cell agglutination.

    Science.gov (United States)

    Lee, Dong-Woo; Kim, Taehoon; Park, Il-Soo; Huang, Zhegang; Lee, Myongsoo

    2012-09-12

    Control of the size and shape of molecular assemblies on the nanometer scale in aqueous solutions is very important for the regulation of biological functions. Among the well-defined supramolecular structures of organic amphiphiles, one-dimensional nanofibers have attracted much attention because of their potential applications in biocompatible materials. Although much progress has been made in the field of self-assembled nanofibers, the ability to control the fiber length remains limited. The approach for control of the fiber length presented herein overcomes this limitation through the coassembly of amphiphilic rod-coil molecules in which the crystallinity of the aromatic segment can be regulated by π-π stacking interactions. The introduction of carbohydrate segments into the fiber exterior endows the nanofibers with the ability to adhere to bacterial cells. Notably, the fiber length systematically regulates the agglutination and proliferation of bacterial cells exposed to these fibers.

  17. The interaction of bacterial magnetosomes and human liver cancer cells in vitro

    Science.gov (United States)

    Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Yue; Pan, Weidong; Song, Tao

    2017-04-01

    As the biogenic magnetic nanomaterial, bacterial magnetic nanoparticles, namely magnetosomes, provide many advantages for potential biomedical applications. As such, interactions among magnetosomes and target cells should be elucidated to develop their bioapplications and evaluate their biocompatibilities. In this study, the interaction of magnetosomes and human liver cancer HepG2 cells was examined. Prussian blue staining revealed numerous stained particles in or on the cells. Intracellular iron concentrations, measured through inductively coupled plasma optical emission spectroscopy, increased with the increasing concentration of the magnetosomes. Transmission electron microscopy images showed that magnetosomes could be internalized in cells, mainly encapsulated in membrane vesicles, such as endosomes and lysosomes, and partly found as free particles in the cytosol. Some of the magnetosomes on cellular surfaces were encapsulated through cell membrane ruffling, which is the initiating process of endocytosis. Applying low temperature treatment and using specific endocytic inhibitors, we validated that macropinocytosis and clathrin-mediated endocytosis were involved in magnetosome uptake by HepG2 cells. Consequently, we revealed the interaction and intrinsic endocytic mechanisms of magnetosomes and HepG2 cells. This study provides a basis for the further research on bacterial magnetosome applications in liver diseases.

  18. Influence of molecular noise on the growth of single cells and bacterial populations.

    Directory of Open Access Journals (Sweden)

    Mischa Schmidt

    Full Text Available During the last decades experimental studies have revealed that single cells of a growing bacterial population are significantly exposed to molecular noise. Important sources for noise are low levels of metabolites and enzymes that cause significant statistical variations in the outcome of biochemical reactions. In this way molecular noise affects biological processes such as nutrient uptake, chemotactic tumbling behavior, or gene expression of genetically identical cells. These processes give rise to significant cell-to-cell variations of many directly observable quantities such as protein levels, cell sizes or individual doubling times. In this study we theoretically explore if there are evolutionary benefits of noise for a growing population of bacteria. We analyze different situations where noise is either suppressed or where it affects single cell behavior. We consider two specific examples that have been experimentally observed in wild-type Escherichia coli cells: (i the precision of division site placement (at which molecular noise is highly suppressed and (ii the occurrence of noise-induced phenotypic variations in fluctuating environments. Surprisingly, our analysis reveals that in these specific situations both regulatory schemes [i.e. suppression of noise in example (i and allowance of noise in example (ii] do not lead to an increased growth rate of the population. Assuming that the observed regulatory schemes are indeed caused by the presence of noise our findings indicate that the evolutionary benefits of noise are more subtle than a simple growth advantage for a bacterial population in nutrient rich conditions.

  19. Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count

    NARCIS (Netherlands)

    Koop, G.; Dik, N.; Nielen, M.; Lipman, L.J.A.

    2010-01-01

    The aims of this study were to assess how different bacterial groups in bulk milk are related to bulk milk somatic cell count (SCC), bulk milk total bacterial count (TBC), and bulk milk standard plate count (SPC) and to measure the repeatability of bulk milk culturing. On 53 Dutch dairy goat farms,

  20. Instrumental analysis of bacterial cells using vibrational and emission Moessbauer spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Kamnev, Alexander A. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation)]. E-mail: aakamnev@ibppm.sgu.ru; Tugarova, Anna V. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Antonyuk, Lyudmila P. [Laboratory of Biochemistry of Plant-Bacterial Symbioses, Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, 410049 Saratov (Russian Federation); Tarantilis, Petros A. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Kulikov, Leonid A. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Perfiliev, Yurii D. [Laboratory of Nuclear Chemistry Techniques, Department of Radiochemistry, Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119992 Moscow (Russian Federation); Polissiou, Moschos G. [Laboratory of Chemistry, Department of Science, Agricultural University of Athens, 11855 Athens (Greece); Gardiner, Philip H.E. [Division of Chemistry, School of Science and Mathematics, Sheffield Hallam University, Sheffield S1 1WB (United Kingdom)

    2006-07-28

    In biosciences and biotechnology, the expanding application of physicochemical approaches using modern instrumental techniques is an efficient strategy to obtain valuable and often unique information at the molecular level. In this work, we applied a combination of vibrational (Fourier transform infrared (FTIR), FT-Raman) spectroscopic techniques, useful in overall structural and compositional analysis of bacterial cells of the rhizobacterium Azospirillum brasilense, with {sup 57}Co emission Moessbauer spectroscopy (EMS) used for sensitive monitoring of metal binding and further transformations in live bacterial cells. The information obtained, together with ICP-MS analyses for metals taken up by the bacteria, is useful in analysing the impact of the environmental conditions (heavy metal stress) on the bacterial metabolism and some differences in the heavy metal stress-induced behaviour of non-endophytic (Sp7) and facultatively endophytic (Sp245) strains. The results show that, while both strains Sp7 and Sp245 take up noticeable and comparable amounts of heavy metals from the medium (0.12 and 0.13 mg Co, 0.48 and 0.44 mg Cu or 4.2 and 2.1 mg Zn per gram of dry biomass, respectively, at a metal concentration of 0.2 mM in the medium), their metabolic responses differ essentially. Whereas for strain Sp7 the FTIR measurements showed significant accumulation of polyhydroxyalkanoates as storage materials involved in stress endurance, strain Sp245 did not show any major changes in cellular composition. Nevertheless, EMS measurements showed rapid binding of cobalt(II) by live bacterial cells (chemically similar to metal binding by dead bacteria) and its further transformation in the live cells within an hour.

  1. Biomechanical Loading Modulates Proinflammatory and Bone Resorptive Mediators in Bacterial-Stimulated PDL Cells

    Directory of Open Access Journals (Sweden)

    Andressa Vilas Boas Nogueira

    2014-01-01

    Full Text Available The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS of low (CTSL and high (CTSH magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2 and prostaglandin E2 (PGE2 was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG and receptor activator of nuclear factor kappa-B ligand (RANKL were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P<0.05 increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

  2. Targeted delivery of an ADP-ribosylating bacterial toxin into cancer cells

    Science.gov (United States)

    Zahaf , N.-I.; Lang, A. E.; Kaiser, L.; Fichter, C. D.; Lassmann, S.; McCluskey, A.; Augspach, A.; Aktories, K.; Schmidt, G.

    2017-01-01

    The actin cytoskeleton is an attractive target for bacterial toxins. The ADP-ribosyltransferase TccC3 from the insect bacterial pathogen Photorhabdus luminescence modifies actin to force its aggregation. We intended to transport the catalytic part of this toxin preferentially into cancer cells using a toxin transporter (Protective antigen, PA) which was redirected to Epidermal Growth Factor Receptors (EGFR) or to human EGF receptors 2 (HER2), which are overexpressed in several cancer cells. Protective antigen of anthrax toxin forms a pore through which the two catalytic parts (lethal factor and edema factor) or other proteins can be transported into mammalian cells. Here, we used PA as a double mutant (N682A, D683A; mPA) which cannot bind to the two natural anthrax receptors. Each mutated monomer is fused either to EGF or to an affibody directed against the human EGF receptor 2 (HER2). We established a cellular model system composed of two cell lines representing HER2 overexpressing esophageal adenocarcinomas (EACs) and EGFR overexpressing esophageal squamous cell carcinomas (ESCCs). We studied the specificity and efficiency of the re-directed anthrax pore for transport of TccC3 toxin and established Photorhabdus luminescence TccC3 as a toxin suitable for the development of a targeted toxin selectively killing cancer cells. PMID:28128281

  3. Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms.

    Science.gov (United States)

    Khasriya, Rajvinder; Sathiananthamoorthy, Sanchutha; Ismail, Salim; Kelsey, Michael; Wilson, Mike; Rohn, Jennifer L; Malone-Lee, James

    2013-07-01

    Chronic lower urinary tract symptoms (LUTS), such as urgency and incontinence, are common, especially among the elderly, but their etiology is often obscure. Recent studies of acute urinary tract infections implicated invasion by Escherichia coli into the cytoplasm of urothelial cells, with persistence of long-term bacterial reservoirs, but the role of infection in chronic LUTS is unknown. We conducted a large prospective study with eligible patients with LUTS and controls over a 3-year period, comparing routine urine cultures of planktonic bacteria with cultures of shed urothelial cells concentrated in centrifuged urinary sediments. This comparison revealed large numbers of bacteria undetected by routine cultures. Next, we typed the bacterial species cultured from patient and control sediments under both aerobic and anaerobic conditions, and we found that the two groups had complex but significantly distinct profiles of bacteria associated with their shed bladder epithelial cells. Strikingly, E. coli, the organism most responsible for acute urinary tract infections, was not the only or even the main offending pathogen in this more-chronic condition. Antibiotic protection assays with shed patient cells and in vitro infection studies using patient-derived strains in cell culture suggested that LUTS-associated bacteria are within or extremely closely associated with shed epithelial cells, which explains how routine cultures might fail to detect them. These data have strong implications for the need to rethink our common diagnoses and treatments of chronic urinary tract symptoms.

  4. Visualization of sialic acid produced on bacterial cell surfaces by lectin staining.

    Science.gov (United States)

    Kajiwara, Hitomi; Toda, Munetoyo; Mine, Toshiki; Nakada, Hiroshi; Wariishi, Hiroyuki; Yamamoto, Takeshi

    2010-01-01

    Oligosaccharides containing N-acetylneuraminic acid on the cell surface of some pathogenic bacteria are important for host-microbe interactions. N-acetylneuraminic acid (Neu5Ac) plays a major role in the pathogenicity of bacterial pathogens. For example, cell surface sialyloligosaccharide moieties of the human pathogen Haemophilus influenzae are involved in virulence and adhesion to host cells. In this study, we have established a method of visualizing Neu5Ac linked to a glycoconjugate on the bacterial cell surface based on lectin staining. Photobacterium damselae strain JT0160, known to produce a-2,6-sialyltransferase, was revealed to possess Neu5Ac by HPLC. Using the strain, a strong Sambucus sieboldiana lectin-binding signal was detected. The bacteria producing α-2,6-sialyltransferases could be divided into two groups: those with a lot of α-2,6-linked Neu5Ac on the cell surface and those with a little. In the present study, we developed a useful method for evaluating the relationship between Neu5Ac expression on the cell surface and the degree of virulence of marine bacteria.

  5. Humidity-Dependent Bacterial Cells Functional Morphometry Investigations Using Atomic Force Microscope

    Directory of Open Access Journals (Sweden)

    Hike Nikiyan

    2010-01-01

    Full Text Available The effect of a relative humidity (RH in a range of 93–65% on morphological and elastic properties of Bacillus cereus and Escherichia coli cells was evaluated using atomic force microscopy. It is shown that gradual dehumidification of bacteria environment has no significant effect on cell dimensional features and considerably decreases them only at 65% RH. The increasing of the bacteria cell wall roughness and elasticity occurs at the same time. Observed changes indicate that morphological properties of B. cereus are rather stable in wide range of relative humidity, whereas E. coli are more sensitive to drying, significantly increasing roughness and stiffness parameters at RH ≤ 84% RH. It is discussed the dependence of the response features on differences in cell wall structure of gram-positive and gram-negative bacterial cells.

  6. Temporal expression of bacterial proteins instructs host CD4 T cell expansion and Th17 development.

    Directory of Open Access Journals (Sweden)

    Seung-Joo Lee

    2012-01-01

    Full Text Available Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.

  7. Neuronal cells' behavior on polypyrrole coated bacterial nanocellulose three-dimensional (3D) scaffolds.

    Science.gov (United States)

    Muller, D; Silva, J P; Rambo, C R; Barra, G M O; Dourado, F; Gama, F M

    2013-01-01

    In this work, polypyrrole (PPy) was in situ polymerized onto the surface of bacterial nanocellulose (BNC) produced by Gluconacetobacter xylinus, by chemical oxidation in aqueous medium using ammonium persulfate. Composites (BNC/PPy) were produced with varying concentrations of pyrrole (Py). The produced BNC/PPy membranes were used as a template for the seeding of PC12 rat neuronal cells. Cell suspensions were directly seeded onto the surfaces of the BNC/PPy membranes. The Py concentration affected the behavior of neuronal cells that adhered and grew significantly more on BNC/PPy comparatively to BNC. Scanning electron microscopy (SEM) micrographs revealed that PC12 cells adhered on the surface of the BNC and BNC/PPy membranes. Conductive PPy coatings on nanofibers acting as an active interface for tissue engineering may be used to regulate cell activity through electrical stimulations.

  8. Imaging of Bacterial and Fungal Cells Using Fluorescent Carbon Dots Prepared from Carica papaya Juice.

    Science.gov (United States)

    Kasibabu, Betha Saineelima B; D'souza, Stephanie L; Jha, Sanjay; Kailasa, Suresh Kumar

    2015-07-01

    In this paper, we have described a simple hydrothermal method for preparation of fluorescent carbon dots (C-dots) using Carica papaya juice as a precursor. The synthesized C-dots show emission peak at 461 nm with a quantum yield of 7.0 %. The biocompatible nature of C-dots was confirmed by a cytotoxicity assay on E. coli. The C-dots were used as fluorescent probes for imaging of bacterial (Bacillus subtilis) and fungal (Aspergillus aculeatus) cells and emitted green and red colors under different excitation wavelengths, which indicates that the C-dots can be used as a promising material for cell imaging.

  9. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.

    Directory of Open Access Journals (Sweden)

    Sandra Schwarz

    Full Text Available Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs of Burkholderia thailandensis (B. thai in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

  10. Immobilization of cells with nitrilase activity from a thermophilic bacterial strain.

    Science.gov (United States)

    Kabaivanova, L; Dobreva, E; Dimitrov, P; Emanuilova, E

    2005-01-01

    Cells of the moderately thermophilic Bacillus sp. UG-5B strain, producing nitrilase (EC3.5.5.1), which converts nitriles directly to the corresponding acid and ammonia, were immobilized using different types of matrices and techniques. A variety of sol-gel silica hybrids were tested for entrapment and adsorption of bacterial cells as well as chemical binding on polysulphone membranes. Activation of the matrix surface with formaldehyde led to an increase in immobilization efficiency and operational stability of the biocatalysts. Among the supports screened, membranes gave the best results for enzyme activity and especially operational stability, with retention of 100% activity after eight reaction cycles.

  11. Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines☆

    Science.gov (United States)

    Alberdi, M. Pilar; Dalby, Matthew J.; Rodriguez-Andres, Julio; Fazakerley, John K.; Kohl, Alain; Bell-Sakyi, Lesley

    2012-01-01

    As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed ‘tick-only’ viruses inhabiting tick cell lines. PMID:22743047

  12. Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines.

    Science.gov (United States)

    Alberdi, M Pilar; Dalby, Matthew J; Rodriguez-Andres, Julio; Fazakerley, John K; Kohl, Alain; Bell-Sakyi, Lesley

    2012-06-01

    As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed 'tick-only' viruses inhabiting tick cell lines.

  13. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    Science.gov (United States)

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  14. Condensation of FtsZ filaments can drive bacterial cell division.

    Science.gov (United States)

    Lan, Ganhui; Daniels, Brian R; Dobrowsky, Terrence M; Wirtz, Denis; Sun, Sean X

    2009-01-06

    Forces are important in biological systems for accomplishing key cell functions, such as motility, organelle transport, and cell division. Currently, known force generation mechanisms typically involve motor proteins. In bacterial cells, no known motor proteins are involved in cell division. Instead, a division ring (Z-ring) consists of mostly FtsZ, FtsA, and ZipA is used to exerting a contractile force. The mechanism of force generation in bacterial cell division is unknown. Using computational modeling, we show that Z-ring formation results from the colocalization of FtsZ and FtsA mediated by the favorable alignment of FtsZ polymers. The model predicts that the Z-ring undergoes a condensation transition from a low-density state to a high-density state and generates a sufficient contractile force to achieve division. FtsZ GTP hydrolysis facilitates monomer turnover during the condensation transition, but does not directly generate forces. In vivo fluorescence measurements show that FtsZ density increases during division, in accord with model results. The mechanism is akin to van der Waals picture of gas-liquid condensation, and shows that organisms can exploit microphase transitions to generate mechanical forces.

  15. The periplasmic enzyme, AnsB, of Shigella flexneri modulates bacterial adherence to host epithelial cells.

    Directory of Open Access Journals (Sweden)

    Divya T George

    Full Text Available S. flexneri strains, most frequently linked with endemic outbreaks of shigellosis, invade the colonic and rectal epithelium of their host and cause severe tissue damage. Here we have attempted to elucidate the contribution of the periplasmic enzyme, L-asparaginase (AnsB to the pathogenesis of S. flexneri. Using a reverse genetic approach we found that ansB mutants showed reduced adherence to epithelial cells in vitro and attenuation in two in vivo models of shigellosis, the Caenorhabditis elegans and the murine pulmonary model. To investigate how AnsB affects bacterial adherence, we compared the proteomes of the ansB mutant with its wild type parental strain using two dimensional differential in-gel electrophoresis and identified the outer membrane protein, OmpA as up-regulated in ansB mutant cells. Bacterial OmpA, is a prominent outer membrane protein whose activity has been found to be required for bacterial pathogenesis. Overexpression of OmpA in wild type S. flexneri serotype 3b resulted in decreasing the adherence of this virulent strain, suggesting that the up-regulation of OmpA in ansB mutants contributes to the reduced adherence of this mutant strain. The data presented here is the first report that links the metabolic enzyme AnsB to S. flexneri pathogenesis.

  16. Comparison of bacterial cells and amine-functionalized abiotic surfaces as support for Pd nanoparticle synthesis

    DEFF Research Database (Denmark)

    De Corte, Simon; Bechstein, Stefanie; Lokanathan, Arcot R.

    2013-01-01

    An increasing demand for catalytic Pd nanoparticles has motivated the search for sustainable production methods. An innovative approach uses bacterial cells as support material for synthesizing Pd nanoparticles by reduction of Pd(II) with e.g. hydrogen or formate. Nevertheless, drawbacks...... nanoparticles, and that abiotic surfaces could support the Pd particle synthesis as efficiently as bacteria. In this study, we explore the possibility of replacing bacteria with amine-functionalized materials, and we compare different functionalization strategies. Pd nanoparticles formed on the support...... of microbially supported Pd catalysts are the low catalytic activity compared to conventional Pd nanocatalysts and the possible poisoning of the catalyst surface by sulfur originating from bacterial proteins. A recent study showed that amine groups were a key component in surface-supported synthesis of Pd...

  17. Modeling quorum sensing trade-offs between bacterial cell density and system extension from open boundaries

    Science.gov (United States)

    Marenda, Mattia; Zanardo, Marina; Trovato, Antonio; Seno, Flavio; Squartini, Andrea

    2016-12-01

    Bacterial communities undergo collective behavioural switches upon producing and sensing diffusible signal molecules; a mechanism referred to as Quorum Sensing (QS). Exemplarily, biofilm organic matrices are built concertedly by bacteria in several environments. QS scope in bacterial ecology has been debated for over 20 years. Different perspectives counterpose the role of density reporter for populations to that of local environment diffusivity probe for individual cells. Here we devise a model system where tubes of different heights contain matrix-embedded producers and sensors. These tubes allow non-limiting signal diffusion from one open end, thereby showing that population spatial extension away from an open boundary can be a main critical factor in QS. Experimental data, successfully recapitulated by a comprehensive mathematical model, demonstrate how tube height can overtake the role of producer density in triggering sensor activation. The biotic degradation of the signal is found to play a major role and to be species-specific and entirely feedback-independent.

  18. Behind the lines–actions of bacterial type III effector proteins in plant cells

    Science.gov (United States)

    Büttner, Daniela

    2016-01-01

    Pathogenicity of most Gram-negative plant-pathogenic bacteria depends on the type III secretion (T3S) system, which translocates bacterial effector proteins into plant cells. Type III effectors modulate plant cellular pathways to the benefit of the pathogen and promote bacterial multiplication. One major virulence function of type III effectors is the suppression of plant innate immunity, which is triggered upon recognition of pathogen-derived molecular patterns by plant receptor proteins. Type III effectors also interfere with additional plant cellular processes including proteasome-dependent protein degradation, phytohormone signaling, the formation of the cytoskeleton, vesicle transport and gene expression. This review summarizes our current knowledge on the molecular functions of type III effector proteins with known plant target molecules. Furthermore, plant defense strategies for the detection of effector protein activities or effector-triggered alterations in plant targets are discussed. PMID:27526699

  19. Enhancement of bacterial denitrification for nitrate removal in groundwater with electrical stimulation from microbial fuel cells

    Science.gov (United States)

    Zhang, Baogang; Liu, Ye; Tong, Shuang; Zheng, Maosheng; Zhao, Yinxin; Tian, Caixing; Liu, Hengyuan; Feng, Chuanping

    2014-12-01

    Electricity generated from the microbial fuel cell (MFC) is applied to the bioelectrical reactor (BER) directly as electrical stimulation means for enhancement of bacterial denitrification to remove nitrate effectively from groundwater. With maximum power density of 502.5 mW m-2 and voltage outputs ranging from 500 mV to 700 mV, the nitrate removal is accelerated, with less intermediates accumulation, compared with control sets without electrical stimulation. Denitrification bacteria proliferations and activities are promoted as its number and Adenosine-5'-triphosphate (ATP) concentration increased one order of magnitude (3.5 × 107 in per milliliter biofilm solution) and about 1.5 folds, respectively. Effects of electricity from MFCs on enhancement of bacterial behaviors are demonstrated for the first time. These results indicate that MFCs can be applied in the in-situ bioremediation of nitrate polluted groundwater for efficiency improvement.

  20. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter.

    Science.gov (United States)

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei

    2015-04-01

    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

  1. INFLUENCE OF THE PHYSICAL STATE OF THE BACTERIAL CELL MEMBRANE UPON THE RATE OF RESPIRATION.

    Science.gov (United States)

    HENNEMAN, D H; UMBREIT, W W

    1964-06-01

    Henneman, Dorothy H. (Rutgers, The State University, New Brunswick, N.J.), and W. W. Umbreit. Influence of the physical state of the bacterial cell membrane upon the rate of respiration. J. Bacteriol. 87:1274-1280. 1964.-NaCl and KCl in concentrations of the order of 0.2 to 0.5 m inhibit the respiration of Escherichia coli B and other gram-negative organisms. Cell-free enzymes concerned in respiration and prepared from the same organisms are not inhibited by these salts, whereas these same enzymes tested in intact cells are. The physical state of the cell membrane appears to be a factor controlling its respiratory activity.

  2. Computer models of bacterial cells: from generalized coarsegrained to genome-specific modular models

    Science.gov (United States)

    Nikolaev, Evgeni V.; Atlas, Jordan C.; Shuler, Michael L.

    2006-09-01

    We discuss a modular modelling framework to rapidly develop mathematical models of bacterial cells that would explicitly link genomic details to cell physiology and population response. An initial step in this approach is the development of a coarse-grained model, describing pseudo-chemical interactions between lumped species. A hybrid model of interest can then be constructed by embedding genome-specific detail for a particular cellular subsystem (e.g. central metabolism), called here a module, into the coarse-grained model. Specifically, a new strategy for sensitivity analysis of the cell division limit cycle is introduced to identify which pseudo-molecular processes should be delumped to implement a particular biological function in a growing cell (e.g. ethanol overproduction or pathogen viability). To illustrate the modeling principles and highlight computational challenges, the Cornell coarsegrained model of Escherichia coli B/r-A is used to benchmark the proposed framework.

  3. Long acting β2-agonist and corticosteroid restore airway glandular cell function altered by bacterial supernatant

    Directory of Open Access Journals (Sweden)

    Nawrocki-Raby Béatrice

    2010-01-01

    Full Text Available Abstract Background Staphylococcus aureus releases virulence factors (VF that may impair the innate protective functions of airway cells. The aim of this study was to determine whether a long-acting β2 adrenergic receptor agonist (salmeterol hydroxynaphthoate, Sal combined with a corticosteroid (fluticasone propionate, FP was able to regulate ion content and cytokine expression by airway glandular cells after exposure to S. aureus supernatant. Methods A human airway glandular cell line was incubated with S. aureus supernatant for 1 h and then treated with the combination Sal/FP for 4 h. The expression of actin and CFTR proteins was analyzed by immunofluorescence. Videomicroscopy was used to evaluate chloride secretion and X-ray microanalysis to measure the intracellular ion and water content. The pro-inflammatory cytokine expression was assessed by RT-PCR and ELISA. Results When the cells were incubated with S. aureus supernatant and then with Sal/FP, the cellular localisation of CFTR was apical compared to the cytoplasmic localisation in cells incubated with S. aureus supernatant alone. The incubation of airway epithelial cells with S. aureus supernatant reduced by 66% the chloride efflux that was fully restored by Sal/FP treatment. We also observed that Sal/FP treatment induced the restoration of ion (Cl and S and water content within the intracellular secretory granules of airway glandular cells and reduced the bacterial supernatant-dependent increase of pro-inflammatory cytokines IL8 and TNFα. Conclusions Our results demonstrate that treatment with the combination of a corticosteroid and a long-acting β2 adrenergic receptor agonist after bacterial infection restores the airway glandular cell function. Abnormal mucus induced by defective ion transport during pulmonary infection could benefit from treatment with a combination of β2 adrenergic receptor agonist and glucocorticoid.

  4. Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations.

    Science.gov (United States)

    Helfrich, Stefan; Pfeifer, Eugen; Krämer, Christina; Sachs, Christian Carsten; Wiechert, Wolfgang; Kohlheyer, Dietrich; Nöh, Katharina; Frunzke, Julia

    2015-11-01

    Almost all bacterial genomes contain DNA of viral origin, including functional prophages or degenerated phage elements. A frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (SPI) even in the absence of an external stimulus. In this study, we have analyzed SPI of the large, degenerated prophage CGP3 (187 kbp), which is integrated into the genome of the Gram-positive Corynebacterium glutamicum ATCC 13032. Time-lapse fluorescence microscopy of fluorescent reporter strains grown in microfluidic chips revealed the sporadic induction of the SOS response as a prominent trigger of CGP3 SPI but also displayed a considerable fraction (∼30%) of RecA-independent SPI. Whereas approx. 20% of SOS-induced cells recovered from this stress and resumed growth, the spontaneous induction of CGP3 always led to a stop of growth and likely cell death. A carbon source starvation experiment clearly emphasized that SPI only occurs in actively proliferating cells, whereas sporadic SOS induction was still observed in resting cells. These data highlight the impact of sporadic DNA damage on the activity of prophage elements and provide a time-resolved, quantitative description of SPI as general phenomenon of bacterial populations.

  5. Total bacterial count and somatic cell count in refrigerated raw milk stored in communal tanks

    Directory of Open Access Journals (Sweden)

    Edmar da Costa Alves

    2014-09-01

    Full Text Available The current industry demand for dairy products with extended shelf life has resulted in new challenges for milk quality maintenance. The processing of milk with high bacterial counts compromises the quality and performance of industrial products. The study aimed to evaluate the total bacteria counts (TBC and somatic cell count (SCC in 768 samples of refrigerated raw milk, from 32 communal tanks. Samples were collected in the first quarter of 2010, 2011, 2012 and 2013 and analyzed by the Laboratory of Milk Quality - LQL. Results showed that 62.5%, 37.5%, 15.6% and 27.1% of the means for TBC in 2010, 2011, 2012 and 2013, respectively, were above the values established by legislation. However, we observed a significant reduction in the levels of total bacterial count (TBC in the studied periods. For somatic cell count, 100% of the means indicated values below 600.000 cells/mL, complying with the actual Brazilian legislation. The values found for the somatic cell count suggests the adoption of effective measures for the sanitary control of the herd. However, the results must be considered with caution as it highlights the need for quality improvements of the raw material until it achieves reliable results effectively.

  6. Increased expression of BDNF and proliferation of dentate granule cells after bacterial meningitis.

    Science.gov (United States)

    Tauber, Simone C; Stadelmann, Christine; Spreer, Annette; Brück, Wolfgang; Nau, Roland; Gerber, Joachim

    2005-09-01

    Proliferation and differentiation of neural progenitor cells is increased after bacterial meningitis. To identify endogenous factors involved in neurogenesis, expression of brain-derived neurotrophic factor (BDNF), TrkB, nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) was investigated. C57BL/6 mice were infected by intracerebral injection of Streptococcus pneumoniae. Mice were killed 30 hours later or treated with ceftriaxone and killed 4 days after infection. Hippocampal BDNF mRNA levels were increased 2.4-fold 4 days after infection (p = 0.026). Similarly, BDNF protein levels in the hippocampal formation were higher in infected mice than in control animals (p = 0.0003). This was accompanied by an elevated proliferation of dentate granule cells (p = 0.0002). BDNF protein was located predominantly in the hippocampal CA3/4 area and the hilus of the dentate gyrus. The density of dentate granule cells expressing the BDNF receptor TrkB as well as mRNA levels of TrkB in the hippocampal formation were increased 4 days after infection (p = 0.027 and 0.0048, respectively). Conversely, NGF mRNA levels at 30 hours after infection were reduced by approximately 50% (p = 0.004). No significant changes in GDNF expression were observed. In conclusion, increased synthesis of BDNF and TrkB suggests a contribution of this neurotrophic factor to neurogenesis after bacterial meningitis.

  7. Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptides.

    Science.gov (United States)

    Malanovic, Nermina; Lohner, Karl

    2016-05-01

    A number of cationic antimicrobial peptides, effectors of innate immunity, are supposed to act at the cytoplasmic membrane leading to permeabilization and eventually membrane disruption. Thereby, interaction of antimicrobial peptides with anionic membrane phospholipids is considered to be a key factor in killing of bacteria. Recently, evidence was provided that killing takes place only when bacterial cell membranes are completely saturated with peptides. This adds to an ongoing debate, which role cell wall components such as peptidoglycan, lipoteichoic acid and lipopolysaccharide may play in the killing event, i.e. if they rather entrap or facilitate antimicrobial peptides access to the cytoplasmic membrane. Therefore, in this review we focused on the impact of Gram-positive cell wall components for the mode of action and activity of antimicrobial peptides as well as in innate immunity. This led us to conclude that interaction of antimicrobial peptides with peptidoglycan may not contribute to a reduction of their antimicrobial activity, whereas interaction with anionic lipoteichoic acids may reduce the local concentration of antimicrobial peptides on the cytoplasmic membrane necessary for sufficient destabilization of the membranes and bacterial killing. Further affinity studies of antimicrobial peptides toward the different cell wall as well as membrane components will be needed to address this problem on a quantitative level. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

  8. Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Van Der Hofstadt, M. [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Hüttener, M.; Juárez, A. [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Departament de Microbiologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona (Spain); Gomila, G., E-mail: ggomila@ibecbarcelona.eu [Institut de Bioenginyeria de Catalunya (IBEC), C/ Baldiri i Reixac 11-15, 08028 Barcelona (Spain); Departament d' Electronica, Universitat de Barcelona, C/ Marti i Franqués 1, 08028 Barcelona (Spain)

    2015-07-15

    With the use of the atomic force microscope (AFM), the Nanomicrobiology field has advanced drastically. Due to the complexity of imaging living bacterial processes in their natural growing environments, improvements have come to a standstill. Here we show the in situ nanoscale imaging of the growth and division of single bacterial cells on planar substrates with the atomic force microscope. To achieve this, we minimized the lateral shear forces responsible for the detachment of weakly adsorbed bacteria on planar substrates with the use of the so called dynamic jumping mode with very soft cantilever probes. With this approach, gentle imaging conditions can be maintained for long periods of time, enabling the continuous imaging of the bacterial cell growth and division, even on planar substrates. Present results offer the possibility to observe living processes of untrapped bacteria weakly attached to planar substrates. - Highlights: • Gelatine coatings used to weakly attach bacterial cells onto planar substrates. • Use of the dynamic jumping mode as a non-perturbing bacterial imaging mode. • Nanoscale resolution imaging of unperturbed single living bacterial cells. • Growth and division of single bacteria cells on planar substrates observed.

  9. A STUDY ON BACTERIAL CONTAMINATION OF CELL PHONES OF HEALTH CARE WORKERS (HCW AT KIMS HOSPITAL, AMALAPURAM

    Directory of Open Access Journals (Sweden)

    Padmaja

    2014-04-01

    Full Text Available A random 100 samples of cell phones of HCWs workers and Doctors working in various wards, OPDs and laboratories, Blood Bank, Causality, ICU of KIMS Hospital were subjected to bacterial analysis by conventional methods to know about the Bacterial contamination of the cell phones from July 2011 to December 2011. The commonest organism isolated from the contaminated cell phone is MRSA - 20%, followed by MSSA - 5%, CONS - 10 %, Micrococcus - 15 %. The obvious observation is none of the doctors’ cell phones are contaminated. In the present study, the contamination rate is compared to the controlled group consisting patients, relatives attending in OPD’s and not working in health care setting.

  10. A dynamin-like protein involved in bacterial cell membrane surveillance under environmental stress.

    Science.gov (United States)

    Sawant, Prachi; Eissenberger, Kristina; Karier, Laurence; Mascher, Thorsten; Bramkamp, Marc

    2016-09-01

    In ever-changing natural environments, bacteria are continuously challenged with numerous biotic and abiotic stresses. Accordingly, they have evolved both specific and more general mechanisms to counteract stress-induced damage and ensure survival. In the soil habitat of Bacillus subtilis, peptide antibiotics and bacteriophages are among the primary stressors that affect the integrity of the cytoplasmic membrane. Dynamin-like proteins (DLPs) play a major role in eukaryotic membrane re-modelling processes, including antiviral activities, but the function of the corresponding bacterial homologues was so far poorly understood. Here, we report on the protective function of a bacterial DLP, DynA from B. subtilis. We provide evidence that DynA plays an important role in a membrane surveillance system that counteracts membrane pore formation provoked by antibiotics and phages. In unstressed cells, DynA is a highly dynamic membrane-associated protein. Upon membrane damage, DynA localizes into large and static assemblies, where DynA acts locally to counteract stress-induced pores, presumably by inducing lipid bilayer fusion and sealing membrane gaps. Thus, lack of DynA increases the sensitivity to antibiotic exposure and phage infection. Taken together, our work suggests that DynA, and potentially other bacterial DLPs, contribute to the innate immunity of bacteria against membrane stress.

  11. Liver dendritic cells present bacterial antigens and produce cytokines upon Salmonella encounter.

    Science.gov (United States)

    Johansson, Cecilia; Wick, Mary Jo

    2004-02-15

    The capacity of murine liver dendritic cells (DC) to present bacterial Ags and produce cytokines after encounter with Salmonella was studied. Freshly isolated, nonparenchymal liver CD11c(+) cells had heterogeneous expression of MHC class II and CD11b and a low level of CD40 and CD86 expression. Characterization of liver DC subsets revealed that CD8alpha(-)CD4(-) double negative cells constituted the majority of liver CD11c(+) ( approximately 85%) with few cells expressing CD8alpha or CD4. Flow cytometry analysis of freshly isolated CD11c(+) cells enriched from the liver and cocultured with Salmonella expressing green fluorescent protein (GFP) showed that CD11c(+) MHC class II(high) cells had a greater capacity to internalize Salmonella relative to CD11c(+) MHC class II(low) cells. Moreover, both CD8alpha(-) and CD8alpha(+) liver DC internalized bacteria with similar efficiency after both in vitro and in vivo infection. CD11c(+) cells enriched from the liver could also process Salmonella for peptide presentation on MHC class I and class II to primary, Ag-specific T cells after internalization requiring actin cytoskeletal rearrangements. Flow cytometry analysis of liver CD11c(+) cells infected with Salmonella expressing GFP showed that both CD8alpha(-) and CD8alpha(+) DC produced IL-12p40 and TNF-alpha. The majority of cytokine-positive cells did not contain bacteria (GFP(-)) whereas only a minor fraction of cytokine-positive cells were GFP(+). Furthermore, only approximately 30-50% of liver DC containing bacteria (GFP(+)) produced cytokines. Thus, liver DC can internalize and process Salmonella for peptide presentation to CD4(+) and CD8(+) T cells and elicit proinflammatory cytokine production upon Salmonella encounter, suggesting that DC in the liver may contribute to immunity against hepatotropic bacteria.

  12. The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

    Directory of Open Access Journals (Sweden)

    Oliveira S.C.

    1998-01-01

    Full Text Available Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer

  13. Staying in Shape: the Impact of Cell Shape on Bacterial Survival in Diverse Environments.

    Science.gov (United States)

    Yang, Desirée C; Blair, Kris M; Salama, Nina R

    2016-03-01

    Bacteria display an abundance of cellular forms and can change shape during their life cycle. Many plausible models regarding the functional significance of cell morphology have emerged. A greater understanding of the genetic programs underpinning morphological variation in diverse bacterial groups, combined with assays of bacteria under conditions that mimic their varied natural environments, from flowing freshwater streams to diverse human body sites, provides new opportunities to probe the functional significance of cell shape. Here we explore shape diversity among bacteria, at the levels of cell geometry, size, and surface appendages (both placement and number), as it relates to survival in diverse environments. Cell shape in most bacteria is determined by the cell wall. A major challenge in this field has been deconvoluting the effects of differences in the chemical properties of the cell wall and the resulting cell shape perturbations on observed fitness changes. Still, such studies have begun to reveal the selective pressures that drive the diverse forms (or cell wall compositions) observed in mammalian pathogens and bacteria more generally, including efficient adherence to biotic and abiotic surfaces, survival under low-nutrient or stressful conditions, evasion of mammalian complement deposition, efficient dispersal through mucous barriers and tissues, and efficient nutrient acquisition.

  14. Real-time Bacterial Detection by Single Cell Based Sensors UsingSynchrotron FTIR Spectromicroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Veiseh, Mandana; Veiseh, Omid; Martin, Michael C.; Bertozzi,Carolyn; Zhang, Miqin

    2005-08-10

    Microarrays of single macrophage cell based sensors weredeveloped and demonstrated for real time bacterium detection bysynchrotron FTIR microscopy. The cells were patterned on gold-SiO2substrates via a surface engineering technique by which the goldelectrodes were immobilized with fibronectin to mediate cell adhesion andthe silicon oxide background were passivated with PEG to resist proteinadsorption and cell adhesion. Cellular morphology and IR spectra ofsingle, double, and triple cells on gold electrodes exposed tolipopolysaccharide (LPS) of different concentrations were compared toreveal the detection capabilities of these biosensors. The single-cellbased sensors were found to generate the most significant IR wave numbervariation and thus provide the highest detection sensitivity. Changes inmorphology and IR spectrum for single cells exposed to LPS were found tobe time- and concentration-dependent and correlated with each other verywell. FTIR spectra from single cell arrays of gold electrodes withsurface area of 25 mu-m2, 100 mu-m2, and 400 mu-m2 were acquired usingboth synchrotron and conventional FTIR spectromicroscopes to study thesensitivity of detection. The results indicated that the developedsingle-cell platform can be used with conventional FTIRspectromicroscopy. This technique provides real-time, label-free, andrapid bacterial detection, and may allow for statistic and highthroughput analyses, and portability.

  15. Bacterial host and reporter gene optimization for genetically encoded whole cell biosensors.

    Science.gov (United States)

    Brutesco, Catherine; Prévéral, Sandra; Escoffier, Camille; Descamps, Elodie C T; Prudent, Elsa; Cayron, Julien; Dumas, Louis; Ricquebourg, Manon; Adryanczyk-Perrier, Géraldine; de Groot, Arjan; Garcia, Daniel; Rodrigue, Agnès; Pignol, David; Ginet, Nicolas

    2017-01-01

    Whole-cell biosensors based on reporter genes allow detection of toxic metals in water with high selectivity and sensitivity under laboratory conditions; nevertheless, their transfer to a commercial inline water analyzer requires specific adaptation and optimization to field conditions as well as economical considerations. We focused here on both the influence of the bacterial host and the choice of the reporter gene by following the responses of global toxicity biosensors based on constitutive bacterial promoters as well as arsenite biosensors based on the arsenite-inducible Pars promoter. We observed important variations of the bioluminescence emission levels in five different Escherichia coli strains harboring two different lux-based biosensors, suggesting that the best host strain has to be empirically selected for each new biosensor under construction. We also investigated the bioluminescence reporter gene system transferred into Deinococcus deserti, an environmental, desiccation- and radiation-tolerant bacterium that would reduce the manufacturing costs of bacterial biosensors for commercial water analyzers and open the field of biodetection in radioactive environments. We thus successfully obtained a cell survival biosensor and a metal biosensor able to detect a concentration as low as 100 nM of arsenite in D. deserti. We demonstrated that the arsenite biosensor resisted desiccation and remained functional after 7 days stored in air-dried D. deserti cells. We also report here the use of a new near-infrared (NIR) fluorescent reporter candidate, a bacteriophytochrome from the magnetotactic bacterium Magnetospirillum magneticum AMB-1, which showed a NIR fluorescent signal that remained optimal despite increasing sample turbidity, while in similar conditions, a drastic loss of the lux-based biosensors signal was observed.

  16. Phylogenetic and metagenomic analyses of substrate-dependent bacterial temporal dynamics in microbial fuel cells.

    Directory of Open Access Journals (Sweden)

    Husen Zhang

    Full Text Available Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate.

  17. Ion Channels Activated by Mechanical Forces in Bacterial and Eukaryotic Cells.

    Science.gov (United States)

    Sokabe, Masahiro; Sawada, Yasuyuki; Kobayashi, Takeshi

    2015-01-01

    Since the first discovery of mechanosensitive ion channel (MSC) in non-sensory cells in 1984, a variety of MSCs has been identified both in prokaryotic and eukaryotic cells. One of the central issues concerning MSCs is to understand the molecular and biophysical mechanisms of how mechanical forces activate/open MSCs. It has been well established that prokaryotic (mostly bacterial) MSCs are activated exclusively by membrane tension. Thus the problem to be solved with prokaryotic MSCs is the mechanisms how the MSC proteins receive tensile forces from the lipid bilayer and utilize them for channel opening. On the other hand, the activation of many eukaryotic MSCs crucially depends on tension in the actin cytoskeleton. By using the actin cytoskeleton as a force sensing antenna, eukaryotic MSCs have obtained sophisticated functions such as remote force sensing and force-direction sensing, which bacterial MSCs do not have. Actin cytoskeletons also give eukaryotic MSCs an interesting and important function called "active touch sensing", by which cells can sense rigidity of their substrates. The contractile actin cytoskeleton stress fiber (SF) anchors its each end to a focal adhesion (FA) and pulls the substrate to generate substrate-rigidity-dependent stresses in the FA. It has been found that those stresses are sensed by some Ca2+-permeable MSCs existing in the vicinity of FAs, thus the MSCs work as a substrate rigidity sensor that can transduce the rigidity into intracellular Ca2+ levels. This short review, roughly constituting of two parts, deals with molecular and biophysical mechanisms underlying the MSC activation process mostly based on our recent studies; (1) structure-function in bacterial MSCs activation at the atomic level, and (2) roles of actin cytoskeletons in the activation of eukaryotic MSCs.

  18. FACTORS WHICH MODIFY THE EFFECT OF SODIUM AND POTASSIUM ON BACTERIAL CELL MEMBRANES.

    Science.gov (United States)

    HENNEMAN, D H; UMBREIT, W W

    1964-06-01

    Henneman, Dorothy H. (Rutgers, The State University, New Brunswick, N.J.), and W. W. Umbreit. Factors which modify the effect of sodium and potassium on bacterial cell membranes. J. Bacteriol. 87:1266-1273. 1964.-Suspensions of Escherichia coli B, when placed in 0.2 to 0.5 m solutions of NaCl, KCl, or LiCl, show an increased turbidity. With NaCl, this increased turbidity is stable with time; with KCl and LiCl, it is gradually lost. The stability to NaCl with time is due to substances removable from the cell by incubation in phosphate buffer; these materials exist in water washings from such phosphate-incubated cells.

  19. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Mølbak, Lars; Jensen, Tim Kåre

    2005-01-01

    Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells....... By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S r......RNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria...

  20. Probing Induced Structural Changes in Biomimetic Bacterial Cell Membrane Interactions with Divalent Cations

    Energy Technology Data Exchange (ETDEWEB)

    Holt, Allison M [ORNL; Standaert, Robert F [ORNL; Jubb, Aaron M [ORNL; Katsaras, John [ORNL; Johs, Alexander [ORNL

    2017-01-01

    Biological membranes, formed primarily by the self-assembly of complex mixtures of phospholipids, provide a structured scaffold for compartmentalization and structural processes in living cells. The specific physical properties of phospholipid species present in a given membrane play a key role in mediating these processes. Phosphatidylethanolamine (PE), a zwitterionic lipid present in bacterial, yeast, and mammalian cell membranes, is exceptional. In addition to undergoing the standard lipid polymorphic transition between the gel and liquid-crystalline phase, it can also assume an unusual polymorphic state, the inverse hexagonal phase (HII). Divalent cations are among the factors that drive the formation of the HII phase, wherein the lipid molecules form stacked tubular structures by burying the hydrophilic head groups and exposing the hydrophobic tails to the bulk solvent. Most biological membranes contain a lipid species capable of forming the HII state suggesting that such lipid polymorphic structural states play an important role in structural biological processes such as membrane fusion. In this study, the interactions between Mg2+ and biomimetic bacterial cell membranes composed of PE and phosphatidylglycerol (PG) were probed using differential scanning calorimetry (DSC), small-angle x-ray scattering (SAXS), and fluorescence spectroscopy. The lipid phase transitions were examined at varying ratios of PE to PG and upon exposure to physiologically relevant concentrations of Mg2+. An understanding of these basic interactions enhances our understanding of membrane dynamics and how membrane-mediated structural changes may occur in vivo.

  1. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells.

    Science.gov (United States)

    Muir, Elizabeth M; Fyfe, Ian; Gardiner, Sonya; Li, Li; Warren, Philippa; Fawcett, James W; Keynes, Roger J; Rogers, John H

    2010-01-15

    Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury.

  2. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis.

    Science.gov (United States)

    Manuse, Sylvie; Fleurie, Aurore; Zucchini, Laure; Lesterlin, Christian; Grangeasse, Christophe

    2016-01-01

    Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis.

  3. Oncostatin M production by human dendritic cells in response to bacterial products.

    Science.gov (United States)

    Suda, Takafumi; Chida, Kingo; Todate, Akihito; Ide, Kyotaro; Asada, Kazuhiro; Nakamura, Yutaro; Suzuki, Kenichiro; Kuwata, Hirofumi; Nakamura, Hirotoshi

    2002-03-21

    Oncostatin M (OSM) is a pleiomorphic cytokine that belongs to the IL-6 cytokine family. It is produced by activated T cells and monocytes/macrophages and plays an important role in the process of inflammatory responses. Although dendritic cells (DCs) have been shown to secrete a variety of cytokines, it is not elucidated whether DCs are able to produce OSM. To clarify this, using human DCs derived from peripheral blood cells, we measured the protein levels of OSM in the supernatants of DC cultures by ELISA and examined the expression of OSM mRNA by RT-PCR after stimulation with lipopolysaccharide (LPS) or fixed Staphylococcus aureus (SACS). Upon stimulation with bacterial products, DCs secreted a large amount of OSM protein in a dose- and time-dependent manner. Concomitantly, the expression of OSM mRNA by DCs was markedly up-regulated. Compared the ability of DCs to produce OSM with that of monocytes, which are major producers of OSM, DCs released significantly higher amounts of OSM protein in the culture supernatants than monocytes. These findings indicate for the first time that human monocyte-derived DCs can synthesize and secrete large amounts of OSM in response to bacterial products, suggesting that OSM produced by DCs at infectious sites may play a role in modulating inflammatory responses.

  4. Two small GTPases act in concert with the bactofilin cytoskeleton to regulate dynamic bacterial cell polarity.

    Science.gov (United States)

    Bulyha, Iryna; Lindow, Steffi; Lin, Lin; Bolte, Kathrin; Wuichet, Kristin; Kahnt, Jörg; van der Does, Chris; Thanbichler, Martin; Søgaard-Andersen, Lotte

    2013-04-29

    Cell polarity is essential for many bacterial activities, but the mechanisms responsible for its establishment are poorly understood. In Myxococcus xanthus, the type IV pili (T4P) motor ATPases PilB and PilT localize to opposite cell poles and switch poles during cellular reversals. We demonstrate that polar localization of PilB and PilT depends on the small GTPase SofG and BacP, a bactofilin cytoskeletal protein. Polymeric BacP localizes in both subpolar regions. SofG interacts directly with polymeric BacP and associates with one of these patches, forming a cluster that shuttles to the pole to establish localization of PilB and PilT at the same pole. Next, the small GTPase MglA sorts PilB and PilT to opposite poles to establish their correct polarity. During reversals, the Frz chemosensory system induces the inversion of PilB and PilT polarity. Thus, three hierarchically organized systems function in a cascade to regulate dynamic bacterial cell polarity.

  5. Role of Sulfhydryl Sites on Bacterial Cell Walls in the Biosorption, Mobility and Bioavailability of Mercury and Uranium

    Energy Technology Data Exchange (ETDEWEB)

    Myneni, Satish C.; Mishra, Bhoopesh; Fein, Jeremy

    2009-04-01

    The goal of this exploratory study is to provide a quantitative and mechanistic understanding of the impact of bacterial sulfhydryl groups on the bacterial uptake, speciation, methylation and bioavailability of Hg and redox changes of uranium. The relative concentration and reactivity of different functional groups present on bacterial surfaces will be determined, enabling quantitative predictions of the role of biosorption of Hg under the physicochemical conditions found at contaminated DOE sites.The hypotheses we propose to test in this investigation are as follows- 1) Sulfhydryl groups on bacterial cell surfaces modify Hg speciation and solubility, and play an important role, specifically in the sub-micromolar concentration ranges of metals in the natural and contaminated systems. 2) Sulfhydryl binding of Hg on bacterial surfaces significantly influences Hg transport into the cell and the methylation rates by the bacteria. 3) Sulfhydryls on cell membranes can interact with hexavalent uranium and convert to insoluble tetravalent species. 4) Bacterial sulfhydryl surface groups are inducible by the presence of metals during cell growth. Our studies focused on the first hypothesis, and we examined the nature of sulfhydryl sites on three representative bacterial species: Bacillus subtilis, a common gram-positive aerobic soil species; Shewanella oneidensis, a facultative gram-negative surface water species; and Geobacter sulfurreducens, an anaerobic iron-reducing gram-negative species that is capable of Hg methylation; and at a range of Hg concentration (and Hg:bacterial concentration ratio) in which these sites become important. A summary of our findings is as follows- Hg adsorbs more extensively to bacteria than other metals. Hg adsorption also varies strongly with pH and chloride concentration, with maximum adsorption occurring under circumneutral pH conditions for both Cl-bearing and Cl-free systems. Under these conditions, all bacterial species tested exhibit

  6. Microarray Analysis of Human Vascular Smooth Muscle Cell Responses to Bacterial Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Joe Minta

    2007-01-01

    Full Text Available Accumulating evidence suggest a causal role of bacterial and viral infections in atherogenesis. Bacterial lipopolysaccharide (LPS has been shown to stimulate resting vascular smooth muscle cells (SMC with the production of inflammatory cytokines and modulation of quiescent cells to the proliferative and synthetic phenotype. To comprehensively identify biologically important genes associated with LPS-induced SMC phenotype modulation, we compared the transcriptomes of quiescent human coronary artery SMC and cells treated with LPS for 4 and 22 h. The SMCs responded robustly to LPS treatment by the differential regulation of several genes involved in chromatin remodeling, transcription regulation, translation, signal transduction, metabolism, host defense, cell proliferation, apoptosis, matrix formation, adhesion and motility and suggest that the induction of clusters of genes involved in cell proliferation, migration and ECM production may be the main force that drives the LPS-induced phenotypic modulation of SMC rather than the differential expression of a single gene or a few genes. An interesting observation was the early and dramatic induction of four tightly clustered interferon-induced genes with tetratricopeptide repeats (IFIT1, 2, 4, 5. siRNA knock-down of IFIT1 in SMC was found to be associated with a remarkable up-regulation of TP53, CDKN1A and FOS, suggesting that IFIT1 may play a role in cell proliferation. Our data provide a comprehensive list of genes involved in LPS biology and underscore the important role of LPS in SMC activation and phenotype modulation which is a pivotal event in the onset of atherogenesis.

  7. Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide.

    Science.gov (United States)

    Nocker, Andreas; Camper, Anne K

    2006-03-01

    The distinction between viable and dead bacterial cells poses a major challenge in microbial diagnostics. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based quantification methods overestimate the number of viable cells in mixed populations or even lead to false-positive results in the absence of viable cells. On the other hand, RNA-based diagnostic methods, which circumvent this problem, are technically demanding and suffer from some drawbacks. A promising and easy-to-use alternative utilizing the DNA-intercalating dye ethidium monoazide bromide (EMA) was published recently. This chemical is known to penetrate only into "dead" cells with compromised cell membrane integrity. Subsequent photoinduced cross-linking was reported to inhibit PCR amplification of DNA from dead cells. We provide evidence here that in addition to inhibition of amplification, most of the DNA from dead cells is actually lost during the DNA extraction procedure, probably together with cell debris which goes into the pellet fraction. Exposure of bacteria to increasing stress and higher proportions of dead cells in defined populations led to increasing loss of genomic DNA. Experiments were performed using Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium as model pathogens and using real-time PCR for their quantification. Results showed that EMA treatment of mixed populations of these two species provides a valuable tool for selective removal of DNA of nonviable cells by using conventional extraction protocols. Furthermore, we provide evidence that prior to denaturing gradient gel electrophoresis, EMA treatment of a mature mixed-population drinking-water biofilm containing a substantial proportion of dead cells can result in community fingerprints dramatically different from those for an untreated biofilm. The interpretation of such fingerprints can have important implications in the field of microbial ecology.

  8. Cooperative role for tetraspanins in adhesin-mediated attachment of bacterial species to human epithelial cells.

    Science.gov (United States)

    Green, Luke R; Monk, Peter N; Partridge, Lynda J; Morris, Paul; Gorringe, Andrew R; Read, Robert C

    2011-06-01

    The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria.

  9. Metabolic activity of bacterial cell enumerated by direct viable count. [Escherichia coli; Salmonella enteritidis

    Energy Technology Data Exchange (ETDEWEB)

    Roszak, D.B.; Colwell, R.R.

    1987-12-01

    The direct viable count (DVC) method was modified by incorporation radiolabeled substrates in microautoradiographic analyses to assess bacterial survival in controlled laboratory microcosms. The DVC method, which permits enumeration of culturable and nonculturable cells, discriminates those cells that are responsive to added nutrients but in which division is inhibited by the addition of nalidixic acid. The resulting elongated cells represent all viable cells; this includes those that are culturable on routine media and those that are not. Escherichia coli and Salmonella enteritidis were employed in the microcosm studies, and radiolabeled substrates included (methyl-/sup 3/H) thymidine or (U-/sup 14/C) glutamic acid. Samples taken at selected intervals during the survival experiments were examined by epifluorescence microscopy to enumerate cells by the DVC and acridine orange direct count methods, as well as by culture methods. Good correlation was obtained for cell-associated metabolic activity, measured by microautoradiography and substrate responsiveness (by the DVC method) at various stages of survival. Of the cells responsive to nutrients by the DVC method, ca. 90% were metabolically active by the microautoradiographic method. No significant difference was observed between DVC enumerations with or without added radiolabeled substrate.

  10. Accuracy of the automated cell counters for management of spontaneous bacterial peritonitis

    Institute of Scientific and Technical Information of China (English)

    Oliviero Riggio; Stefania Angeloni; Antonella Parente; Cinzia Leboffe; Giorgio Pinto; Teresa Aronne; Manuela Merli

    2008-01-01

    AIM: To evaluate the accuracy of automated blood cell counters for ascitic polymorphonuclear (PMN)determination for: (1) diagnosis,(2) efficacy of the ongoing antibiotic therapy,and (3) resolution of spontaneous bacterial peritonitis (SBP).METHODS: One hundred and twelve ascitic fluid samples were collected from 52 consecutive cirrhotic patients,16 of them with SBR The agreement between the manual and the automated method for PMN count was assessed.The sensitivity/specificity and the positive/negative predictive value of the automated blood cell counter were also calculated by considering the manual method as the "gold standard"RESULTS: The mean + SD of the difference between manual and automated measurements was 7.8±58cells/mm3,while the limits of agreement were +124 cells/mm3 [95% confidence interval (CI): +145 to +103] and -108 cells/mm3 (95% CI: -87 to -129).The automated cell counter had a sensitivity of 100% and a specificity of 97.7% in diagnosing SBP,and a sensitivity of 91% and a specificity of 100% for the efficacy of the ongoing antibiotic therapy.The two methods showed a complete agreement for the resolution of infection.CONCLUSION: Automated cell counters not only have a good diagnostic accuracy,but are also very effective in monitoring the antibiotic treatment in patients with SBP.Because of their quicker performance,they should replace the manual counting for PMN determination in the ascitic fluid of patients with SBR

  11. Fate study of water-borne gram positive vegetative bacterial cells with Raman microscopy

    Science.gov (United States)

    Guicheteau, Jason; Tripathi, Ashish; Minter, Jennifer; Wilcox, Phillip; Christesen, Steven

    2010-04-01

    We present an initial bacterial fate study of Gram positive vegetative cells suspended in water and stored at ambient room temperature via Raman spectroscopy monitoring. Two types of cells were considered for this study: vegetative cells of Bacillus cereus, Bacillus thuringiensis which contain the polyhydroxybutyric acid (PHBA) as an energy storage compound and Bacillus subtlilis cells which do not. The cells were cultured specifically for this project. Immediately following the culturing phase, the bacteria were extracted, cleaned and at the onset of the study were suspended in de-ionized water and stored at room temperature. Aliquots of suspensions were deposited onto aluminum slides at different times and allowed to dry for Raman analysis. Spectra from multiple regions of each dried spot and each deposit time were acquired along with the bright-field and fluorescence images. Results were examined to investigate the effect of suspension time on the spectral signatures as well as the fate behavior of the three types of cells investigated. The cells were monitored daily for over a 14 period during which time the onset of starvation induced sporulation was observed.

  12. Peripheral T Cell Apoptosis and Its Role in Generalized Bacterial Infections: A Minireview.

    Science.gov (United States)

    Chernykh, Helen R.; Norkin, Maxim N.; Leplina, Olga Yu.; Khonina, Nataliya A.; Tihonova, Marina A.; Ostanin, Alexander A.

    2001-07-01

    In the present review we have attempted to analyze recent findings concerning apoptosis of mature peripheral T cells. The great attention is made to the factors underlying resistance or sensitivity of mature T lymphocytes to activation-induced cell death. The role of preactivation and altered costimulation is discussed in this regard. Besides, the possible role of cytokines in the modulation of T cell apoptosis is emphasized. Particular attention is paid to the studies of apoptosis disorders in the pathogenesis of generalized bacterial infections. In this connection some own results are summarized as well. To characterize T cell death and its role in the pathogenesis of bacterial infections an anti-CD3-mAb or Con A-induced apoptosis in patients with severe and generalized forms of surgical infections have been investigated. We have found a significant increase of activation-induced lymphocyte apoptosis and a high level of apoptosis in freshly isolated lymphocytes in patients with surgical infections. Alternatively, peripheral blood mononuclear cells from surgical patients without infectious complications did not exhibit a marked enhancement of activation-induced cell death. Activation-induced T cell death in surgical infections appeared to be Fas-dependent, involved reactive oxygen intermediates and was partly prevented by pro-inflammatory cytokines, among which IL-2 exhibited the most pronounced anti-apoptotic activity. Likewise, APACHE II score, as a marker of the infection severity, directly correlated with a rate of activation-induced T cell apoptosis. Accelerated T cell apoptosis at the early stage of infection was revealed in survivors and non-survivors, that appears to designate a common pathway for the restriction of systemic inflammation. At the late stage of infection altered T cell apoptosis could account for different outcomes, since the patients with lethal outcome showed 2-fold increase in activation-induced cell death compared to the opposite group

  13. Regulatory T cell suppressive potency dictates the balance between bacterial proliferation and clearance during persistent Salmonella infection.

    Directory of Open Access Journals (Sweden)

    Tanner M Johanns

    Full Text Available The pathogenesis of persistent infection is dictated by the balance between opposing immune activation and suppression signals. Herein, virulent Salmonella was used to explore the role and potential importance of Foxp3-expressing regulatory T cells in dictating the natural progression of persistent bacterial infection. Two distinct phases of persistent Salmonella infection are identified. In the first 3-4 weeks after infection, progressively increasing bacterial burden was associated with delayed effector T cell activation. Reciprocally, at later time points after infection, reductions in bacterial burden were associated with robust effector T cell activation. Using Foxp3(GFP reporter mice for ex vivo isolation of regulatory T cells, we demonstrate that the dichotomy in infection tempo between early and late time points is directly paralleled by drastic changes in Foxp3(+ Treg suppressive potency. In complementary experiments using Foxp3(DTR mice, the significance of these shifts in Treg suppressive potency on infection outcome was verified by enumerating the relative impacts of regulatory T cell ablation on bacterial burden and effector T cell activation at early and late time points during persistent Salmonella infection. Moreover, Treg expression of CTLA-4 directly paralleled changes in suppressive potency, and the relative effects of Treg ablation could be largely recapitulated by CTLA-4 in vivo blockade. Together, these results demonstrate that dynamic regulation of Treg suppressive potency dictates the course of persistent bacterial infection.

  14. Simultaneous selection of soil electroactive bacterial communities associated to anode and cathode in a two-chamber Microbial Fuel Cell

    Science.gov (United States)

    Chiellini, Carolina; Bacci, Giovanni; Fani, Renato; Mocali, Stefano

    2016-04-01

    Different bacteria have evolved strategies to transfer electrons over their cell surface to (or from) their extracellular environment. This electron transfer enables the use of these bacteria in bioelectrochemical systems (BES) such as Microbial Fuel Cells (MFCs). In MFC research the biological reactions at the cathode have long been a secondary point of interest. However, bacterial biocathodes in MFCs represent a potential advantage compared to traditional cathodes, for both their low costs and their low impact on the environment. The main challenge in biocathode set-up is represented by the selection of a bacterial community able to efficiently accept electrons from the electrode, starting from an environmental matrix. In this work, a constant voltage was supplied on a two-chamber MFC filled up with soil over three weeks in order to simultaneously select an electron donor bacterial biomass on the anode and an electron acceptor biomass on the cathode, starting from the same soil. Next Generation Sequencing (NGS) analysis was performed to characterize the bacterial community of the initial soil, in the anode, in the cathode and in the control chamber not supplied with any voltage. Results highlighted that both the MFC conditions and the voltage supply affected the soil bacterial communities, providing a selection of different bacterial groups preferentially associated to the anode (Betaproteobacteria, Bacilli and Clostridia) and to the cathode (Actinobacteria and Alphaproteobacteria). These results confirmed that several electroactive bacteria are naturally present within a top soil and, moreover, different soil bacterial genera could provide different electrical properties.

  15. Repeatability of differential goat bulk milk culture and associations with somatic cell count, total bacterial count, and standard plate count

    OpenAIRE

    Koop, G.; Dik, N; Nielen, M; Lipman, L. J. A.

    2010-01-01

    The aims of this study were to assess how different bacterial groups in bulk milk are related to bulk milk somatic cell count (SCC), bulk milk total bacterial count (TBC), and bulk milk standard plate count (SPC) and to measure the repeatability of bulk milk culturing. On 53 Dutch dairy goat farms, 3 bulk milk samples were collected at intervals of 2 wk. The samples were cultured for SPC, coliform count, and staphylococcal count and for the presence of Staphylococcus aureus. Furthermore, SCC ...

  16. A Pro-Drug Approach for Selective Modulation of AI-2-Mediated Bacterial Cell-to-Cell Communication

    Directory of Open Access Journals (Sweden)

    Herman O. Sintim

    2012-03-01

    Full Text Available The universal quorum sensing autoinducer, AI-2, is utilized by several bacteria. Analogs of AI-2 have the potential to modulate bacterial behavior. Selectively quenching the communication of a few bacteria, in the presence of several others in an ecosystem, using analogs of AI-2 is non-trivial due to the ubiquity of AI-2 processing receptors in many bacteria that co-exist. Herein, we demonstrate that when an AI-2 analog, isobutyl DPD (which has been previously shown to be a quorum sensing, QS, quencher in both Escherichia coli and Salmonella typhimurium is modified with ester groups, which get hydrolyzed once inside the bacterial cells, only QS in E. coli, but not in S. typhimurium, is inhibited. The origin of this differential QS inhibition could be due to differences in analog permeation of the bacterial membranes or ester hydrolysis rates. Such differences could be utilized to selectively target QS in specific bacteria amongst a consortium of other species that also use AI-2 signaling.

  17. Spatiotemporal development of the bacterial community in a tubular longitudinal microbial fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Rae; Premier, Giuliano C. [Glamorgan Univ., Pontypridd (United Kingdom). Faculty of Advnaced Technology; Beecroft, Nelli J.; Avignone-Rossa, Claudio [Surrey Univ., Guildford (United Kingdom). Microbial Sciences; Varcoe, John R.; Slade, Robert C.T. [Surrey Univ., Guildford (United Kingdom). Chemical Sciences; Dinsdale, Richard M.; Guwy, Alan J. [Glamorgan Univ., Pontypridd (United Kingdom). Faculty of Health, Sport and Science; Thumser, Alfred [Surrey Univ., Guildford (United Kingdom). Biochemical Sciences

    2011-05-15

    The spatiotemporal development of a bacterial community in an exoelectrogenic biofilm was investigated in sucrose-fed longitudinal tubular microbial fuel cell reactors, consisting of two serially connected modules. The proportional changes in the microbial community composition were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and DNA sequencing in order to relate them to the performance and stability of the bioelectrochemical system. The reproducibility of duplicated reactors, evaluated by cluster analysis and Jaccard's coefficient, shows 80-90% similarity in species composition. Biofilm development through fed-batch start-up and subsequent stable continuous operation results in a population shift from {gamma}-Proteobacteria- and Bacteroidetes- to Firmicutes-dominated communities, with other diverse species present at much lower relative proportions. DGGE patterns were analysed by range-weighted richness (Rr) and Pareto-Lorenz evenness distribution curves to investigate the evolution of the bacterial community. The first modules shifted from dominance by species closely related to Bacteroides graminisolvens, Raoultella ornithinolytica and Klebsiella sp. BM21 at the start of continuous-mode operation to a community dominated by Paludibacter propionicigenes-, Lactococcus sp.-, Pantoea agglomerans- and Klebsiella oxytoca-related species with stable power generation (6.0 W/m{sup 3}) at day 97. Operational strategies that consider the dynamics of the population will provide useful parameters for evaluating system performance in the practical application of microbial fuel cells. (orig.)

  18. Cell cycle arrest and biochemical changes accompanying cell death in harmful dinoflagellates following exposure to bacterial algicide IRI-160AA

    Science.gov (United States)

    Pokrzywinski, Kaytee L.; Tilney, Charles L.; Warner, Mark E.; Coyne, Kathryn J.

    2017-03-01

    Bacteria may play a role in regulating harmful algal blooms, but little is known about the biochemical and physiological changes associated with cell death induced by algicidal bacteria. Previous work characterized an algicidal exudate (IRI-160AA) produced by Shewanella sp. IRI-160 that is effective against dinoflagellates, while having little to no effect on other phytoplankton species in laboratory culture experiments. The objective of this study was to evaluate biochemical changes associated with cell death and impacts on the cell cycle in three dinoflagellate species (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum) after exposure to IRI-160AA. In this study, IRI-160AA induced cell cycle arrest in all dinoflagellates examined. Several indicators for programmed cell death (PCD) that are often observed in phytoplankton in response to a variety of stressors were also evaluated. Cell death was accompanied by significant increases in DNA degradation, intra- and extracellular ROS concentrations and DEVDase (caspase-3 like) protease activity, which have been associated with PCD in other phytoplankton species. Overall, results of this investigation provide strong evidence that treatment with the bacterial algicide, IRI-160AA results in cell cycle arrest and induces biochemical changes consistent with stress-related cell death responses observed in other phytoplankton.

  19. The balance of apoptotic and necrotic cell death in Mycobacterium tuberculosis infected macrophages is not dependent on bacterial virulence.

    OpenAIRE

    2012-01-01

    BACKGROUND: An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission. METHODS: We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and ...

  20. Plug-and-playable fluorescent cell imaging modular toolkits using the bacterial superglue, SpyTag/SpyCatcher.

    Science.gov (United States)

    Moon, Hyojin; Bae, Yoonji; Kim, Hansol; Kang, Sebyung

    2016-11-29

    Simple plug-and-playable fluorescent cell imaging modular toolkits are established using the bacterial superglue SpyTag/SpyCatcher protein ligation system. A variety of affibody-fluorescent protein conjugates (AFPCs) are post-translationally generated via the isopeptide bond formation, and each AFPC effectively recognizes and binds to its targeting cells, visualizing them with selective colors on demand.

  1. Nutritional stress induces exchange of cell material and energetic coupling between bacterial species.

    Science.gov (United States)

    Benomar, Saida; Ranava, David; Cárdenas, María Luz; Trably, Eric; Rafrafi, Yan; Ducret, Adrien; Hamelin, Jérôme; Lojou, Elisabeth; Steyer, Jean-Philippe; Giudici-Orticoni, Marie-Thérèse

    2015-02-23

    Knowledge of the behaviour of bacterial communities is crucial for understanding biogeochemical cycles and developing environmental biotechnology. Here we demonstrate the formation of an artificial consortium between two anaerobic bacteria, Clostridium acetobutylicum (Gram-positive) and Desulfovibrio vulgaris Hildenborough (Gram-negative, sulfate-reducing) in which physical interactions between the two partners induce emergent properties. Molecular and cellular approaches show that tight cell-cell interactions are associated with an exchange of molecules, including proteins, which allows the growth of one partner (D. vulgaris) in spite of the shortage of nutrients. This physical interaction induces changes in expression of two genes encoding enzymes at the pyruvate crossroads, with concomitant changes in the distribution of metabolic fluxes, and allows a substantial increase in hydrogen production without requiring genetic engineering. The stress induced by the shortage of nutrients of D. vulgaris appears to trigger the interaction.

  2. The Gut Microbiota and Human Health with an Emphasis on the Use of Microencapsulated Bacterial Cells

    Directory of Open Access Journals (Sweden)

    Satya Prakash

    2011-01-01

    Full Text Available The gut microbiota plays a crucial role in maintaining health. Alterations of the gut bacterial population have been associated with a number of diseases. Past and recent studies suggest that one can positively modify the contents of the gut microbiota by introducing prebiotics, probiotics, synbiotics, and other therapeutics. This paper focuses on probiotic modulation of the gut microbiota by their delivery to the lower gastrointestinal tract (GIT. There are numerous obstacles to overcome before microorganisms can be utilized as therapeutics. One important limitation is the delivery of viable cells to the lower GIT without a significant loss of cell viability and metabolic features through the harsh conditions of the upper GIT. Microencapsulation has been shown to overcome this, with various types of microcapsules available for resolving this limitation. This paper discusses the gut microbiota and its role in disease, with a focus on microencapsulated probiotics and their potentials and limitations.

  3. Analysis of a stochastic model for bacterial growth and the lognormality in the cell-size distribution

    CERN Document Server

    Yamamoto, Ken

    2016-01-01

    This paper theoretically analyzes a phenomenological stochastic model for bacterial growth. This model comprises cell divisions and linear growth of cells, where growth rates and cell cycles are drawn from lognormal distributions. We derive that the cell size is expressed as a sum of independent lognormal variables. We show numerically that the quality of the lognormal approximation greatly depends on the distributions of the growth rate and cell cycle. Furthermore, we show that actual parameters of the growth rate and cell cycle take values which give good lognormal approximation, so the experimental cell-size distribution is in good agreement with a lognormal distribution.

  4. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    Science.gov (United States)

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-06-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases.

  5. Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

    Science.gov (United States)

    Schüürmann, Jan; Quehl, Paul; Festel, Gunter; Jose, Joachim

    2014-10-01

    Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

  6. Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion

    Science.gov (United States)

    van Wijk, Xander M.; Döhrmann, Simon; Hallström, Björn M.; Li, Shangzhong; Voldborg, Bjørn G.; Meng, Brandon X.; McKee, Karen K.; van Kuppevelt, Toin H.; Yurchenco, Peter D.; Palsson, Bernhard O.; Lewis, Nathan E.; Nizet, Victor

    2017-01-01

    ABSTRACT To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit α2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-α2β1γ1/laminin-α2β2γ1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin α2 is important for cellular invasion by a number of bacterial pathogens. PMID:28074024

  7. Binding domains of Bacillus anthracis phage endolysins recognize cell culture age-related features on the bacterial surface.

    Science.gov (United States)

    Paskaleva, Elena E; Mundra, Ruchir V; Mehta, Krunal K; Pangule, Ravindra C; Wu, Xia; Glatfelter, Willing S; Chen, Zijing; Dordick, Jonathan S; Kane, Ravi S

    2015-01-01

    Bacteriolytic enzymes often possess a C-terminal binding domain that recognizes specific motifs on the bacterial surface and a catalytic domain that cleaves covalent linkages within the cell wall peptidoglycan. PlyPH, one such lytic enzyme of bacteriophage origin, has been reported to be highly effective against Bacillus anthracis, and can kill up to 99.99% of the viable bacteria. The bactericidal activity of this enzyme, however, appears to be strongly dependent on the age of the bacterial culture. Although highly bactericidal against cells in the early exponential phase, the enzyme is substantially less effective against stationary phase cells, thus limiting its application in real-world settings. We hypothesized that the binding domain of PlyPH may differ in affinity to cells in different Bacillus growth stages and may be primarily responsible for the age-restricted activity. We therefore employed an in silico approach to identify phage lysins differing in their specificity for the bacterial cell wall. Specifically we focused our attention on Plyβ, an enzyme with improved cell wall-binding ability and age-independent bactericidal activity. Although PlyPH and Plyβ have dissimilar binding domains, their catalytic domains are highly homologous. We characterized the biocatalytic mechanism of Plyβ by identifying the specific bonds cleaved within the cell wall peptidoglycan. Our results provide an example of the diversity of phage endolysins and the opportunity for these biocatalysts to be used for broad-based protection from bacterial pathogens.

  8. Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jennifer C Regan

    2013-10-01

    Full Text Available Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of

  9. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    Science.gov (United States)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  10. Effects of zinc oxide nanoparticles on Kupffer cell phagosomal motility, bacterial clearance, and liver function

    Directory of Open Access Journals (Sweden)

    Watson CY

    2015-06-01

    Full Text Available Christa Y Watson, Ramon M Molina, Andressa Louzada, Kimberly M Murdaugh, Thomas C Donaghey, Joseph D BrainCenter for Nanotechnology and Nanotoxicology, Molecular and Integrative Physiological Sciences Program, Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, USABackground: Zinc oxide engineered nanoparticles (ZnO ENPs have potential as nanomedicines due to their inherent properties. Studies have described their pulmonary impact, but less is known about the consequences of ZnO ENP interactions with the liver. This study was designed to describe the effects of ZnO ENPs on the liver and Kupffer cells after intravenous (IV administration.Materials and methods: First, pharmacokinetic studies were conducted to determine the tissue distribution of neutron-activated 65ZnO ENPs post-IV injection in Wistar Han rats. Then, a noninvasive in vivo method to assess Kupffer cell phagosomal motility was employed using ferromagnetic iron particles and magnetometry. We also examined whether prior IV injection of ZnO ENPs altered Kupffer cell bactericidal activity on circulating Pseudomonas aeruginosa. Serum and liver tissues were collected to assess liver-injury biomarkers and histological changes, respectively.Results: We found that the liver was the major site of initial uptake of 65ZnO ENPs. There was a time-dependent decrease in tissue levels of 65Zn in all organs examined, reflecting particle dissolution. In vivo magnetometry showed a time-dependent and transient reduction in Kupffer cell phagosomal motility. Animals challenged with P. aeruginosa 24 hours post-ZnO ENP injection showed an initial (30 minutes delay in vascular bacterial clearance. However, by 4 hours, IV-injected bacteria were cleared from the blood, liver, spleen, lungs, and kidneys. Seven days post-ZnO ENP injection, creatine phosphokinase and aspartate aminotransferase levels in serum were significantly increased. Histological evidence of

  11. Identification of Bacterial Surface Antigens by Screening Peptide Phage Libraries Using Whole Bacteria Cell-Purified Antisera

    Science.gov (United States)

    Hu, Yun-Fei; Zhao, Dun; Yu, Xing-Long; Hu, Yu-Li; Li, Run-Cheng; Ge, Meng; Xu, Tian-Qi; Liu, Xiao-Bo; Liao, Hua-Yuan

    2017-01-01

    Bacterial surface proteins can be good vaccine candidates. In the present study, we used polyclonal antibodies purified with intact Erysipelothrix rhusiopthiae to screen phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries, which led to the identification of mimotopes. Homology search of the mimotope sequences against E. rhusiopthiae-encoded ORF sequences revealed 14 new antigens that may localize on the surface of E. rhusiopthiae. When these putative surface proteins were used to immunize mice, 9/11 antigens induced protective immunity. Thus, we have demonstrated that a combination of using the whole bacterial cells to purify antibodies and using the phage-displayed peptide libraries to determine the antigen specificities of the antibodies can lead to the discovery of novel bacterial surface antigens. This can be a general approach for identifying surface antigens for other bacterial species. PMID:28184219

  12. Effects of mimosine on Wolbachia in mosquito cells: cell cycle suppression reduces bacterial abundance.

    Science.gov (United States)

    Fallon, Ann M

    2015-10-01

    The plant allelochemical L-mimosine (β-[N-(3-hydroxy-4-pyridone)]-α-aminopropionic acid; leucenol) resembles the nonessential amino acid, tyrosine. Because the obligate intracellular alphaproteobacterium, Wolbachia pipientis, metabolizes amino acids derived from host cells, the effects of mimosine on infected and uninfected mosquito cells were investigated. The EC50 for mimosine was 6-7 μM with Aedes albopictus C7-10 and C/wStr cell lines, and was not influenced by infection status. Mosquito cells responded to concentrations of mimosine substantially lower than those used to synchronize the mammalian cell cycle; at concentrations of 30-35 μM, mimosine reversibly arrested the mosquito cell cycle at the G1/S boundary and inhibited growth of Wolbachia strain wStr. Although lower concentrations of mimosine slightly increased wStr abundance, concentrations that suppressed the cell cycle reduced Wolbachia levels.

  13. Structural reorganization of the bacterial cell-division protein FtsZ from Staphylococcus aureus.

    Science.gov (United States)

    Matsui, Takashi; Yamane, Junji; Mogi, Nobuyuki; Yamaguchi, Hiroto; Takemoto, Hiroshi; Yao, Min; Tanaka, Isao

    2012-09-01

    FtsZ is a key molecule in bacterial cell division. In the presence of GTP, it polymerizes into tubulin-like protofilaments by head-to-tail association. Protofilaments of FtsZ seem to adopt a straight or a curved conformation in relation to the bound nucleotide. However, although several bacterial and archaeal FtsZ structures have been determined, all of the structures reported previously are considered to have a curved conformation. In this study, structures of FtsZ from Staphylococcus aureus (SaFtsZ) were determined in apo, GDP-bound and inhibitor-complex forms and it was found that SaFtsZ undergoes marked conformational changes. The accumulated evidence suggests that the GDP-bound structure has the features of the straight form. The structural change between the curved and straight forms shows intriguing similarity to the eukaryotic cytoskeletal protein tubulin. Furthermore, the structure of the apo form showed an unexpectedly large conformational change in the core region. FtsZ has also been recognized as a novel target for antibacterial drugs. The structure of the complex with the inhibitor PC190723, which has potent and selective antistaphylococcal activity, indicated that the inhibitor binds at the cleft between the two subdomains.

  14. Antibiotic discovery: combatting bacterial resistance in cells and in biofilm communities.

    Science.gov (United States)

    Penesyan, Anahit; Gillings, Michael; Paulsen, Ian T

    2015-03-24

    Bacterial resistance is a rapidly escalating threat to public health as our arsenal of effective antibiotics dwindles. Therefore, there is an urgent need for new antibiotics. Drug discovery has historically focused on bacteria growing in planktonic cultures. Many antibiotics were originally developed to target individual bacterial cells, being assessed in vitro against microorganisms in a planktonic mode of life. However, towards the end of the 20th century it became clear that many bacteria live as complex communities called biofilms in their natural habitat, and this includes habitats within a human host. The biofilm mode of life provides advantages to microorganisms, such as enhanced resistance towards environmental stresses, including antibiotic challenge. The community level resistance provided by biofilms is distinct from resistance mechanisms that operate at a cellular level, and cannot be overlooked in the development of novel strategies to combat infectious diseases. The review compares mechanisms of antibiotic resistance at cellular and community levels in the light of past and present antibiotic discovery efforts. Future perspectives on novel strategies for treatment of biofilm-related infectious diseases are explored.

  15. Antibiotic Discovery: Combatting Bacterial Resistance in Cells and in Biofilm Communities

    Directory of Open Access Journals (Sweden)

    Anahit Penesyan

    2015-03-01

    Full Text Available Bacterial resistance is a rapidly escalating threat to public health as our arsenal of effective antibiotics dwindles. Therefore, there is an urgent need for new antibiotics. Drug discovery has historically focused on bacteria growing in planktonic cultures. Many antibiotics were originally developed to target individual bacterial cells, being assessed in vitro against microorganisms in a planktonic mode of life. However, towards the end of the 20th century it became clear that many bacteria live as complex communities called biofilms in their natural habitat, and this includes habitats within a human host. The biofilm mode of life provides advantages to microorganisms, such as enhanced resistance towards environmental stresses, including antibiotic challenge. The community level resistance provided by biofilms is distinct from resistance mechanisms that operate at a cellular level, and cannot be overlooked in the development of novel strategies to combat infectious diseases. The review compares mechanisms of antibiotic resistance at cellular and community levels in the light of past and present antibiotic discovery efforts. Future perspectives on novel strategies for treatment of biofilm-related infectious diseases are explored.

  16. SLiCE: a novel bacterial cell extract-based DNA cloning method.

    Science.gov (United States)

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-04-01

    We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

  17. Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages.

    Science.gov (United States)

    Veszelka, Szilvia; Pásztói, Mária; Farkas, Attila E; Krizbai, István; Ngo, Thi Khue Dung; Niwa, Masami; Abrahám, Csongor S; Deli, Mária A

    2007-01-01

    Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood-brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and beta-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.

  18. Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.

    Science.gov (United States)

    Uprety, Rajendra; Luo, Ji; Liu, Jihe; Naro, Yuta; Samanta, Subhas; Deiters, Alexander

    2014-08-18

    We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.

  19. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

    Directory of Open Access Journals (Sweden)

    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  20. Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

    Science.gov (United States)

    Close, Dan M.; Patterson, Stacey S.; Ripp, Steven; Baek, Seung J.; Sanseverino, John; Sayler, Gary S.

    2010-01-01

    Background The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Methodology/Principal Findings Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. Conclusions/Significance The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies. PMID:20805991

  1. Increased electrical output when a bacterial ABTS oxidizer is used in a microbial fuel cell.

    Science.gov (United States)

    Hunter, William J; Manter, Daniel K

    2011-02-01

    Microbial fuel cells (MFCs) are a technology that provides electrical energy from the microbial oxidation of organic compounds. Most MFCs use oxygen as the oxidant in the cathode chamber. This study examined the formation in culture of an unidentified bacterial oxidant and investigated the performance of this oxidant in a two-chambered MFC with a proton exchange membrane and an uncoated carbon cathode. DNA, FAME profile and characterization studies identified the microorganism that produced the oxidant as Burkholderia cenocepacia. The oxidant was produced by log phase cells, oxidized the dye 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), had a mass below 1 kD, was heat stable (121°C) and was soluble in ethanol. In a MFC with a 1000 Ω load and ABTS as a mediator, the oxidizer increased cell voltage 11 times higher than atmospheric oxygen and 2.9 times higher than that observed with ferricyanide in the cathode chamber. No increase in cell voltage was observed when no mediator was present. Organisms that produce and release oxidizers into the media may prove useful as bio-cathodes by improving the electrical output of MFCs.

  2. Elucidation of the molecular recognition of bacterial cell wall by modular pneumococcal phage endolysin CPL-1.

    Science.gov (United States)

    Pérez-Dorado, Inmaculada; Campillo, Nuria E; Monterroso, Begoña; Hesek, Dusan; Lee, Mijoon; Páez, Juan A; García, Pedro; Martínez-Ripoll, Martín; García, José L; Mobashery, Shahriar; Menéndez, Margarita; Hermoso, Juan A

    2007-08-24

    Pneumococcal bacteriophage-encoded lysins are modular proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) in treatment of streptococcal infections. The first x-ray crystal structures of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1, in complex with three bacterial cell wall peptidoglycan (PG) analogues are reported herein. The Cpl-1 structure is folded in two well defined modules, one responsible for anchoring to the pneumococcal cell wall and the other, a catalytic module, that hydrolyzes the PG. Conformational rearrangement of Tyr-127 is a critical event in molecular recognition of a stretch of five saccharide rings of the polymeric peptidoglycan (cell wall). The PG is bound at a stretch of the surface that is defined as the peptidoglycan-binding sites 1 and 2, the juncture of which catalysis takes place. The peptidoglycan-binding site 1 binds to a stretch of three saccharides of the peptidoglycan in a conformation essentially identical to that of the peptidoglycan in solution. In contrast, binding of two peptidoglycan saccharides at the peptidoglycan-binding site 2 introduces a kink into the solution structure of the peptidoglycan, en route to catalytic turnover. These findings provide the first structural evidence on recognition of the peptidoglycan and shed light on the discrete events of cell wall degradation by Cpl-1.

  3. Coinfection of tick cell lines has variable effects on replication of intracellular bacterial and viral pathogens

    Science.gov (United States)

    Moniuszko, Anna; Rückert, Claudia; Alberdi, M. Pilar; Barry, Gerald; Stevenson, Brian; Fazakerley, John K.; Kohl, Alain; Bell-Sakyi, Lesley

    2014-01-01

    Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72 h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen–pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence

  4. Coinfection of tick cell lines has variable effects on replication of intracellular bacterial and viral pathogens.

    Science.gov (United States)

    Moniuszko, Anna; Rückert, Claudia; Alberdi, M Pilar; Barry, Gerald; Stevenson, Brian; Fazakerley, John K; Kohl, Alain; Bell-Sakyi, Lesley

    2014-06-01

    Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen-pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence.

  5. Detection and quantification of bacterial autofluorescence at the single-cell level by a laboratory-built high-sensitivity flow cytometer.

    Science.gov (United States)

    Yang, Lingling; Zhou, Yingxing; Zhu, Shaobin; Huang, Tianxun; Wu, Lina; Yan, Xiaomei

    2012-02-07

    Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents.

  6. CAP-D3 Promotes Bacterial Clearance in Human Intestinal Epithelial Cells by Repressing Expression of Amino Acid Transporters

    Science.gov (United States)

    Kemp, Jacqueline R.; Nickerson, Kourtney P.; Deutschman, Emily; Kim, Yeojung; West, Gail; Sadler, Tammy; Stylianou, Eleni; Krokowski, Dawid; Hatzoglou, Maria; de la Motte, Carol; Rubin, Brian P.; Fiocchi, Claudio

    2015-01-01

    BACKGROUND & AIMS Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The chromosome-associated protein D3 (dCAP-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, whose products heterodimerize to form an amino acid transporter in HT-29 cells following bacterial infection; levels of SLC7A5–SLC3A2 were increased in tissues from patients with UC, compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5–SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3– deficient cells. CONCLUSIONS CAP-D3 downregulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with

  7. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Favi, Pelagie M.; Benson, Roberto S. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Neilsen, Nancy R. [Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Hammonds, Ryan L. [Department of Materials Science and Engineering, College of Engineering, University of Tennessee, Knoxville, TN 37996 (United States); Bates, Cassandra C. [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Stephens, Christopher P. [Department of Surgery, Graduate School of Medicine, University of Tennessee, Knoxville, TN 37996 (United States); Center for Materials Processing, University of Tennessee, Knoxville, TN 37996 (United States); Dhar, Madhu S., E-mail: mdhar@utk.edu [Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996 (United States)

    2013-05-01

    The culture of multipotent mesenchymal stem cells on natural biopolymers holds great promise for treatments of connective tissue disorders such as osteoarthritis. The safety and performance of such therapies relies on the systematic in vitro evaluation of the developed stem cell-biomaterial constructs prior to in vivo implantation. This study evaluates bacterial cellulose (BC), a biocompatible natural polymer, as a scaffold for equine-derived bone marrow mesenchymal stem cells (EqMSCs) for application in bone and cartilage tissue engineering. An equine model was chosen due to similarities in size, load and types of joint injuries suffered by horses and humans. Lyophilized and critical point dried BC hydrogel scaffolds were characterized using scanning electron microscopy (SEM) to confirm nanostructure morphology which demonstrated that critical point drying induces fibre bundling unlike lyophilisation. EqMSCs positively expressed the undifferentiated pluripotent mesenchymal stem cell surface markers CD44 and CD90. The BC scaffolds were shown to be cytocompatible, supporting cellular adhesion and proliferation, and allowed for osteogenic and chondrogenic differentiation of EqMSCs. The cells seeded on the BC hydrogel were shown to be viable and metabolically active. These findings demonstrate that the combination of a BC hydrogel and EqMSCs are promising constructs for musculoskeletal tissue engineering applications. - Highlights: ► Critical point drying induces fibre bundling unlike lyophilisation. ► Cells positively expressed undifferentiated pluripotent stem cell markers. ► BCs were cytocompatible, supported cell adhesion, proliferation and differentiation ► Cells seeded on BC scaffolds were viable and metabolically active. ► Findings demonstrate that BC and EqMSCs are promising tissue engineered constructs.

  8. Direct measurement of cell wall stress-stiffening and turgor pressure in live bacterial cells

    CERN Document Server

    Deng, Yi; Shaevitz, Joshua W

    2011-01-01

    The mechanical properties of gram-negative bacteria are governed by a rigid peptidoglycan (PG) cell wall and the turgor pressure generated by the large concentration of solutes in the cytoplasm. The elasticity of the PG has been measured in bulk and in isolated sacculi and shown to be compliant compared to the overall stiffness of the cell itself. However, the stiffness of the cell wall in live cells has not been measured. In particular, the effects that pressure-induced stress might have on the stiffness of the mesh-like PG network have not been addressed even though polymeric materials often exhibit large amounts of stress-stiffening. We study bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. We find strong evidence of power-law stress-stiffening in the E. coli cell wall, with an exponent of $1.07 \\pm 0.25$, such that the wall is significantly stiffer in live cells ($E\\sim32\\pm10$ MPa) than in unpres...

  9. Influence of setup and carbon source on the bacterial community of biocathodes in microbial electrolysis cells.

    Science.gov (United States)

    Croese, Elsemiek; Jeremiasse, Adriaan W; Marshall, Ian P G; Spormann, Alfred M; Euverink, Gert-Jan W; Geelhoed, Jeanine S; Stams, Alfons J M; Plugge, Caroline M

    2014-01-01

    The microbial electrolysis cell (MEC) biocathode has shown great potential as alternative for expensive metals as catalyst for H2 synthesis. Here, the bacterial communities at the biocathode of five hydrogen producing MECs using molecular techniques were characterized. The setups differed in design (large versus small) including electrode material and flow path and in carbon source provided at the cathode (bicarbonate or acetate). A hydrogenase gene-based DNA microarray (Hydrogenase Chip) was used to analyze hydrogenase genes present in the three large setups. The small setups showed dominant groups of Firmicutes and two of the large setups showed dominant groups of Proteobacteria and Bacteroidetes. The third large setup received acetate but no sulfate (no sulfur source). In this setup an almost pure culture of a Promicromonospora sp. developed. Most of the hydrogenase genes detected were coding for bidirectional Hox-type hydrogenases, which have shown to be involved in cytoplasmatic H2 production.

  10. Bacterial nanocellulose/Nafion composite membranes for low temperature polymer electrolyte fuel cells

    Science.gov (United States)

    Jiang, Gao-peng; Zhang, Jing; Qiao, Jin-li; Jiang, Yong-ming; Zarrin, Hadis; Chen, Zhongwei; Hong, Feng

    2015-01-01

    Novel nanocomposite membranes aimed for both proton-exchange membrane fuel cell (PEMFC) and direct methanol fuel cell (DMFC) are presented in this work. The membranes are based on blending bacterial nanocellulose pulp and Nafion (abbreviated as BxNy, where x and y indicates the mass ratio of bacterial cellulose to Nafion). The structure and properties of BxNy membranes are characterized by FTIR, SEM, TG, DMA and EIS, along with water uptake, swelling behavior and methanol permeability tests. It is found that the BxNy composite membranes with reinforced concrete-like structure show excellent mechanical and thermal stability regardless of annealing. The water uptake plus area and volume swelling ratios are all decreased compared to Nafion membranes. The proton conductivities of pristine and annealed B1N9 are 0.071 and 0.056 S cm-1, respectively, at 30 °C and 100% humidity. Specifically, annealed B1N1 exhibited the lowest methanol permeability of 7.21 × 10-7 cm2 s-1. Through the selectivity analysis, pristine and annealed B1N7 are selected to assemble the MEAs. The performances of annealed B1N7 in PEMFC and DMFC show the maximum power densities of 106 and 3.2 mW cm-2, respectively, which are much higher than those of pristine B1N7 at 25 °C. The performances of the pristine and annealed B1N7 reach a level as high as 21.1 and 20.4 mW cm-2 at 80 °C in DMFC, respectively.

  11. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2012-06-01

    Full Text Available Abstract Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG 25 and diazo-dye Acid Red (AR 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l with relative decolorization values of 91.2% (3 h and 97.1% (18 h, as well as high activity to AR18 (1 g/l by 80.5% (3 h and 89.0% (18 h, was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l. No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved

  12. Improved method for bacterial cell capture after flow cytometry cell sorting.

    Science.gov (United States)

    Guillebault, D; Laghdass, M; Catala, P; Obernosterer, I; Lebaron, P

    2010-11-01

    Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification.

  13. The contribution of cell-cell signaling and motility to bacterial biofilm formation

    DEFF Research Database (Denmark)

    Shrout, Joshua D; Tolker-Nielsen, Tim; Givskov, Michael;

    2011-01-01

    Many bacteria grow attached to a surface as biofilms. Several factors dictate biofilm formation, including responses by the colonizing bacteria to their environment. Here we review how bacteria use cell-cell signaling (also called quorum sensing) and motility during biofilm formation. Specificall...

  14. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    Science.gov (United States)

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  15. Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification.

    Science.gov (United States)

    Manti, Anita; Boi, Paola; Amalfitano, Stefano; Puddu, Alberto; Papa, Stefano

    2011-12-01

    Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.

  16. Phenotypic T cell exhaustion in a murine model of bacterial infection in the setting of pre-existing malignancy.

    Directory of Open Access Journals (Sweden)

    Rohit Mittal

    Full Text Available While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8+ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with Listeria monocytogenes, and antigen-specific CD8+ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8+ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8+ T cells and total CD4+ and CD8+ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8+ T cells in the cancer group. Taken together, these data suggest that cancer's immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8+ T cell functionality and differentiation.

  17. Sensing the structural differences in cellulose from apple and bacterial cell wall materials by Raman and FT-IR spectroscopy.

    Science.gov (United States)

    Szymańska-Chargot, Monika; Cybulska, Justyna; Zdunek, Artur

    2011-01-01

    Raman and Fourier Transform Infrared (FT-IR) spectroscopy was used for assessment of structural differences of celluloses of various origins. Investigated celluloses were: bacterial celluloses cultured in presence of pectin and/or xyloglucan, as well as commercial celluloses and cellulose extracted from apple parenchyma. FT-IR spectra were used to estimate of the I(β) content, whereas Raman spectra were used to evaluate the degree of crystallinity of the cellulose. The crystallinity index (X(C)(RAMAN)%) varied from -25% for apple cellulose to 53% for microcrystalline commercial cellulose. Considering bacterial cellulose, addition of xyloglucan has an impact on the percentage content of cellulose I(β). However, addition of only xyloglucan or only pectins to pure bacterial cellulose both resulted in a slight decrease of crystallinity. However, culturing bacterial cellulose in the presence of mixtures of xyloglucan and pectins results in an increase of crystallinity. The results confirmed that the higher degree of crystallinity, the broader the peak around 913 cm(-1). Among all bacterial celluloses the bacterial cellulose cultured in presence of xyloglucan and pectin (BCPX) has the most similar structure to those observed in natural primary cell walls.

  18. Sensing the Structural Differences in Cellulose from Apple and Bacterial Cell Wall Materials by Raman and FT-IR Spectroscopy

    Directory of Open Access Journals (Sweden)

    Artur Zdunek

    2011-05-01

    Full Text Available Raman and Fourier Transform Infrared (FT-IR spectroscopy was used for assessment of structural differences of celluloses of various origins. Investigated celluloses were: bacterial celluloses cultured in presence of pectin and/or xyloglucan, as well as commercial celluloses and cellulose extracted from apple parenchyma. FT-IR spectra were used to estimate of the Iβ content, whereas Raman spectra were used to evaluate the degree of crystallinity of the cellulose. The crystallinity index (XCRAMAN% varied from −25% for apple cellulose to 53% for microcrystalline commercial cellulose. Considering bacterial cellulose, addition of xyloglucan has an impact on the percentage content of cellulose Iβ. However, addition of only xyloglucan or only pectins to pure bacterial cellulose both resulted in a slight decrease of crystallinity. However, culturing bacterial cellulose in the presence of mixtures of xyloglucan and pectins results in an increase of crystallinity. The results confirmed that the higher degree of crystallinity, the broader the peak around 913 cm−1. Among all bacterial celluloses the bacterial cellulose cultured in presence of xyloglucan and pectin (BCPX has the most similar structure to those observed in natural primary cell walls.

  19. Ascites bacterial burden and immune cell profile are associated with poor clinical outcomes in the absence of overt infection.

    Directory of Open Access Journals (Sweden)

    Kevin J Fagan

    Full Text Available Bacterial infections, most commonly spontaneous bacterial peritonitis in patients with ascites, occur in one third of admitted patients with cirrhosis, and account for a 4-fold increase in mortality. Bacteria are isolated from less than 40% of ascites infections by culture, necessitating empirical antibiotic treatment, but culture-independent studies suggest bacteria are commonly present, even in the absence of overt infection. Widespread detection of low levels of bacteria in ascites, in the absence of peritonitis, suggests immune impairment may contribute to higher susceptibility to infection in cirrhotic patients. However, little is known about the role of ascites leukocyte composition and function in this context. We determined ascites bacterial composition by quantitative PCR and 16S rRNA gene sequencing in 25 patients with culture-negative, non-neutrocytic ascites, and compared microbiological data with ascites and peripheral blood leukocyte composition and phenotype. Bacterial DNA was detected in ascitic fluid from 23 of 25 patients, with significant positive correlations between bacterial DNA levels and poor 6-month clinical outcomes (death, readmission. Ascites leukocyte composition was variable, but dominated by macrophages or T lymphocytes, with lower numbers of B lymphocytes and natural killer cells. Consistent with the hypothesis that impaired innate immunity contributes to susceptibility to infection, high bacterial DNA burden was associated with reduced major histocompatibility complex class II expression on ascites (but not peripheral blood monocytes/macrophages. These data indicate an association between the presence of ascites bacterial DNA and early death and readmission in patients with decompensated cirrhosis. They further suggest that impairment of innate immunity contributes to increased bacterial translocation, risk of peritonitis, or both.

  20. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    Energy Technology Data Exchange (ETDEWEB)

    Economou, Nicoleta J.; Zentner, Isaac J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States); Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian [Brookhaven National Laboratory, Upton, NY 11973 (United States); Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J. [Drexel University College of Medicine, 245 North 15th Street, Philadelphia, PA 19102 (United States)

    2013-04-01

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance.

  1. Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Dalton, Helen M.; Angels, Mark;

    1997-01-01

    A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells: a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of Fluorescently labelled oligonucleotide probes to rRNA. The method a...

  2. X-ray crystallography and its impact on understanding bacterial cell wall remodeling processes.

    Science.gov (United States)

    Büttner, Felix Michael; Renner-Schneck, Michaela; Stehle, Thilo

    2015-02-01

    The molecular structure of matter defines its properties and function. This is especially true for biological macromolecules such as proteins, which participate in virtually all biochemical processes. A three dimensional structural model of a protein is thus essential for the detailed understanding of its physiological function and the characterization of essential properties such as ligand binding and reaction mechanism. X-ray crystallography is a well-established technique that has been used for many years, but it is still by far the most widely used method for structure determination. A particular strength of this technique is the elucidation of atomic details of molecular interactions, thus providing an invaluable tool for a multitude of scientific projects ranging from the structural classification of macromolecules over the validation of enzymatic mechanisms or the understanding of host-pathogen interactions to structure-guided drug design. In the first part of this review, we describe essential methodological and practical aspects of X-ray crystallography. We provide some pointers that should allow researchers without a background in structural biology to assess the overall quality and reliability of a crystal structure. To highlight its potential, we then survey the impact X-ray crystallography has had on advancing an understanding of a class of enzymes that modify the bacterial cell wall. A substantial number of different bacterial amidase structures have been solved, mostly by X-ray crystallography. Comparison of these structures highlights conserved as well as divergent features. In combination with functional analyses, structural information on these enzymes has therefore proven to be a valuable template not only for understanding their mechanism of catalysis, but also for targeted interference with substrate binding.

  3. Electricity producing property and bacterial community structure in microbial fuel cell equipped with membrane electrode assembly.

    Science.gov (United States)

    Rubaba, Owen; Araki, Yoko; Yamamoto, Shuji; Suzuki, Kei; Sakamoto, Hisatoshi; Matsuda, Atsunori; Futamata, Hiroyuki

    2013-07-01

    It is important for practical use of microbial fuel cells (MFCs) to not only develop electrodes and proton exchange membranes but also to understand the bacterial community structure related to electricity generation. Four lactate fed MFCs equipped with different membrane electrode assemblies (MEAs) were constructed with paddy field soil as inoculum. The MEAs significantly affected the electricity-generating properties of the MFCs. MEA-I was made with Nafion 117 solution and the other MEAs were made with different configurations of three kinds of polymers. MFC-I equipped with MEA-I exhibited the highest performance with a stable current density of 55 ± 3 mA m⁻². MFC-III equipped with MEA-III with the highest platinum concentration, exhibited the lowest performance with a stable current density of 1.7 ± 0.1 mA m⁻². SEM observation revealed that there were cracks on MEA-III. These results demonstrated that it is significantly important to prevent oxygen-intrusion for improved MFC performance. By comparing the data of DGGE and phylogenetic analyzes, it was suggested that the dominant bacterial communities of MFC-I were constructed with lactate-fermenters and Fe(III)-reducers, which consisted of bacteria affiliated with the genera of Enterobacter, Dechlorosoma, Pelobacter, Desulfovibrio, Propioniferax, Pelosinus, and Firmicutes. A bacterium sharing 100% similarity to one of the DGGE bands was isolated from MFC-I. The 16S rRNA gene sequence of the isolate shared 98% similarity to gram-positive Propioniferax sp. P7 and it was confirmed that the isolate produced electricity in an MFC. These results suggested that these bacteria are valuable for constructing the electron transfer network in MFC.

  4. Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope

    Directory of Open Access Journals (Sweden)

    Tran Ngat

    2011-05-01

    Full Text Available Abstract Background A decline in the discovery of new antibacterial drugs, coupled with a persistent rise in the occurrence of drug-resistant bacteria, has highlighted antibiotics as a diminishing resource. The future development of new drugs with novel antibacterial activities requires a detailed understanding of adaptive responses to existing compounds. This study uses Streptomyces coelicolor A3(2 as a model system to determine the genome-wide transcriptional response following exposure to three antibiotics (vancomycin, moenomycin A and bacitracin that target distinct stages of cell wall biosynthesis. Results A generalised response to all three antibiotics was identified which involves activation of transcription of the cell envelope stress sigma factor σE, together with elements of the stringent response, and of the heat, osmotic and oxidative stress regulons. Attenuation of this system by deletion of genes encoding the osmotic stress sigma factor σB or the ppGpp synthetase RelA reduced resistance to both vancomycin and bacitracin. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. Sensitivity studies using mutants constructed on the basis of the transcriptome profiling confirmed a role for several such genes in antibiotic resistance, validating the usefulness of the approach. Conclusions Antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses. Both can be exploited to increase antibiotic susceptibility. Regulatory networks known to govern responses to environmental and nutritional stresses are also at the core of the common antibiotic response, and likely help cells survive until any specific resistance mechanisms are fully functional.

  5. Microencapsulation for the Therapeutic Delivery of Drugs, Live Mammalian and Bacterial Cells, and Other Biopharmaceutics: Current Status and Future Directions

    OpenAIRE

    Catherine Tomaro-Duchesneau; Shyamali Saha; Meenakshi Malhotra; Imen Kahouli; Satya Prakash

    2013-01-01

    Microencapsulation is a technology that has shown significant promise in biotherapeutics, and other applications. It has been proven useful in the immobilization of drugs, live mammalian and bacterial cells and other cells, and other biopharmaceutics molecules, as it can provide material structuration, protection of the enclosed product, and controlled release of the encapsulated contents, all of which can ensure efficient and safe therapeutic effects. This paper is a comprehensive review of ...

  6. Modeling nucleotide excision repair and its impact on UV-induced mutagenesis during SOS-response in bacterial cells.

    Science.gov (United States)

    Bugay, Aleksandr N; Krasavin, Evgeny A; Parkhomenko, Aleksandr Yu; Vasilyeva, Maria A

    2015-01-01

    A model of the UV-induced mutation process in Escherichia coli bacteria has been developed taking into account the whole sequence of molecular events starting from initial photo-damage and finishing with the fixation of point mutations. The wild-type phenotype bacterial cells are compared with UV-sensitive repair-deficient mutant cells. Attention is mainly paid to excision repair system functioning as regards induced mutagenesis.

  7. Construction and application of riboswitch-based sensors that detect metabolites within bacterial cells.

    Science.gov (United States)

    Fowler, Casey C; Li, Yingfu

    2014-01-01

    A riboswitch is an RNA element that detects the level of a specific metabolite within the cell and regulates the expression of co-transcribed genes. By fusing a riboswitch to a reporter protein in a carefully designed and tested construct, this ability can be exploited to create an intracellular sensor that detects the level of a particular small molecule within live bacterial cells. There is a great deal of flexibility in the design of such a sensor and factors such as the molecule to be detected and the downstream experiments in which the sensor will be applied should guide the specific blueprint of the final construct. The completed sensor plasmid needs to be rigorously tested with appropriate controls to ensure that its dynamic range, signal strength, sensitivity and specificity are suitable for its intended applications. In this chapter, methods for the design, assessment and use of riboswitch sensors are provided along with those for one example application for which riboswitch sensors are ideally suited.

  8. Recruitment of dendritic cells to the cerebrospinal fluid in bacterial neuroinfections.

    Science.gov (United States)

    Pashenkov, Mikhail; Teleshova, Natalia; Kouwenhoven, Mathilde; Smirnova, Tatiana; Jin, Ya Ping; Kostulas, Vasilios; Huang, Yu Min; Pinegin, Boris; Boiko, Alexey; Link, Hans

    2002-01-01

    Dendritic cells (DC) accumulate in the CNS during inflammation and may contribute to local immune responses. Two DC subsets present in human cerebrospinal fluid (CSF) are probably recruited from myeloid (CD11c(+)CD123(dim)) and plasmacytoid (CD11c(-)CD123(high)) blood DC. In bacterial meningitis and especially in Lyme meningoencephalitis, numbers of myeloid and plasmacytoid DC in CSF were increased, compared to non-inflammatory neurological diseases, and correlated with chemotactic activity of CSF for immature monocyte-derived DC (moDC). Multiple DC chemoattractants, including macrophage inflammatory protein (MIP)-1beta, monocyte chemotactic protein (MCP)-1, MCP-3, RANTES and stromal cell-derived factor (SDF)-1alpha were elevated in CSF in these two neuroinfections. Chemotaxis of immature moDC induced by these CSFs could be partially inhibited by mAbs against CXCR4, the receptor for SDF-1alpha, and CD88, the receptor for C5a. SDF-1alpha present in CSF also chemoattracted mature moDC, which in vivo could correspond to a diminished migration of antigen-bearing DC from the CSF to secondary lymphoid organs. Regulation of DC trafficking to and from the CSF may represent a mechanism of controlling the CNS inflammation.

  9. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

    Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting Ab......SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti......-polysaccharide AbSC of the IgG isotype, the increase was as high as 7.4-11.8 times. Evidence is presented that the pronounced improvement in the detection of the latter is due to the presence of aggregating anti-IgG antibody from the beginning of the assay. It is proposed that in the case of low affinity of anti...

  10. Modified bacterial cellulose scaffolds for localized doxorubicin release in human colorectal HT-29 cells.

    Science.gov (United States)

    Cacicedo, Maximiliano L; León, Ignacio E; Gonzalez, Jimena S; Porto, Luismar M; Alvarez, Vera A; Castro, Guillermo R

    2016-04-01

    Bacterial cellulose (BC) films modified by the in situ method with the addition of alginate (Alg) during the microbial cultivation of Gluconacetobacter hansenii under static conditions increased the loading of doxorubicin by at least three times. Biophysical analysis of BC-Alg films by scanning electron microscopy, thermogravimetry, X-ray diffraction and FTIR showed a highly homogeneous interpenetrated network scaffold without changes in the BC crystalline structure but with an increased amorphous phase. The main molecular interactions determined by FTIR between both biopolymers clearly suggest high compatibility. These results indicate that alginate plays a key role in the biophysical properties of the hybrid BC matrix. BC-Alg scaffold analysis by nitrogen adsorption isotherms revealed by the Brunauer-Emmett-Teller (BET) method an increase in surface area of about 84% and in pore volume of more than 200%. The Barrett-Joyner-Halenda (BJH) model also showed an increase of about 25% in the pore size compared to the BC film. Loading BC-Alg scaffolds with different amounts of doxorubicin decreased the cell viability of HT-29 human colorectal adenocarcinoma cell line compared to the free Dox from around 95-53% after 24h and from 63% to 37% after 48 h. Dox kinetic release from the BC-Alg nanocomposite displayed hyperbolic curves related to the different amounts of drug payload and was stable for at least 14 days. The results of the BC-Alg nanocomposites show a promissory potential for anticancer therapies of solid tumors.

  11. Structural insights into alginate binding by bacterial cell-surface protein.

    Science.gov (United States)

    Temtrirath, Kanate; Murata, Kousaku; Hashimoto, Wataru

    2015-03-02

    A gram-negative Sphingomonas sp. strain A1 inducibly forms a mouth-like pit on the cell surface in the presence of alginate and directly incorporates polymers into the cytoplasm via the pit and ABC transporter. Among the bacterial proteins involved in import of alginate, a cell-surface EfeO-like Algp7 shows an ability to bind alginate, suggesting its contribution to accumulate alginate in the pit. Here, we show identification of its positively charged cluster involved in alginate binding using X-ray crystallography, docking simulation, and site-directed mutagenesis. The tertiary structure of Algp7 was determined at a high resolution (1.99Å) by molecular replacement, although no alginates were included in the structure. Thus, an in silico model of Algp7/oligoalginate was constructed by docking simulation using atomic coordinates of Algp7 and alginate oligosaccharides, where some charged residues were found to be potential candidates for alginate binding. Site-directed mutagenesis was conducted and five purified mutants K68A, K69A, E194A, N221A, and K68A/K69A were subjected to a binding assay. UV absorption difference spectroscopy along with differential scanning fluorimetry analysis indicated that K68A/K69A exhibited a significant reduction in binding affinity with alginate than wild-type Algp7. Based on these data, Lys68/Lys69 residues of Algp7 probably play an important role in binding alginate.

  12. The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets

    Directory of Open Access Journals (Sweden)

    Yao Liu

    2016-08-01

    Full Text Available Peptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied extensively over the last twenty years. The pathway starts in the cytoplasm, continues in the cytoplasmic membrane and finishes in the periplasmic space, where the precursor is polymerized into the peptidoglycan layer. A number of proteins involved in this pathway, such as the Mur enzymes and the penicillin binding proteins (PBPs, have been studied and regarded as good targets for antibiotics. The present review focuses on the membrane steps of peptidoglycan synthesis that involve two enzymes, MraY and MurG, the inhibitors of these enzymes and the inhibition mechanisms. We also discuss the challenges of targeting these two cytoplasmic membrane (associated proteins in bacterial cells and the perspectives on how to overcome the issues.

  13. Structure of Ristocetin A in Complex with a Bacterial Cell-wall Mimetic

    Energy Technology Data Exchange (ETDEWEB)

    Nahoum, V.; Spector, S; Loll, P

    2009-01-01

    Antimicrobial drug resistance is a serious public health problem and the development of new antibiotics has become an important priority. Ristocetin A is a class III glycopeptide antibiotic that is used in the diagnosis of von Willebrand disease and which has served as a lead compound for the development of new antimicrobial therapeutics. The 1.0 A resolution crystal structure of the complex between ristocetin A and a bacterial cell-wall peptide has been determined. As is observed for most other glycopeptide antibiotics, it is shown that ristocetin A forms a back-to-back dimer containing concave binding pockets that recognize the cell-wall peptide. A comparison of the structure of ristocetin A with those of class I glycopeptide antibiotics such as vancomycin and balhimycin identifies differences in the details of dimerization and ligand binding. The structure of the ligand-binding site reveals a likely explanation for ristocetin A's unique anticooperativity between dimerization and ligand binding.

  14. Relationship among specific bacterial counts and total bacterial and somatic cell counts and factors influencing their variation in ovine bulk tank milk.

    Science.gov (United States)

    de Garnica, M L; Linage, B; Carriedo, J A; De La Fuente, L F; García-Jimeno, M C; Santos, J A; Gonzalo, C

    2013-02-01

    To analyze the relationship among the counts of different organisms and total bacterial count (BTTBC) and somatic cell count (BTSCC) as determined in dairy laboratories in ovine bulk tank milk, 751 bulk tank milk samples from 205 dairy sheep flocks belonging to Consortium for Ovine Promotion (CPO) were collected between January and December 2011. Four samplings were carried out in each flock, once per season, throughout 1 yr. Variables analyzed were bulk tank counts of thermoduric, psychrotrophic, coliform, and gram-positive catalase-negative cocci (GPCNC) bacterial groups. Thermoduric, psychrotrophic, and coliform species were significantly related to BTTBC, whereas GPCNC were correlated with both BTTBC and BTSCC variables. Highest counts were for psychrotroph and coliform groups, and a moderate to high correlation (r=0.51) was found between both variables, indicating that poor cleaning practices in the flocks tend to select for less-resistant organisms, such as gram-negative rods. In addition, BTTBC correlated with BTSCC (r=0.42). Some variation factors for specific bacterial counts, such as breed, season, milking type, dry therapy, and milk yield, were also analyzed. Flock information was collected from flock books, annual audits, and the CPO traceability system. Psychrotrophs and coliforms had elevated counts in winter, whereas GPCNC were higher in summer and in hand-milked flocks. Dry therapy contributed to the reduction in psychrotrophic bacteria; therefore, some strains of mammary pathogens could also be psychrotrophic bacteria. Results of this study would be helpful for troubleshooting milk quality problems and developing premium payment systems in dairy sheep.

  15. Interference of bacterial cell-to-cell communication: A new concept of antimicrobial chemotherapy breaks antibiotic

    Directory of Open Access Journals (Sweden)

    Hidetada eHirakawa

    2013-05-01

    Full Text Available Bacteria use a cell-to-cell communication activity termed Quorum sensing to coordinate group behaviors in a cell-density dependent manner. Quorum sensing influences the expression profile of diverse genes, including antibiotic tolerance and virulence determinants, via specific chemical compounds called Auto-inducers. During quorum sensing, Gram-negative bacteria typically use an acylated homoserine lactone (AHL called auto-inducer 1 (AI-1. Since the first discovery of quorum sensing in a marine bacterium, it has been recognized that more than 100 species possess this mechanism of cell-to-cell communication. In addition to being of interest from a biological standpoint, quorum sensing is a potential target for antimicrobial chemotherapy. This unique concept of antimicrobial control relies on reducing the burden of virulence rather than killing the bacteria. It is believed that this approach will not only suppress the development of antibiotic resistance, but will also improve the treatment of refractory infections triggered by multi-drug resistant (MDR pathogens. In this paper, we review and track recent progress in studies on AHL inhibitors/modulators from a biological standpoint. It has been discovered that both natural and synthetic compounds can disrupt quorum sensing by a variety of means, such as jamming signal transduction, inhibition of signal production and break-down and trapping of signal compounds. We also focus on the regulatory elements that attenuate quorum sensing activities and discuss their unique properties. Understanding the biological roles of regulatory elements might be useful in developing inhibitor applications and understanding how quorum sensing is controlled.

  16. Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion

    Directory of Open Access Journals (Sweden)

    Carlson Petteri

    2010-10-01

    Full Text Available Abstract Background Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD, which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition. Results Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities. Conclusions These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

  17. Vigorous, but differential mononuclear cell response of cirrhotic patients to bacterial ligands

    Institute of Scientific and Technical Information of China (English)

    Varenka J Barbero-Becerra; María Concepción Gutiérrez-Ruiz; Carmen Maldonado-Bernal; Félix I Téllez-Avila; Roberto Alfaro-Lara; Florencia Vargas-Vorácková

    2011-01-01

    AIM: To study the role of gram-positive and gram-negative bacteria in the pathogenesis of liver injury, specifically the activation of inflammatory mediators. METHODS: Peripheral blood mononuclear cells of 20 out-patients were studied, 10 of them with cirrhosis. Peripheral blood mononuclear cells were isolated and exposed to lipopolysaccharide or lipoteichoic acid. CD14, Toll-like receptor 2 and 4 expression was determined by flow cytometry, and tumor necrosis factor (TNF) α, interleukin (IL)-1β, IL-6, IL-12 and IL-10 secretion in supernatants was determined by ELISA. RESULTS: Higher CD14, Toll-like receptor 2 and 4 expression was observed in peripheral blood mononuclear cells from cirrhotic patients, (P < 0.01, P < 0.006, P < 0.111) respectively. Lipopolysaccharide and lipoteichoic acid induced a further increase in CD14 expression (P < 0.111 lipopolysaccharide, P < 0.013 lipoteichoic acid), and a decrease in Toll-like receptor 2 (P < 0.008 lipopolysaccharide, P < 0.008 lipoteichoic acid) and Toll-like receptor 4 (P < 0.008 lipopolysaccharide, P < 0.028 lipoteichoic acid) expression. With the exception of TNFα, absolute cytokine secretion of peripheral blood mononuclear cells was lower in cirrhotic patients under nonexposure conditions (P < 0.070 IL-6, P < 0.009 IL-1β, P < 0.022 IL-12). Once exposed to lipopolysaccharide or lipoteichoic acid, absolute cytokine secretion of peripheral blood mononuclear cells was similar in cirrhotic and non-cirrhotic patients, determining a more vigorous response in the former (P < 0.005 TNFα, IL-1β, IL-6, IL-2 and IL-10 lipopolysaccharide; P < 0.037 TNFα; P < 0.006 IL-1β; P < 0.005 IL-6; P < 0.007 IL-12; P < 0.014 IL-10 lipoteichoic acid). Response of peripheral blood mononuclear cells was more intense after lipopolysaccharide than after lipoteichoic acid exposure. CONCLUSION: Peripheral blood mononuclear cells of cirrhotic patients are able to respond to a sudden bacterial ligand exposure, particularly lipopolysaccharide

  18. Studies on interfacial interactions of TiO2 nanoparticles with bacterial cells under light and dark conditions

    Indian Academy of Sciences (India)

    Swayamprava Dalai; Sunandan Pakrashi; Sujay Chakravarty; Shamima Hussain; N Chandrasekaran; Amitava Mukherjee

    2014-05-01

    The probable underlying mechanism(s) of bacterial cell–TiO2 nanoparticles (TiO2 NPs) interaction in the absence of photo-irradiation has been less studied since most of the prior cytotoxicity studies focused on irradiated TiO2. The present study draws attention to the possible role of cell surface–TiO2 NP interactions under dark conditions, through an array of spectroscopic and microscopic investigations. A dominant freshwater bacterial isolate, Bacillus licheniformis, which interacted with environmentally relevant concentrations of TiO2 NPs (1 g/mL), was analysed and compared under both light and dark conditions. Aggregation of cells upon NP interaction and adsorption of NPs onto the cell membrane was evident from the scanning electron micrographs under both light and dark conditions. The FT–IR and FT–Raman spectra suggested stress response of bacterial cells by elevated protein and polysaccharide content in the cell–NP interaction. The X-ray photoelectron spectroscopic data substantiated the reduction of titanium from Ti(IV) to Ti(III) species which might have contributed to the redox interactions on the cell surface under light as well as dark conditions. The internalization of NPs in the cytoplasm were obvious from the transmission electron micrographs. The consequent cell death/damage was confirmed through fluorescence spectroscopy and microscopy. To conclude, the current study established the substantial role of interfacial interactions in cytotoxicity of the TiO2 NPs irrespective of the irradiation conditions.

  19. Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells.

    Science.gov (United States)

    Hayward, Richard D; Cain, Robert J; McGhie, Emma J; Phillips, Neil; Garner, Matthew J; Koronakis, Vassilis

    2005-05-01

    A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.

  20. Bone marrow-derived cells participate in stromal remodeling of the lung following acute bacterial pneumonia in mice.

    Science.gov (United States)

    Serikov, Vladimir B; Mikhaylov, Viatcheslav M; Krasnodembskay, Anna D; Matthay, Michael A

    2008-01-01

    Bone marrow-derived cells (BMDC) have been shown to graft injured tissues, differentiate in specialized cells, and participate in repair. The importance of these processes in acute lung bacterial inflammation and development of fibrosis is unknown. The goal of this study was to investigate the temporal sequence and lineage commitment of BMDC in mouse lungs injured by bacterial pneumonia. We transplanted GFP-tagged BMDC into 5-Gy-irradiated C57BL/6 mice. After 3 months of recovery, mice were subjected to LD(50) intratracheal instillation of live E. coli (controls received saline) which produced pneumonia and subsequent areas of fibrosis. Lungs were investigated by immunohistology for up to 6 months. At the peak of lung inflammation, the predominant influx of BMDC were GFP(+) leukocytes. Postinflammatory foci of lung fibrosis were evident after 1-2 months. The fibrotic foci in lung stroma contained clusters of GFP(+) CD45(+) cells, GFP(+) vimentin-positive cells, and GFP(+) collagen I-positive fibroblasts. GFP(+) endothelial or epithelial cells were not identified. These data suggest that following 5-Gy irradiation and acute bacterial pneumonia, BMDC may temporarily participate in lung postinflammatory repair and stromal remodeling without long-term engraftment as specialized endothelial or epithelial cells.

  1. Connecting the dots of the bacterial cell cycle: Coordinating chromosome replication and segregation with cell division.

    Science.gov (United States)

    Hajduk, Isabella V; Rodrigues, Christopher D A; Harry, Elizabeth J

    2016-05-01

    Proper division site selection is crucial for the survival of all organisms. What still eludes us is how bacteria position their division site with high precision, and in tight coordination with chromosome replication and segregation. Until recently, the general belief, at least in the model organisms Bacillus subtilis and Escherichia coli, was that spatial regulation of division comes about by the combined negative regulatory mechanisms of the Min system and nucleoid occlusion. However, as we review here, these two systems cannot be solely responsible for division site selection and we highlight additional regulatory mechanisms that are at play. In this review, we put forward evidence of how chromosome replication and segregation may have direct links with cell division in these bacteria and the benefit of recent advances in chromosome conformation capture techniques in providing important information about how these three processes mechanistically work together to achieve accurate generation of progenitor cells.

  2. Bacterial β-(1,3)-glucan prevents DSS-induced IBD by restoring the reduced population of regulatory T cells.

    Science.gov (United States)

    Lee, Kwang-Ho; Park, Min; Ji, Kon-Young; Lee, Hwa-Youn; Jang, Ji-Hun; Yoon, Il-Joo; Oh, Seung-Su; Kim, Su-Man; Jeong, Yun-Hwa; Yun, Chul-Ho; Kim, Mi-Kyoung; Lee, In-Young; Choi, Ha-Rim; Ko, Ki-sung; Kang, Hyung-Sik

    2014-10-01

    Bacterial β-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi. We have previously developed a novel and high-yield β-(1,3)-glucan produced by Agrobacterium sp. R259. This study aimed to elucidate the functional mechanism and therapeutic efficacy of bacterial β-(1,3)-glucan in dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD).Mice were orally pretreated with bacterial β-(1,3)-glucan at daily doses of 2.5 or 5mg/kg for 2 weeks. After 6 days of DSS treatment, clinical assessment of IBD severity and expression of pro-inflammatory cytokines were evaluated. In vivo cell proliferation was examined by immunohistochemistry using Ki-67 and ER-TR7 antibodies. The frequency of regulatory T cells (Tregs) was analyzed by flow cytometry. Natural killer (NK) activity and IgA level were evaluated using NK cytotoxicity assay and ELISA.The deterioration of body weight gain, colonic architecture, disease score and histological score was recovered in DSS-induced IBD mice when pretreated with bacterial β-(1,3)-glucan. The recruitment of macrophages and the gene expression of proinflammatory cytokines, such as IL-1β, IL-6 and IL-17A/F, were markedly decreased in the colon of β-(1,3)-glucan-pretreated mice. β-(1,3)-Glucan induced the recovery of Tregs in terms of their frequency in DSS-induced IBD mice. Intriguingly, β-(1,3)-glucan reversed the functional defects of NK cells and excessive IgA production in DSS-induced IBD mice.We conclude that bacterial β-(1,3)-glucan prevented the progression of DSS-induced IBD by recovering the reduction of Tregs, functional defect of NK cells and excessive IgA production.

  3. In vitro behaviors of rat mesenchymal stem cells on bacterial celluloses with different moduli

    Energy Technology Data Exchange (ETDEWEB)

    Taokaew, Siriporn [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906 (United States); Phisalaphong, Muenduen [Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Zhang Newby, Bi-min, E-mail: bimin@uakron.edu [Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH 44325-3906 (United States)

    2014-05-01

    Compressive moduli of bacteria-synthesized cellulose (BC) were altered by two drying techniques: ambient-air drying and freeze drying. While no significant differences in dry weight were found, their cross-sectional structures and thickness varied greatly. Freeze dried BCs had loose cross-sectional structures and a thickness of ∼ 4.7 mm, whereas air dried BCs had more compacted cross-sectional structures and a thickness of ∼ 0.1 mm. The compressive moduli of the rehydrated freeze dried and rehydrated air dried BCs were measured to be 21.06 ± 0.22 kPa and 90.09 ± 21.07 kPa, respectively. When rat mesenchymal stem cells (rMSCs) were seeded on these BCs, they maintained a round morphology in the first 3 days of cultivation. More spread-out morphology and considerable proliferation on freeze dried BCs were observed in 7 days, but not on air-dried BCs. The cells were further grown for 3 weeks in the absence and presence of differentiation agents. Without using any differentiation agents, no detectable differentiation was noticed for rMSCs further cultivated on both types of BC. With differentiation inducing agents, chondrogenic differentiation, visualized by histological staining, was observed in some area of the rehydrated freeze dried BCs; while osteogenic differentiation was noticed on the stiffer rehydrated air dried BCs. - Graphical abstract: In the presence of induction agents, rat mesenchymal stem cells (rMSCs) preferentially differentiated into osteocytes on stiffer air dried BC films. - Highlights: • Bacterial cellulose (BC) sheets with different moduli generated by drying differently • Air-dried BC exhibited a modulus similar to that of bone. • Freeze-dried BC showed a modulus in the range of that of muscle. • Air-dried BC promoted the differentiation of rMSCs into osteocytes. • Freeze-dried BC promoted the differentiation of rMSCs into chondrocytes.

  4. Dissociation of a population of Pectobacterium atrosepticum SCRI1043 in tobacco plants: formation of bacterial emboli and dormant cells.

    Science.gov (United States)

    Gorshkov, Vladimir; Daminova, Amina; Ageeva, Marina; Petrova, Olga; Gogoleva, Natalya; Tarasova, Nadezhda; Gogolev, Yuri

    2014-05-01

    The population dynamics of Pectobacterium atrosepticum SCRI1043 (Pba) within tobacco plants was monitored from the time of inoculation until after long-term preservation of microorganisms in the remnants of dead plants. We found and characterised peculiar structures that totally occlude xylem vessels, which we have named bacterial emboli. Viable but non-culturable (VBN) Pba cells were identified in the remnants of dead plants, and the conditions for resuscitation of these VBN cells were established. Our investigation shows that dissociation of the integrated bacterial population during plant colonisation forms distinct subpopulations and cell morphotypes, which are likely to perform specific functions that ensure successful completion of the life cycle within the plant.

  5. Anhydride-functional silane immobilized onto titanium surfaces induces osteoblast cell differentiation and reduces bacterial adhesion and biofilm formation

    Energy Technology Data Exchange (ETDEWEB)

    Godoy-Gallardo, Maria, E-mail: maria.godoy.gallardo@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Guillem-Marti, Jordi, E-mail: jordi.guillem.marti@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Sevilla, Pablo, E-mail: psevilla@euss.es [Department of Mechanics, Escola Universitària Salesiana de Sarrià (EUSS), C/ Passeig de Sant Bosco, 42, 08017 Barcelona (Spain); Manero, José M., E-mail: jose.maria.manero@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); Gil, Francisco J., E-mail: francesc.xavier.gil@upc.edu [Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Metallurgy, Technical University of Catalonia (UPC), ETSEIB, Av. Diagonal 647, 08028 Barcelona (Spain); Centre for Research in NanoEngineering (CRNE) — UPC, C/ Pascual i Vila 15, 08028 Barcelona (Spain); and others

    2016-02-01

    Bacterial infection in dental implants along with osseointegration failure usually leads to loss of the device. Bioactive molecules with antibacterial properties can be attached to titanium surfaces with anchoring molecules such as silanes, preventing biofilm formation and improving osseointegration. Properties of silanes as molecular binders have been thoroughly studied, but research on the biological effects of these coatings is scarce. The aim of the present study was to determine the in vitro cell response and antibacterial effects of triethoxysilypropyl succinic anhydride (TESPSA) silane anchored on titanium surfaces. X-ray photoelectron spectroscopy confirmed a successful silanization. The silanized surfaces showed no cytotoxic effects. Gene expression analyses of Sarcoma Osteogenic (SaOS-2) osteoblast-like cells cultured on TESPSA silanized surfaces reported a remarkable increase of biochemical markers related to induction of osteoblastic cell differentiation. A manifest decrease of bacterial adhesion and biofilm formation at early stages was observed on treated substrates, while favoring cell adhesion and spreading in bacteria–cell co-cultures. Surfaces treated with TESPSA could enhance a biological sealing on implant surfaces against bacteria colonization of underlying tissues. Furthermore, it can be an effective anchoring platform of biomolecules on titanium surfaces with improved osteoblastic differentiation and antibacterial properties. - Highlights: • TESPSA silane induces osteoblast differentiation. • TESPSA reduces bacterial adhesion and biofilm formation. • TESPSA is a promising anchoring platform of biomolecules onto titanium.

  6. Cytotoxic responses to 405nm light exposure in mammalian and bacterial cells: Involvement of reactive oxygen species.

    Science.gov (United States)

    Ramakrishnan, Praveen; Maclean, Michelle; MacGregor, Scott J; Anderson, John G; Grant, M Helen

    2016-06-01

    Light at wavelength 405 nm is an effective bactericide. Previous studies showed that exposing mammalian cells to 405 nm light at 36 J/cm(2) (a bactericidal dose) had no significant effect on normal cell function, although at higher doses (54 J/cm(2)), mammalian cell death became evident. This research demonstrates that mammalian and bacterial cell toxicity induced by 405 nm light exposure is accompanied by reactive oxygen species production, as detected by generation of fluorescence from 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate. As indicators of the resulting oxidative stress in mammalian cells, a decrease in intracellular reduced glutathione content and a corresponding increase in the efflux of oxidised glutathione were observed from 405 nm light treated cells. The mammalian cells were significantly protected from dying at 54 J/cm(2) in the presence of catalase, which detoxifies H2O2. Bacterial cells were significantly protected by sodium pyruvate (H2O2 scavenger) and by a combination of free radical scavengers (sodium pyruvate, dimethyl thiourea (OH scavenger) and catalase) at 162 and 324 J/cm(2). Results therefore suggested that the cytotoxic mechanism of 405 nm light in mammalian cells and bacteria could be oxidative stress involving predominantly H2O2 generation, with other ROS contributing to the damage.

  7. An ethanol biosensor based on a bacterial cell-immobilized eggshell membrane

    Institute of Scientific and Technical Information of China (English)

    Guang Ming Wen; Shao Min Shuang; Chuan Dong; Martin M.F. Choi

    2012-01-01

    An ethanol biosensor was fabricated based on a Methylobacterium organophilium-immobilized eggshell membrane and an oxygen (O2) electrode.A linear response for ethanol was obtained in the range of 0.050-7.5 mmol/L with a detection limit of 0.025 mmol/L (S/N =3) and a R.S.D.of 2.1%.The response time was less than 100 s at room temperature and ambient pressure.The optimal loading of bacterial cells on the biosensor membrane is 40 mg (wet weight).The optimal working conditions for the microbial biosensor are pH 7.0 phosphate buffer (50 mmol/L) at 20-25 ℃.The interference test,operational and storage stability of the biosensor are studied in detail.Finally,the biosensor is applied to determine the ethanol contents in various alcohol samples and the results are comparable to that obtained by gas chromatographic method and the results are satisfactory.Our proposed biosensor provides a convenient,simple and reliable method to determine ethanol content in alcoholic drinks.

  8. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays.

    Science.gov (United States)

    Henriksen, S T; Liu, J; Estiu, G; Oltvai, Z N; Wiest, O

    2010-07-15

    The rapid spread on multidrug-resistant strains of Staphylococcus aureus requires not just novel treatment options, but the development of faster methods for the identification of new hits for drug development. The exponentially increasing speed of computational methods makes a more extensive use in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking, followed by ensemble rescoring, that is sufficiently accurate to justify immediate testing of the identified compounds by whole-cell assays, avoiding the need for time-consuming and often difficult intermediary enzyme assays. This novel strategy is demonstrated for three key enzymes of the S. aureus histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of approximately 10(6) compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus- and two Escherichia coli strains in standard disk inhibition assays. Thirteen compounds are inhibitors of some or all of the S. aureus strains, while 14 compounds weakly inhibit growth in one or both E. coli strains. The high hit rate obtained from a fast virtual screen demonstrates the applicability of this novel strategy to the histidine biosynthesis pathway.

  9. Interaction of Gram-negative bacteria with cationic proteins: Dependence on the surface characteristics of the bacterial cell

    Directory of Open Access Journals (Sweden)

    Isabella R Prokhorenko

    2009-03-01

    Full Text Available Isabella R Prokhorenko1, Svetlana V Zubova1, Alexandr Yu Ivanov2, Sergey V Grachev31Laboratory of Molecular Biomedicine, Institute of Basic Biological Problems; 2Institute of Cell Biophysics, Russian Academy of Sciences, Moscow, Russia; 3I.M. Sechenov’s Moscow Medical Academy, Moscow, Russia Abstract: Gram-negative bacteria can enter the bloodstream and interact with serum cationic proteins. The character of interaction will depend on the surface characteristics of bacterial cells, which are determined by bacterial chemotype and density of lipopolysaccharide (LPS packing in the cell wall. It was shown that the lysozyme treatment resulted in the increase sensitivity to hypotonic shock. Signifi cant differences to this effect were found between Escherichia coli strain D21 and D21f2 under treatment with physiological protein concentration. On the basis of electrokinetic measurements and studies of the interaction of cells with lysozyme, the hypothesis was formed that the cell wall of the E. coli strain D21f2 contains more LPS and has a higher density of their packing than the cell wall of the E. coli D21 cells. The effect of lysozyme and lactoferrin on the viability of E. coli cells of two different strains was examined. Lysozyme was found to more effectively inhibit the growth of the E. coli D21 bacteria, and lactoferrin suppressed mainly the growth of the E. coli D21f2 bacteria. These results indicate that the differences in LPS core structure of bacterial R-chemotype, which determines surface charge and density of LPS packing, plays an essential role in the mechanisms of interaction of the cationic proteins with the cell wall.Keywords: lipopolysaccharide, Escherichia coli, chemotype, lysozyme, lactoferrin, colony-forming units

  10. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Salamon, Achim; Adam, Stefanie; Herzmann, Nicole [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Taubenheim, Jan [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Peters, Kirsten [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

    2013-11-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.

  11. Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells.

    Science.gov (United States)

    Fiedler, Tomas; Salamon, Achim; Adam, Stefanie; Herzmann, Nicole; Taubenheim, Jan; Peters, Kirsten

    2013-11-01

    Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions.

  12. Bacterial CD1d-restricted glycolipids induce IL-10 production by human regulatory T cells upon cross-talk with invariant NKT cells.

    Science.gov (United States)

    Venken, Koen; Decruy, Tine; Aspeslagh, Sandrine; Van Calenbergh, Serge; Lambrecht, Bart N; Elewaut, Dirk

    2013-09-01

    Invariant NKT (iNKT) cells and CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) are important immune regulatory T cells with Ag reactivity to glycolipids and peptides, respectively. However, the functional interplay between these cells in humans is poorly understood. We show that Tregs suppress iNKT cell proliferation induced by CD1d-restricted glycolipids, including bacterial-derived diacylglycerols, as well as by innate-like activation. Inhibition was related to the potency of iNKT agonists, making diacylglycerol iNKT responses very prone to suppression. Cytokine production by iNKT cells was differentially modulated by Tregs because IL-4 production was reduced more profoundly compared with IFN-γ. A compelling observation was the significant production of IL-10 by Tregs after cell contact with iNKT cells, in particular in the presence of bacterial diacylglycerols. These iNKT-primed Tregs showed increased FOXP3 expression and superior suppressive function. Suppression of iNKT cell responses, but not conventional T cell responses, was IL-10 dependent, suggesting that there is a clear difference in mechanism between the Treg-mediated inhibition of these cell types. Our data highlight a physiologically relevant interaction between human iNKT and Tregs upon pathogen-derived glycolipid recognition that has a significant impact on the design of iNKT cell-based therapeutics.

  13. The balance of apoptotic and necrotic cell death in Mycobacterium tuberculosis infected macrophages is not dependent on bacterial virulence.

    Directory of Open Access Journals (Sweden)

    Rachel E Butler

    Full Text Available BACKGROUND: An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission. METHODS: We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and M.tuberculosis strains including H37Rv, and isogenic virulent and avirulent strains of the Beijing lineage isolate GC1237. RESULTS: We show that bacterial virulence influences the dynamics of caspase activation and the total level of cytotoxicity. We show that the powerful ability of M.tuberculosis to inhibit exogenously stimulated apoptosis is abrogated by loss of virulence. However, loss of virulence did not influence the balance of macrophage apoptosis and necrosis--both virulent and avirulent isogenic strains of GC1237 induced predominantly necrotic cell death compared to H37Rv which induced a higher relative level of apoptosis. CONCLUSIONS: This reveals that macrophage necrosis and apoptosis are independently regulated during M. tuberculosis infection of macrophages. Virulence affects the level of host cell death and ability to inhibit apoptosis but other strain-specific characteristics influence the ultimate mode of host cell death and alter the balance of apoptosis and necrosis.

  14. Effects of season, milking routine and cow cleanliness on bacterial and somatic cell counts of bulk tank milk.

    Science.gov (United States)

    Zucali, Maddalena; Bava, Luciana; Tamburini, Alberto; Brasca, Milena; Vanoni, Laura; Sandrucci, Anna

    2011-11-01

    The aim of the study was to investigate the effects of season, cow cleanliness and milking routine on bacterial and somatic cell counts of bulk tank milk. A total of 22 dairy farms in Lombardy (Italy) were visited three times in a year in different seasons. During each visit, samples of bulk tank milk were taken for bacterial and somatic cell counts; swabs from the teat surface of a group of cows were collected after teat cleaning and before milking. Cow cleanliness was assessed by scoring udder, flanks and legs of all milking cows using a 4-point scale system. Season affected cow cleanliness with a significantly higher percentage of non-clean (NC) cows during Cold compared with Mild season. Standard plate count (SPC), laboratory pasteurization count (LPC), coliform count (CC) and somatic cell count, expressed as linear score (LS), in milk significantly increased in Hot compared with Cold season. Coagulase-positive staphylococci on teat swabs showed higher counts in Cold season in comparison with the other ones. The effect of cow cleanliness was significant for SPC, psychrotrophic bacterial count (PBC), CC and Escherichia coli in bulk tank milk. Somatic cell count showed a relationship with udder hygiene score. Milking operation routine strongly affected bacterial counts and LS of bulk tank milk: farms that accomplished a comprehensive milking scheme including two or more operations among forestripping, pre-dipping and post-dipping had lower teat contamination and lower milk SPC, PBC, LPC, CC and LS than farms that did not carry out any operation.

  15. A mixture of bacterial mechanical lysates is more efficient than single strain lysate and of bacterial-derived soluble products for the induction of an activating phenotype in human dendritic cells.

    Science.gov (United States)

    Morandi, Barbara; Agazzi, Alessia; D'Agostino, Antonella; Antonini, Francesca; Costa, Gregorio; Sabatini, Federica; Ferlazzo, Guido; Melioli, Giovanni

    2011-07-01

    Dendritic cells (DCs), following an optimal maturation, are able to drive an efficient immune-response. For this, both co-stimulatory molecules (CD80 and CD86), activation molecules (CD83) and peptide presenting molecules (HLA) are over-expressed. The in vitro treatment of immature DC with fragments of bacterial strains, obtained by using a mechanical lysis as well as with bacterial-derived molecules (such as lipopolysaccharide and protido-glycan), induced the maturation of DCs and the secretion of a panel of cytokines and chemokines. Of note, ex vivo treated circulating DCs and plasmacytoid DCs were also activated by these bacterial bodies. However, while the particulate fraction of single bacterial strains or soluble bacterial-derived molecules induced a sub-optimal maturation (as evaluated by the expression of an activating phenotype on DCs and the amount of cytokine secretion), the addition of the mixture of the particulate fractions of the different bacterial strains was able to mediate an optimal maturation. These results were also confirmed by using the secretion of both cytokines and chemokines as markers of DC activation. All these findings suggest that the particulate fraction of bacterial lysate mixtures, because of their ability to interact with different surface structures, might be exploited not only as an immunogen, but also as an adjuvant treatment to boost an immune-response to poorly "antigenic" proteins, such as cancer antigens or allergens.

  16. Yeast cell wall extract induces disease resistance against bacterial and fungal pathogens in Arabidopsis thaliana and Brassica crop.

    Directory of Open Access Journals (Sweden)

    Mari Narusaka

    Full Text Available Housaku Monogatari (HM is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods.

  17. Bacterial Adhesion of Streptococcus suis to Host Cells and Its Inhibition by Carbohydrate Ligands

    Directory of Open Access Journals (Sweden)

    Sauli Haataja

    2013-07-01

    Full Text Available Streptococcus suis is a Gram-positive bacterium, which causes sepsis and meningitis in pigs and humans. This review examines the role of known S. suis virulence factors in adhesion and S. suis carbohydrate-based adhesion mechanisms, as well as the inhibition of S. suis adhesion by anti-adhesion compounds in in vitro assays. Carbohydrate-binding specificities of S. suis have been identified, and these studies have shown that many strains recognize Galα1-4Gal-containing oligosaccharides present in host glycolipids. In the era of increasing antibiotic resistance, new means to treat infections are needed. Since microbial adhesion to carbohydrates is important to establish disease, compounds blocking adhesion could be an alternative to antibiotics. The use of oligosaccharides as drugs is generally hampered by their relatively low affinity (micromolar to compete with multivalent binding to host receptors. However, screening of a library of chemically modified Galα1-4Gal derivatives has identified compounds that inhibit S. suis adhesion in nanomolar range. Also, design of multivalent Galα1-4Gal-containing dendrimers has resulted in a significant increase of the inhibitory potency of the disaccharide. The S. suis adhesin binding to Galα1-4Gal-oligosaccharides, Streptococcal adhesin P (SadP, was recently identified. It has a Galα1-4Gal-binding N-terminal domain and a C-terminal LPNTG-motif for cell wall anchoring. The carbohydrate-binding domain has no homology to E. coli P fimbrial adhesin, which suggests that these Gram-positive and Gram-negative bacterial adhesins recognizing the same receptor have evolved by convergent evolution. SadP adhesin may represent a promising target for the design of anti-adhesion ligands for the prevention and treatment of S. suis infections.

  18. Denitrifying bacterial communities affect current production and nitrous oxide accumulation in a microbial fuel cell.

    Directory of Open Access Journals (Sweden)

    Ariadna Vilar-Sanz

    Full Text Available The biocathodic reduction of nitrate in Microbial Fuel Cells (MFCs is an alternative to remove nitrogen in low carbon to nitrogen wastewater and relies entirely on microbial activity. In this paper the community composition of denitrifiers in the cathode of a MFC is analysed in relation to added electron acceptors (nitrate and nitrite and organic matter in the cathode. Nitrate reducers and nitrite reducers were highly affected by the operational conditions and displayed high diversity. The number of retrieved species-level Operational Taxonomic Units (OTUs for narG, napA, nirS and nirK genes was 11, 10, 31 and 22, respectively. In contrast, nitrous oxide reducers remained virtually unchanged at all conditions. About 90% of the retrieved nosZ sequences grouped in a single OTU with a high similarity with Oligotropha carboxidovorans nosZ gene. nirS-containing denitrifiers were dominant at all conditions and accounted for a significant amount of the total bacterial density. Current production decreased from 15.0 A · m(-3 NCC (Net Cathodic Compartment, when nitrate was used as an electron acceptor, to 14.1 A · m(-3 NCC in the case of nitrite. Contrarily, nitrous oxide (N2O accumulation in the MFC was higher when nitrite was used as the main electron acceptor and accounted for 70% of gaseous nitrogen. Relative abundance of nitrite to nitrous oxide reducers, calculated as (qnirS+qnirK/qnosZ, correlated positively with N2O emissions. Collectively, data indicate that bacteria catalysing the initial denitrification steps in a MFC are highly influenced by main electron acceptors and have a major influence on current production and N2O accumulation.

  19. Measuring bacterial adaptation dynamics at the single-cell level using a microfluidic chemostat and time-lapse fluorescence microscopy.

    Science.gov (United States)

    Long, Zhicheng; Olliver, Anne; Brambilla, Elisa; Sclavi, Bianca; Lagomarsino, Marco Cosentino; Dorfman, Kevin D

    2014-10-21

    We monitored the dynamics of cell dimensions and reporter GFP expression in individual E. coli cells growing in a microfluidic chemostat using time-lapse fluorescence microscopy. This combination of techniques allows us to study the dynamical responses of single bacterial cells to nutritional shift-down or shift-up for longer times and with more precision over the chemical environment than similar experiments performed on conventional agar pads. We observed two E. coli strains containing different promoter-reporter gene constructs and measured how both their cell dimensions and the GFP expression change after nutritional upshift and downshift. As expected, both strains have similar adaptation dynamics for cell size rearrangement. However, the strain with a ribosomal RNA promoter dependent reporter has a faster GFP production rate than the strain with a constitutive promoter reporter. As a result, the mean GFP concentration in the former strain changes rapidly with the nutritional shift, while that in the latter strain remains relatively stable. These findings characterize the present microfluidic chemostat as a versatile platform for measuring single-cell bacterial dynamics and physiological transitions.

  20. The effects of non-metabolizing bacterial cells on the precipitation of U, Pb and Ca phosphates

    Science.gov (United States)

    Dunham-Cheatham, Sarrah; Rui, Xue; Bunker, Bruce; Menguy, Nicolas; Hellmann, Roland; Fein, Jeremy

    2011-05-01

    In this study, we test the potential for passive cell wall biomineralization by determining the effects of non-metabolizing bacteria on the precipitation of uranyl, lead, and calcium phosphates from a range of over-saturated conditions. Experiments were performed using Gram-positive Bacillus subtilis and Gram-negative Shewanella oneidensis MR-1. After equilibration, the aqueous phases were sampled and the remaining metal and P concentrations were analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES); the solid phases were collected and analyzed using X-ray diffractometry (XRD), transmission electron microscopy (TEM), and X-ray absorption spectroscopy (XAS). At the lower degrees of over-saturation studied, bacterial cells exerted no discernable effect on the mode of precipitation of the metal phosphates, with homogeneous precipitation occurring exclusively. However, at higher saturation states in the U system, we observed heterogeneous mineralization and extensive nucleation of hydrogen uranyl phosphate (HUP) mineralization throughout the fabric of the bacterial cell walls. This mineral nucleation effect was observed in both B. subtilis and S. oneidensis cells. In both cases, the biogenic mineral precipitates formed under the higher saturation state conditions were significantly smaller than those that formed in the abiotic controls. The cell wall nucleation effects that occurred in some of the U systems were not observed under any of the saturation state conditions studied in the Pb or Ca systems. The presence of B. subtilis significantly decreased the extent of precipitation in the U system, but had little effect in the Pb and Ca systems. At least part of this effect is due to higher solubility of the nanoscale HUP precipitate relative to macroscopic HUP. This study documents several effects of non-metabolizing bacterial cells on the nature and extent of metal phosphate precipitation. Each of these effects likely contributes to higher

  1. A man-made ATP-binding protein evolved independent of nature causes abnormal growth in bacterial cells.

    Directory of Open Access Journals (Sweden)

    Joshua M Stomel

    Full Text Available Recent advances in de novo protein evolution have made it possible to create synthetic proteins from unbiased libraries that fold into stable tertiary structures with predefined functions. However, it is not known whether such proteins will be functional when expressed inside living cells or how a host organism would respond to an encounter with a non-biological protein. Here, we examine the physiology and morphology of Escherichia coli cells engineered to express a synthetic ATP-binding protein evolved entirely from non-biological origins. We show that this man-made protein disrupts the normal energetic balance of the cell by altering the levels of intracellular ATP. This disruption cascades into a series of events that ultimately limit reproductive competency by inhibiting cell division. We now describe a detailed investigation into the synthetic biology of this man-made protein in a living bacterial organism, and the effect that this protein has on normal cell physiology.

  2. Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

    Directory of Open Access Journals (Sweden)

    Rafael Nisa-Martínez

    Full Text Available Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

  3. Effect of Bacterial Lipopolysaccharide Contamination on Gutta Percha- versus Resilon-Induced Human Monocyte Cell Line Toxicity.

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    Jamshid Hadjati

    2015-04-01

    Full Text Available Cytotoxic effects of obturation materials were tested in presence and absence of endotoxin on human monocytes in vitro.Human monocytes from THP-1 cell line were cultured. Three millimeters from the tip of each Resilon and gutta percha points were cut and directly placed at the bottom of the culture wells. Cultured cells were exposed to gutta percha (groups G1 and G2 and Resilon (R1 and R2. Ten μg/ml bacterial lipopolysaccharide (LPS was added to the culture wells in groups G1 and R1. Positive control included the bacterial LPS without the root canal filling material and the negative control contained the cells in culture medium only. Viability of cells was tested in all groups after 24, 48, and 72 hours using the methylthiazolyldiphenyl-tetrazolium bromide (MTT assay for at least 3 times to obtain reproducible results. Optical density values were read and the data were analyzed using three-way ANOVA and post hoc statistical test.The results showed that cells in G2 had the lowest rate of viability at 24 hours, but the lowest rate of viable cells was recorded in G1 at 48 and 72 hours. The effect of LPS treatment was not statistically significant. Resilon groups showed cell viability values higher than those of gutta percha groups, although statistically non-significant (P=0.105. Cell viability values were lower in gutta percha than Resilon groups when LPS-treated and LPS-untreated groups were compared independently at each time point.It could be concluded that none of the tested root canal filling materials had toxic effects on cultured human monocyte cells whether in presence or absence of LPS contamination.

  4. Vimentin in Bacterial Infections

    DEFF Research Database (Denmark)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate...... filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge...... about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria...

  5. Effect of premilking teat disinfection on mastitis incidence, total bacterial count, cell count and milk yield in three dairy herds.

    Science.gov (United States)

    Blowey, R W; Collis, K

    1992-02-29

    An iodophor teat disinfectant was applied before milking by dip or spray to 50 cows and 50 cows were left untreated in each of three commercial herds. The mean incidence of clinical mastitis was reduced by 57 per cent, the total bacterial count by 70 per cent and the count of thermoduric organisms by 32 per cent. These results were not statistically significant, except that one herd showed a significant decrease in total bacterial count. There was no effect on somatic cell count, milk production or milk iodine residues. Atmospheric iodine concentrations increased in the two herds which applied the treatment as a spray, but the levels attained were not likely to be detrimental to human health.

  6. Aggregatibacter actinomycetemcomitans Omp29 is associated with bacterial entry to gingival epithelial cells by F-actin rearrangement.

    Directory of Open Access Journals (Sweden)

    Mikihito Kajiya

    Full Text Available The onset and progressive pathogenesis of periodontal disease is thought to be initiated by the entry of Aggregatibacter actinomycetemcomitans (Aa into periodontal tissue, especially gingival epithelium. Nonetheless, the mechanism underlying such bacterial entry remains to be clarified. Therefore, this study aimed to investigate the possible role of Aa outer membrane protein 29 kD (Omp29, a homologue of E. coli OmpA, in promoting bacterial entry into gingival epithelial cells. To accomplish this, Omp29 expression vector was incorporated in an OmpA-deficient mutant of E. coli. Omp29(+/OmpA(- E. coli demonstrated 22-fold higher entry into human gingival epithelial line cells (OBA9 than Omp29(-/OmpA(- E. coli. While the entry of Aa and Omp29(+/OmpA(- E. coli into OBA9 cells were inhibited by anti-Omp29 antibody, their adherence to OBA9 cells was not inhibited. Stimulation of OBA9 cells with purified Omp29 increased the phosphorylation of focal adhesion kinase (FAK, a pivotal cell-signaling molecule that can up-regulate actin rearrangement. Furthermore, Omp29 increased the formation of F-actin in OBA9 cells. The internalization of Omp29-coated beads and the entry of Aa into OBA9 were partially inhibited by treatment with PI3-kinase inhibitor (Wortmannin and Rho GTPases inhibitor (EDIN, both known to convey FAK-signaling to actin-rearrangement. These results suggest that Omp29 is associated with the entry of Aa into gingival epithelial cells by up-regulating F-actin rearrangement via the FAK signaling pathway.

  7. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  8. High speed classification of individual bacterial cells using a model-based light scatter system and multivariate statistics

    Science.gov (United States)

    Venkatapathi, Murugesan; Rajwa, Bartek; Ragheb, Kathy; Banada, Padmapriya P.; Lary, Todd; Robinson, J. Paul; Hirleman, E. Daniel

    2008-02-01

    We describe a model-based instrument design combined with a statistical classification approach for the development and realization of high speed cell classification systems based on light scatter. In our work, angular light scatter from cells of four bacterial species of interest, Bacillus subtilis, Escherichia coli, Listeria innocua, and Enterococcus faecalis, was modeled using the discrete dipole approximation. We then optimized a scattering detector array design subject to some hardware constraints, configured the instrument, and gathered experimental data from the relevant bacterial cells. Using these models and experiments, it is shown that optimization using a nominal bacteria model (i.e., using a representative size and refractive index) is insufficient for classification of most bacteria in realistic applications. Hence the computational predictions were constituted in the form of scattering-data-vector distributions that accounted for expected variability in the physical properties between individual bacteria within the four species. After the detectors were optimized using the numerical results, they were used to measure scatter from both the known control samples and unknown bacterial cells. A multivariate statistical method based on a support vector machine (SVM) was used to classify the bacteria species based on light scatter signatures. In our final instrument, we realized correct classification of B. subtilis in the presence of E. coli,L. innocua, and E. faecalis using SVM at 99.1%, 99.6%, and 98.5%, respectively, in the optimal detector array configuration. For comparison, the corresponding values for another set of angles were only 69.9%, 71.7%, and 70.2% using SVM, and more importantly, this improved performance is consistent with classification predictions.

  9. Possible implication of bacterial infection in acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Shigeo eFuji

    2014-04-01

    Full Text Available Graft-versus-host disease (GVHD is still one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (HSCT. In the pathogenesis of acute GVHD, it has been established that donor-derived T cells activated in the recipient play a major role in GVHD in initiation and maintenance within an inflammatory cascade. To reduce the risk of GVHD, intensification of GVHD prophylaxis like T cell depletion is effective, but it inevitably increases the risk of infectious diseases and abrogates beneficial graft-versus-leukemia effects. Although various cytokines are considered to play an important role in the pathogenesis of GVHD, GVHD initiation is such a complex process that cannot be prevented by means of single inflammatory cytokine inhibition. Thus, efficient methods to control the whole inflammatory milieu both on cellular and humoral view are needed. In this context, infectious diseases can theoretically contribute to an elevation of inflammatory cytokines after allogeneic HSCT and activation of various subtypes of immune effector cells, which might in summary lead to an aggravation of acute GVHD. The appropriate treatments or prophylaxis of bacterial infection during the early phase after allogeneic HSCT might be beneficial to reduce not only infectious-related but also GVHD-related mortality. Here, we aim to review the literature addressing the interactions of bacterial infections and GVHD after allogeneic HSCT.

  10. Possible Implication of Bacterial Infection in Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

    Science.gov (United States)

    Fuji, Shigeo; Kapp, Markus; Einsele, Hermann

    2014-01-01

    Graft-versus-host disease (GVHD) is still one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (HSCT). In the pathogenesis of acute GVHD, it has been established that donor-derived T-cells activated in the recipient play a major role in GVHD in initiation and maintenance within an inflammatory cascade. To reduce the risk of GVHD, intensification of GVHD prophylaxis like T-cell depletion is effective, but it inevitably increases the risk of infectious diseases and abrogates beneficial graft-versus-leukemia effects. Although various cytokines are considered to play an important role in the pathogenesis of GVHD, GVHD initiation is such a complex process that cannot be prevented by means of single inflammatory cytokine inhibition. Thus, efficient methods to control the whole inflammatory milieu both on cellular and humoral view are needed. In this context, infectious diseases can theoretically contribute to an elevation of inflammatory cytokines after allogeneic HSCT and activation of various subtypes of immune effector cells, which might in summary lead to an aggravation of acute GVHD. The appropriate treatments or prophylaxis of bacterial infection during the early phase after allogeneic HSCT might be beneficial to reduce not only infectious-related but also GVHD-related mortality. Here, we aim to review the literature addressing the interactions of bacterial infections and GVHD after allogeneic HSCT. PMID:24795865

  11. Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells.

    Science.gov (United States)

    Flaherty, Rebecca A; Lee, Shaun W

    2016-08-19

    Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

  12. Administration of a polyvalent mechanical bacterial lysate to elderly patients with COPD: Effects on circulating T, B and NK cells.

    Science.gov (United States)

    Lanzilli, Giulia; Traggiai, Elisabetta; Braido, Fulvio; Garelli, Valentina; Folli, Chiara; Chiappori, Alessandra; Riccio, Anna Maria; Bazurro, Gyada; Agazzi, Alessia; Magnani, Alessandra; Canonica, Giorgio Walter; Melioli, Giovanni

    2013-01-01

    The modifications of the subsets of circulating lymphocytes were evaluated in a group of patients with COPD undergoing treatment with a polyvalent mechanical bacterial lysate (PMBL), a drug that is able to significantly modify the natural history of these patients. Using multicolor immune-florescence and flow cytometry, T, B subsets and NK cells were extensively studied both in the group of treated patients and in a disease and age matched controls. Despite the age, in treated patients, T and NK cells were significantly increased in numbers of circulating cells, but not in percentages, while B cells remained unmodified. CD3+4+T cells were increased in treated patients, while CD3+CD8T cells were unmodified by the treatment. Activated T cells were increased but Treg, resulted reduced both in percentage than in absolute numbers. Transitional B cells resulted increased (in percentage and in absolute numbers) in their late maturation step (T3), while only early Naïve B cells were increased by the treatment, while other naïve subpopulations were unmodified. Memory B cells were reduced in percentage (but remained unmodified as absolute numbers), while the most immature form of memory B cells was significantly increased. Finally, both switch memory B cells and plasma cells resulted unmodified by the PMBL treatment. These results clearly indicated that the administration of the PMBL, even in elderly patients with COPD, was able to induce a significant immune-stimulation and these results, at cellular level, clearly support the evidence that the mechanism of action of PMBL is strictly related to a direct effect on immune-competent cells.

  13. Nanoscale Cell Wall Deformation Impacts Long-Range Bacterial Adhesion Forces on Surfaces

    NARCIS (Netherlands)

    Chen, Yun; Harapanahalli, Akshay K.; Busscher, Henk J.; Norde, Willem; van der Mei, Henny C.

    2014-01-01

    Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of

  14. Nanoscale cell wall deformation impacts long-range bacterial adhesion forces on surfaces

    NARCIS (Netherlands)

    Chen, Y.; Harapanahalli, A.K.; Busscher, H.J.; Norde, W.; Mei, van der H.C.

    2014-01-01

    Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of

  15. Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

    Science.gov (United States)

    Brosig, Anton; Kuhrt, Heidrun; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2015-01-01

    Purpose The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. Results Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. Conclusions The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit

  16. Spatial distribution of bacterial communities on volumetric and planar anodes in single-chamber air-cathode microbial fuel cells

    KAUST Repository

    Vargas, Ignacio T.

    2013-05-29

    Pyrosequencing was used to characterize bacterial communities in air-cathode microbial fuel cells across a volumetric (graphite fiber brush) and a planar (carbon cloth) anode, where different physical and chemical gradients would be expected associated with the distance between anode location and the air cathode. As expected, the stable operational voltage and the coulombic efficiency (CE) were higher for the volumetric anode than the planar anode (0.57V and CE=22% vs. 0.51V and CE=12%). The genus Geobacter was the only known exoelectrogen among the observed dominant groups, comprising 57±4% of recovered sequences for the brush and 27±5% for the carbon-cloth anode. While the bacterial communities differed between the two anode materials, results showed that Geobacter spp. and other dominant bacterial groups were homogenously distributed across both planar and volumetric anodes. This lends support to previous community analysis interpretations based on a single biofilm sampling location in these systems. © 2013 Wiley Periodicals, Inc.

  17. Effect of Structure on the Interactions between Five Natural Antimicrobial Compounds and Phospholipids of Bacterial Cell Membrane on Model Monolayers

    Directory of Open Access Journals (Sweden)

    Stella W. Nowotarska

    2014-06-01

    Full Text Available Monolayers composed of bacterial phospholipids were used as model membranes to study interactions of the naturally occurring phenolic compounds 2,5-dihydroxybenzaldehyde and 2-hydroxy-5-methoxybenzaldehyde, and the plant essential oil compounds carvacrol, cinnamaldehyde, and geraniol, previously found to be active against both Gram-positive and Gram-negative pathogenic microorganisms. The lipid monolayers consist of 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE, 1,2-dihexa- decanoyl-sn-glycero-3-phospho-(1'-rac-glycerol (DPPG, and 1,1',2,2'-tetratetradecanoyl cardiolipin (cardiolipin. Surface pressure–area (π-A and surface potential–area (Δψ-A isotherms were measured to monitor changes in the thermodynamic and physical properties of the lipid monolayers. Results of the study indicated that the five compounds modified the three lipid monolayer structures by integrating into the monolayer, forming aggregates of antimicrobial –lipid complexes, reducing the packing effectiveness of the lipids, increasing the membrane fluidity, and altering the total dipole moment in the monolayer membrane model. The interactions of the five antimicrobial compounds with bacterial phospholipids depended on both the structure of the antimicrobials and the composition of the monolayers. The observed experimental results provide insight into the mechanism of the molecular interactions between naturally-occurring antimicrobial compounds and phospholipids of the bacterial cell membrane that govern activities.

  18. Microencapsulation for the Therapeutic Delivery of Drugs, Live Mammalian and Bacterial Cells, and Other Biopharmaceutics: Current Status and Future Directions

    Directory of Open Access Journals (Sweden)

    Catherine Tomaro-Duchesneau

    2013-01-01

    Full Text Available Microencapsulation is a technology that has shown significant promise in biotherapeutics, and other applications. It has been proven useful in the immobilization of drugs, live mammalian and bacterial cells and other cells, and other biopharmaceutics molecules, as it can provide material structuration, protection of the enclosed product, and controlled release of the encapsulated contents, all of which can ensure efficient and safe therapeutic effects. This paper is a comprehensive review of microencapsulation and its latest developments in the field. It provides a comprehensive overview of the technology and primary goals of microencapsulation and discusses various processes and techniques involved in microencapsulation including physical, chemical, physicochemical, and other methods involved. It also summarizes the state-of-the-art successes of microencapsulation, specifically with regard to the encapsulation of microorganisms, mammalian cells, drugs, and other biopharmaceutics in various diseases. The limitations and future directions of microencapsulation technologies are also discussed.

  19. Design, synthesis and antibacterial activity of cinnamaldehyde derivatives as inhibitors of the bacterial cell division protein FtsZ.

    Science.gov (United States)

    Li, Xin; Sheng, Juzheng; Huang, Guihua; Ma, Ruixin; Yin, Fengxin; Song, Di; Zhao, Can; Ma, Shutao

    2015-06-05

    In an attempt to discover potential antibacterial agents against the increasing bacterial resistance, novel cinnamaldehyde derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their antibacterial activity against nine significant pathogens using broth microdilution method, and their cell division inhibitory activity against four representative strains. In the in vitro antibacterial activity, the newly synthesized compounds generally displayed better efficacy against Staphylococcus aureus ATCC25923 than the others. In particular, compounds 3, 8 and 10 exerted superior or comparable activity to all the reference drugs. In the cell division inhibitory activity, all the compounds showed the same trend as their in vitro antibacterial activity, exhibiting better activity against S. aureus ATCC25923 than the other strains. Additionally, compounds 3, 6, 7 and 8 displayed potent cell division inhibitory activity with an MIC value of below 1 μg/mL, over 256-fold better than all the reference drugs.

  20. S-layer proteins from Lactobacillus sp. inhibit bacterial infection by blockage of DC-SIGN cell receptor.

    Science.gov (United States)

    Prado Acosta, Mariano; Ruzal, Sandra M; Cordo, Sandra M

    2016-11-01

    Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells.

  1. Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids.

    Science.gov (United States)

    Cava, Felipe; de Pedro, Miguel A; Lam, Hubert; Davis, Brigid M; Waldor, Matthew K

    2011-07-26

    Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities.

  2. N-terminal fusion tags for effective production of g-protein-coupled receptors in bacterial cell-free systems.

    Science.gov (United States)

    Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kulbatskiy, D S; Shulepko, M A; Petrovskaya, L E; Arseniev, A S; Dolgikh, D A; Kirpichnikov, M P

    2012-10-01

    G-protein-coupled receptors (GPCR) constitute one of the biggest families of membrane proteins. In spite of the fact that they are highly relevant to pharmacy, they have remained poorly explored. One of the main bottlenecks encountered in structural-functional studies of GPCRs is the difficulty to produce sufficient amounts of the proteins. Cell-free systems based on bacterial extracts fromE. colicells attract much attention as an effective tool for recombinant production of membrane proteins. GPCR production in bacterial cell-free expression systems is often inefficient because of the problems associated with the low efficiency of the translation initiation process. This problem could be resolved if GPCRs were expressed in the form of hybrid proteins with N-terminal polypeptide fusion tags. In the present work, three new N-terminal fusion tags are proposed for cell-free production of the human β2-adrenergic receptor, human M1 muscarinic acetylcholine receptor, and human somatostatin receptor type 5. It is demonstrated that the application of an N-terminal fragment (6 a.a.) of bacteriorhodopsin fromExiguobacterium sibiricum(ESR-tag), N-terminal fragment (16 а.о.) of RNAse A (S-tag), and Mistic protein fromB. subtilisallows to increase the CF synthesis of the target GPCRs by 5-38 times, resulting in yields of 0.6-3.8 mg from 1 ml of the reaction mixture, which is sufficient for structural-functional studies.

  3. The bacterial elicitor flagellin activates its receptor in tomato cells according to the address-message concept.

    Science.gov (United States)

    Meindl, T; Boller, T; Felix, G

    2000-09-01

    flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, acts as a potent elicitor in plants. Here, we have used an iodinated derivative of flg22 ((125)I-labeled Tyr-flg22) as a molecular probe for the flagellin receptor in tomato cells. This radioligand showed rapid binding to a single class of specific, saturable, high-affinity receptor sites in intact cells and membrane preparations. Binding, although essentially nonreversible under physiological conditions, was not covalent, and chemical cross-linking was required to specifically label a single polypeptide of 115 kD. Intact flagellin and elicitor-active flagellin peptides but not biologically inactive analogs efficiently competed for binding of radioligand. Peptides lacking the C terminus of the conserved domain, previously found to act as competitive antagonists of elicitor action in tomato cells, also competed for binding of radioligand. Thus, this novel, high-affinity binding site exhibited all the characteristics expected of a functional receptor of bacterial flagellin. For a model of receptor activation, we propose a two-step mechanism according to the address-message concept, in which binding of the N terminus (address) is the first step and activation of responses with the C terminus (message) is the second step.

  4. Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

    DEFF Research Database (Denmark)

    Rosenstiel, Philip; Sina, Christian; End, Caroline

    2007-01-01

    for the intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF......-kappaB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology...

  5. Cooperative Role for Tetraspanins in Adhesin-Mediated Attachment of Bacterial Species to Human Epithelial Cells ▿ †

    Science.gov (United States)

    Green, Luke R.; Monk, Peter N.; Partridge, Lynda J.; Morris, Paul; Gorringe, Andrew R.; Read, Robert C.

    2011-01-01

    The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis, the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis. In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus, Neisseria lactamica, Escherichia coli, and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria. PMID:21464080

  6. Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

    Science.gov (United States)

    Choudhary, Swati; Pardo, Alessa; Rosinke, Reinhard; Batra, Janendra K; Barth, Stefan; Verma, Rama S

    2016-01-01

    Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.

  7. Procalcitonin Identifies Cell Injury, Not Bacterial Infection, in Acute Liver Failure.

    Directory of Open Access Journals (Sweden)

    Jody A Rule

    Full Text Available Because acute liver failure (ALF patients share many clinical features with severe sepsis and septic shock, identifying bacterial infection clinically in ALF patients is challenging. Procalcitonin (PCT has proven to be a useful marker in detecting bacterial infection. We sought to determine whether PCT discriminated between presence and absence of infection in patients with ALF.Retrospective analysis of data and samples of 115 ALF patients from the United States Acute Liver Failure Study Group randomly selected from 1863 patients were classified for disease severity and ALF etiology. Twenty uninfected chronic liver disease (CLD subjects served as controls.Procalcitonin concentrations in most samples were elevated, with median values for all ALF groups near or above a 2.0 ng/mL cut-off that generally indicates severe sepsis. While PCT concentrations increased somewhat with apparent liver injury severity, there were no differences in PCT levels between the pre-defined severity groups-non-SIRS and SIRS groups with no documented infections and Severe Sepsis and Septic Shock groups with documented infections, (p = 0.169. PCT values from CLD patients differed from all ALF groups (median CLD PCT value 0.104 ng/mL, (p ≤0.001. Subjects with acetaminophen (APAP toxicity, many without evidence of infection, demonstrated median PCT >2.0 ng/mL, regardless of SIRS features, while some culture positive subjects had PCT values <2.0 ng/mL.While PCT appears to be a robust assay for detecting bacterial infection in the general population, there was poor discrimination between ALF patients with or without bacterial infection presumably because of the massive inflammation observed. Severe hepatocyte necrosis with inflammation results in elevated PCT levels, rendering this biomarker unreliable in the ALF setting.

  8. Topological relationship of prophage lambda to the bacterial chromosome in lysogenic cells.

    Science.gov (United States)

    Freifelder, D; Meselson, M

    1970-01-01

    X irradiation converts bacterial sex factor DNA covalent circles to more slowly sedimenting forms according to first order kinetics. The rate of conversion is greater for sex factors carrying lambda prophage than it is for nonlysogenized sex factors. The magnitude of the difference and the absence of covalent circular DNA from the product show that the prophage DNA is linearly inserted into the sex factor, as predicted by Campbell's model for lysogenization.

  9. Topological Relationship of Prophage λ to the Bacterial Chromosome in Lysogenic Cells

    Science.gov (United States)

    Freifelder, David; Meselson, Matthew

    1970-01-01

    X irradiation converts bacterial sex factor DNA covalent circles to more slowly sedimenting forms according to first order kinetics. The rate of conversion is greater for sex factors carrying λ prophage than it is for nonlysogenized sex factors. The magnitude of the difference and the absence of covalent circular DNA from the product show that the prophage DNA is linearly inserted into the sex factor, as predicted by Campbell's model for lysogenization. PMID:4905668

  10. Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.

    Science.gov (United States)

    Alekseeva, Ludmila; Rault, Lucie; Almeida, Sintia; Legembre, Patrick; Edmond, Valérie; Azevedo, Vasco; Miyoshi, Anderson; Even, Sergine; Taieb, Frédéric; Arlot-Bonnemains, Yannick; Le Loir, Yves; Berkova, Nadia

    2013-01-01

    Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration.

  11. Staphylococcus aureus-induced G2/M phase transition delay in host epithelial cells increases bacterial infective efficiency.

    Directory of Open Access Journals (Sweden)

    Ludmila Alekseeva

    Full Text Available Staphylococcus aureus is a highly versatile, opportunistic pathogen and the etiological agent of a wide range of infections in humans and warm-blooded animals. The epithelial surface is its principal site of colonization and infection. In this work, we investigated the cytopathic effect of S. aureus strains from human and animal origins and their ability to affect the host cell cycle in human HeLa and bovine MAC-T epithelial cell lines. S. aureus invasion slowed down cell proliferation and induced a cytopathic effect, resulting in the enlargement of host cells. A dramatic decrease in the number of mitotic cells was observed in the infected cultures. Flow cytometry analysis revealed an S. aureus-induced delay in the G2/M phase transition in synchronous HeLa cells. This delay required the presence of live S. aureus since the addition of the heat-killed bacteria did not alter the cell cycle. The results of Western blot experiments showed that the G2/M transition delay was associated with the accumulation of inactive cyclin-dependent kinase Cdk1, a key inducer of mitosis entry, and with the accumulation of unphosphorylated histone H3, which was correlated with a reduction of the mitotic cell number. Analysis of S. aureus proliferation in asynchronous, G1- and G2-phase-enriched HeLa cells showed that the G2 phase was preferential for bacterial infective efficiency, suggesting that the G2 phase delay may be used by S. aureus for propagation within the host. Taken together, our results divulge the potential of S. aureus in the subversion of key cellular processes such as cell cycle progression, and shed light on the biological significance of S. aureus-induced host cell cycle alteration.

  12. Molecular mechanism of immune cells activated by bacterial DNA%细菌DNA激活免疫细胞的分子机制

    Institute of Scientific and Technical Information of China (English)

    王良喜; 周红

    2003-01-01

    Bacterial DNA taken up by immune cells in a CpG motif- independent manner is translo-cated into endosome. Endosomal maturation is essential for subsequent bacterial DNA - mediated signal trans-duction. TLR9 is recruited into endosome to recognize bacterial DNA and initiate the TLB/IL- 1R signal transduction pathway. As a result , transcription factors NF - κB and AP- 1 are activated, which, in tum,leads to proinflammatory cytokine expression and induces a strong acute Th1 - like inflammatory response.

  13. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens;

    2008-01-01

    of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs) by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C......-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under...

  14. Abundance and single-cell activity of heterotrophic bacterial groups in the western Arctic Ocean in summer and winter.

    Science.gov (United States)

    Nikrad, Mrinalini P; Cottrell, M T; Kirchman, D L

    2012-04-01

    Environmental conditions in the western Arctic Ocean range from constant light and nutrient depletion in summer to complete darkness and sea ice cover in winter. This seasonal environmental variation is likely to have an effect on the use of dissolved organic matter (DOM) by heterotrophic bacteria in surface water. However, this effect is not well studied and we know little about the activity of specific bacterial clades in the surface oceans. The use of DOM by three bacterial subgroups in both winter and summer was examined by microautoradiography combined with fluorescence in situ hybridization. We found selective use of substrates by these groups, although the abundances of Ant4D3 (Antarctic Gammaproteobacteria), Polaribacter (Bacteroidetes), and SAR11 (Alphaproteobacteria) were not different between summer and winter in the Beaufort and Chukchi Seas. The number of cells taking up glucose within all three bacterial groups decreased significantly from summer to winter, while the percentage of cells using leucine did not show a clear pattern between seasons. The uptake of the amino acid mix increased substantially from summer to winter by the Ant4D3 group, although such a large increase in uptake was not seen for the other two groups. Use of glucose by bacteria, but not use of leucine or the amino acid mix, related strongly to inorganic nutrients, chlorophyll a, and other environmental factors. Our results suggest a switch in use of dissolved organic substrates from summer to winter and that the three phylogenetic subgroups examined fill different niches in DOM use in the two seasons.

  15. Effects of inoculation sources on the enrichment and performance of anode bacterial consortia in sensor typed microbial fuel cells

    Directory of Open Access Journals (Sweden)

    Phuong Tran

    2016-01-01

    Full Text Available Microbial fuel cells are a recently emerging technology that promises a number of applications in energy recovery, environmental treatment and monitoring. In this study, we investigated the effect of inoculating sources on the enrichment of electrochemically active bacterial consortia in sensor-typed microbial fuel cells (MFCs. Several MFCs were constructed, operated with modified artificial wastewater and inoculated with different microbial sources from natural soil, natural mud, activated sludge, wastewater and a mixture of those sources. After enrichment, the MFCs inoculated with the natural soil source generated higher and more stable currents (0.53±0.03 mA, in comparisons with the MFCs inoculated with the other sources. The results from denaturing gradient gel electrophoresis (DGGE showed that there were significant changes in bacterial composition from the original inocula to the enriched consortia. Even more interestingly, Pseudomonas sp. was found dominant in the natural soil source and also in the corresponding enriched consortium. The interactions between Pseudomonas sp. and other species in such a community are probably the key for the effective and stable performance of the MFCs.

  16. Effects of elaidic acid, a predominant industrial trans fatty acid, on bacterial growth and cell surface hydrophobicity of lactobacilli.

    Science.gov (United States)

    Wu, Qinglong; Shah, Nagendra P

    2014-12-01

    The consumption of trans fatty acids (TFAs) increases the risk of cardiovascular diseases and coronary heart disease in human, and there are no effective ways to remove TFAs after consumption. The aim of this study was to investigate the effects of elaidic acid on bacterial growth, cell surface hydrophobicity of lactobacilli, and metabolism of elaidic acid by lactobacilli. Lactobacilli were inoculated in MRS broth containing 0, 100, 200, and 500 mg/L of elaidic acid. Viable cell counts of lactobacilli were enumerated, concentrations of elaidic acid were determined, and cell surface hydrophobicity of lactobacilli was measured. The results showed that the growth of lactobacilli was significantly inhibited by 500 mg/L of elaidic acid, however, a cell count of 8.50 log10 CFU/mL was still reached for tested lactobacilli after 24-h incubation. In particular, a reduction of elaidic acid was found for tested lactobacilli after 24-h incubation as compared to its initial concentration of 200 mg/L. However, cell surface hydrophobicity showed no correlations with the metabolism of elaidic acid by lactobacilli. Moreover, elaidic acid was able to influence cell surface hydrophobicity, and the decrease in hydrophobicity was more obvious in Lactobacillus paracasei and Lactobacillus casei compared with that in other tested lactobacilli. This study suggests that elaidic acid could change physiochemical surface properties of lactobacilli and the lactobacilli have the potential to reduce TFAs.

  17. The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Jansen Sandra

    2011-02-01

    Full Text Available Abstract Background Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure mediates activation of the immune response in bacterial infection of the central nervous system (CNS. The chemotactic G-protein-coupled receptor (GPCR formyl-peptide-receptor like-1 (FPRL1 plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD. Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. Methods Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP and Neisseria meningitides (NM. Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2 phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene expression and signal transduction were determined. Results We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between

  18. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  19. Bacterial hydrodynamics

    CERN Document Server

    Lauga, Eric

    2015-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells yet they represent the bulk of the world's biomass, and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds-number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micron scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically-complex environments. Using hydrodynamics as an organizing framework, we review the biomechanics of bacterial motility and look ahead to future challenges.

  20. Surface charge-switching polymeric nanoparticles for bacterial cell wall-targeted delivery of antibiotics.

    Science.gov (United States)

    Radovic-Moreno, Aleksandar F; Lu, Timothy K; Puscasu, Vlad A; Yoon, Christopher J; Langer, Robert; Farokhzad, Omid C

    2012-05-22

    Bacteria have shown a remarkable ability to overcome drug therapy if there is a failure to achieve sustained bactericidal concentration or if there is a reduction in activity in situ. The latter can be caused by localized acidity, a phenomenon that can occur as a result of the combined actions of bacterial metabolism and the host immune response. Nanoparticles (NP) have shown promise in treating bacterial infections, but a significant challenge has been to develop antibacterial NPs that may be suitable for systemic administration. Herein we develop drug-encapsulated, pH-responsive, surface charge-switching poly(D,L-lactic-co-glycolic acid)-b-poly(L-histidine)-b-poly(ethylene glycol) (PLGA-PLH-PEG) nanoparticles for treating bacterial infections. These NP drug carriers are designed to shield nontarget interactions at pH 7.4 but bind avidly to bacteria in acidity, delivering drugs and mitigating in part the loss of drug activity with declining pH. The mechanism involves pH-sensitive NP surface charge switching, which is achieved by selective protonation of the imidazole groups of PLH at low pH. NP binding studies demonstrate pH-sensitive NP binding to bacteria with a 3.5 ± 0.2- to 5.8 ± 0.1-fold increase in binding to bacteria at pH 6.0 compared to 7.4. Further, PLGA-PLH-PEG-encapsulated vancomycin demonstrates reduced loss of efficacy at low pH, with an increase in minimum inhibitory concentration of 1.3-fold as compared to 2.0-fold and 2.3-fold for free and PLGA-PEG-encapsulated vancomycin, respectively. The PLGA-PLH-PEG NPs described herein are a first step toward developing systemically administered drug carriers that can target and potentially treat Gram-positive, Gram-negative, or polymicrobial infections associated with acidity.

  1. Determinants of eukaryal cell killing by the bacterial ribotoxin PrrC

    OpenAIRE

    Meineke, Birthe; Schwer, Beate; Schaffrath, Raffael; Shuman, Stewart

    2010-01-01

    tRNA damage inflicted by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies an antiviral response to phage T4 infection. PrrC homologs are present in many bacterial proteomes, though their biological activities are uncharted. PrrCs consist of two domains: an N-terminal NTPase module related to the ABC family and a distinctive C-terminal ribonuclease module. In this article, we report that the expression of EcoPrrC in budding yeast is fungicidal, signifying that PrrC is toxic in ...

  2. Addition of selenium nanoparticles to electrospun silk scaffolds improves mammalian cell activity while reducing bacterial growth

    Directory of Open Access Journals (Sweden)

    Stanley Chung

    2016-07-01

    Full Text Available Silk possesses many beneficial wound healing properties, and electrospun scaffolds are especially applicable for skin applications, due to their smaller interstices and higher surface areas compared to non-electrospun equivalents. However, purified silk promotes microbial growth. In contrast, selenium nanoparticles have excellent antibacterial properties and are a novel antimicrobial chemistry. Here, electrospun silk scaffolds were doped with selenium nanoparticles to impart antibacterial properties to the silk scaffolds. Results showed significantly improved bacterial inhibition and improvement in human dermal fibroblast metabolic activity. These results suggest that the addition of selenium nanoparticles to electrospun silk is a promising approach to improve wound healing with reduced infection, without relying on antibiotics.

  3. Identification of novel bacterial histidine biosynthesis inhibitors using docking, ensemble rescoring, and whole-cell assays

    DEFF Research Database (Denmark)

    Henriksen, Signe Teuber; Liu, J.; Estiu, G.;

    2010-01-01

    in the early stages of drug discovery attractive if sufficient accuracy can be achieved. Computational target identification using systems-level methods suggested the histidine biosynthesis pathway as an attractive target against S. aureus. Potential inhibitors for the pathway were identified through docking...... histidine biosynthesis pathway, which is predicted to be essential for bacterial biomass productions. Virtual screening of a library of similar to 10(6) compounds identified 49 potential inhibitors of three enzymes of this pathway. Eighteen representative compounds were directly tested on three S. aureus...... of this novel strategy to the histidine biosynthesis pathway....

  4. Temporal trends in bulk tank somatic cell count and total bacterial count in Irish dairy herds during the past decade.

    Science.gov (United States)

    Berry, D P; O'Brien, B; O'Callaghan, E J; Sullivan, K O; Meaney, W J

    2006-10-01

    The objective of this study was to document temporal trends in bulk tank somatic cell count (SCC) and total bacterial counts (TBC) in Irish dairy herds during the years 1994 to 2004. Three milk processors participated in the study, providing data on 2,754,270 individual bulk tank SCC and 2,056,992 individual bulk tank TBC records from 9,113 herds. Somatic cell counts decreased during the years 1994 to 2000, followed by an annual increase thereafter of more than 2,000 cells/mL. A tendency existed for TBC to decrease over time. Across all years, bulk tank SCC were the lowest in April and highest in November; TBC were the lowest in May and highest in December. The significant seasonal pattern observed in herd SCC and TBC was an artifact of seasonal calving in Ireland. In general, herds selling more milk had lower bulk tank SCC and TBC. Herds having the highest SCC (i.e., > 450,000 cells/mL) and the lowest SCC (i.e., < or = 150,000 cells/mL) both contributed substantially to the mean SCC of the milk pool collected by the milk processors. Derived transition matrices showed that between adjacent years, herds had the greatest probability of remaining in the same annual mean SCC or TBC category.

  5. Mucin biopolymers prevent bacterial aggregation by retaining cells in the free-swimming state

    Science.gov (United States)

    Caldara, Marina; Friedlander, Ronn S.; Kavanaugh, Nicole L.; Aizenberg, Joanna; Foster, Kevin R.; Ribbeck, Katharina

    2013-01-01

    Summary Many species of bacteria form surface-attached communities known as biofilms. Surrounded in secreted polymers, these aggregates are difficult to both prevent and eradicate, posing problems for medicine and industry [1, 2]. Humans play host to hundreds of trillions of microbes that live adjacent to our epithelia and we are typically able to prevent harmful colonization. Mucus, the hydrogel overlying all wet epithelia in the body, can prevent bacterial contact with the underlying tissue. The digestive tract, for example, is lined by a firmly adherent mucus layer that is typically devoid of bacteria, followed by a second, loosely adherent layer that contains numerous bacteria [3]. Here, we investigate mucus's role as a principle arena for host-microbe interactions. Using defined in vitro assays, we found that mucin biopolymers, the main functional constituents of mucus, promote the motility of planktonic bacteria, and prevent their adhesion to underlying surfaces. The deletion of motility genes, however, allows Pseudomonas aeruginosa to overcome the dispersive effects of mucus and form suspended antibiotic-resistant flocs, which mirror the clustered morphology of immotile natural isolates found in the cystic fibrosis lung mucus [4, 5]. Mucus may offer new strategies to target bacterial virulence, such as the design of anti-biofilm coatings for implants. PMID:23142047

  6. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    Science.gov (United States)

    Alquier, M.; Kassim, C.; Bertron, A.; Rafrafi, Y.; Sablayrolles, C.; Albrecht, A.; Erable, B.

    2013-07-01

    Leaching experiments of solid matrices (bitumen and cement pastes) have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.). Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these extreme conditions of pH. The denitrifying activity of Halomonas desiderata was quantified in batch bioreactor in the presence of solid matrices and / or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 hours) and 2 to 3 times slower in the presence of bituminous matrix. Overall, the presence of solid cement promoted the kinetics of denitrification. The observation of solid surfaces at the end of the experiment revealed the presence of a biofilm of Halomonas desiderata on the cement paste surface. These attached bacteria showed a denitrifying activity comparable to planktonic bacterial culture. On the other side, no colonization of bitumen could be highlighted as either by SEM or epifluorescence microscopy. Now, we are currently developing a continuous experimental bioreactor which should allow us a more rational understanding of the bitumen-cement-microbe interactions.

  7. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    Directory of Open Access Journals (Sweden)

    Albrecht A.

    2013-07-01

    Full Text Available Leaching experiments of solid matrices (bitumen and cement pastes have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.. Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these extreme conditions of pH. The denitrifying activity of Halomonas desiderata was quantified in batch bioreactor in the presence of solid matrices and / or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 hours and 2 to 3 times slower in the presence of bituminous matrix. Overall, the presence of solid cement promoted the kinetics of denitrification. The observation of solid surfaces at the end of the experiment revealed the presence of a biofilm of Halomonas desiderata on the cement paste surface. These attached bacteria showed a denitrifying activity comparable to planktonic bacterial culture. On the other side, no colonization of bitumen could be highlighted as either by SEM or epifluorescence microscopy. Now, we are currently developing a continuous experimental bioreactor which should allow us a more rational understanding of the bitumen-cement-microbe interactions.

  8. Alteration of bacterial communities and organic matter in microbial fuel cells (MFCs) supplied with soil and organic fertilizer.

    Science.gov (United States)

    Mocali, Stefano; Galeffi, Carlo; Perrin, Elena; Florio, Alessandro; Migliore, Melania; Canganella, Francesco; Bianconi, Giovanna; Di Mattia, Elena; Dell'Abate, Maria Teresa; Fani, Renato; Benedetti, Anna

    2013-02-01

    The alteration of the organic matter (OM) and the composition of bacterial community in microbial fuel cells (MFCs) supplied with soil (S) and a composted organic fertilizer (A) was examined at the beginning and at the end of 3 weeks of incubation under current-producing as well as no-current-producing conditions. Denaturing gradient gel electrophoresis revealed a significant alteration of the microbial community structure in MFCs generating electricity as compared with no-current-producing MFCs. The genetic diversity of cultivable bacterial communities was assessed by random amplified polymorphic DNA (RAPD) analysis of 106 bacterial isolates obtained by using both generic and elective media. Sequencing of the 16S rRNA genes of the more representative RAPD groups indicated that over 50.4% of the isolates from MFCs fed with S were Proteobacteria, 25.1% Firmicutes, and 24.5% Actinobacteria, whereas in MFCs supplied with A 100% of the dominant species belonged to γ-Proteobacteria. The chemical analysis performed by fractioning the OM and using thermal analysis showed that the amount of total organic carbon contained in the soluble phase of the electrochemically active chambers significantly decreased as compared to the no-current-producing systems, whereas the OM of the solid phase became more humified and aromatic along with electricity generation, suggesting a significant stimulation of a humification process of the OM. These findings demonstrated that electroactive bacteria are commonly present in aerobic organic substrates such as soil or a fertilizer and that MFCs could represent a powerful tool for exploring the mineralization and humification processes of the soil OM.

  9. Focused specificity of intestinal TH17 cells towards commensal bacterial antigens.

    Science.gov (United States)

    Yang, Yi; Torchinsky, Miriam B; Gobert, Michael; Xiong, Huizhong; Xu, Mo; Linehan, Jonathan L; Alonzo, Francis; Ng, Charles; Chen, Alessandra; Lin, Xiyao; Sczesnak, Andrew; Liao, Jia-Jun; Torres, Victor J; Jenkins, Marc K; Lafaille, Juan J; Littman, Dan R

    2014-06-05

    T-helper-17 (TH17) cells have critical roles in mucosal defence and in autoimmune disease pathogenesis. They are most abundant in the small intestine lamina propria, where their presence requires colonization of mice with microbiota. Segmented filamentous bacteria (SFB) are sufficient to induce TH17 cells and to promote TH17-dependent autoimmune disease in animal models. However, the specificity of TH17 cells, the mechanism of their induction by distinct bacteria, and the means by which they foster tissue-specific inflammation remain unknown. Here we show that the T-cell antigen receptor (TCR) repertoire of intestinal TH17 cells in SFB-colonized mice has minimal overlap with that of other intestinal CD4(+) T cells and that most TH17 cells, but not other T cells, recognize antigens encoded by SFB. T cells with antigen receptors specific for SFB-encoded peptides differentiated into RORγt-expressing TH17 cells, even if SFB-colonized mice also harboured a strong TH1 cell inducer, Listeria monocytogenes, in their intestine. The match of T-cell effector function with antigen specificity is thus determined by the type of bacteria that produce the antigen. These findings have significant implications for understanding how commensal microbiota contribute to organ-specific autoimmunity and for developing novel mucosal vaccines.

  10. An X-ray Absorption Fine Structure study of Au adsorbed onto the non-metabolizing cells of two soil bacterial species

    Energy Technology Data Exchange (ETDEWEB)

    Song, Zhen; Kenney, Janice P.L.; Fein, Jeremy B.; Bunker, Bruce A. (Notre)

    2015-02-09

    Gram-positive and Gram-negative bacterial cells can remove Au from Au(III)-chloride solutions, and the extent of removal is strongly pH dependent. In order to determine the removal mechanisms, X-ray Absorption Fine Structure (XAFS) spectroscopy experiments were conducted on non-metabolizing biomass of Bacillus subtilis and Pseudomonas putida with fixed Au(III) concentrations over a range of bacterial concentrations and pH values. X-ray Absorption Near Edge Structure (XANES) and Extended X-ray Absorption Fine Structure (EXAFS) data on both bacterial species indicate that more than 90% of the Au atoms on the bacterial cell walls were reduced to Au(I). In contrast to what has been observed for Au(III) interaction with metabolizing bacterial cells, no Au(0) or Au-Au nearest neighbors were observed in our experimental systems. All of the removed Au was present as adsorbed bacterial surface complexes. For both species, the XAFS data suggest that although Au-chloride-hydroxide aqueous complexes dominate the speciation of Au in solution, Au on the bacterial cell wall is characterized predominantly by binding of Au atoms to sulfhydryl functional groups and amine and/or carboxyl functional groups, and the relative importance of the sulfhydryl groups increases with increasing pH and with decreasing Au loading. The XAFS data for both microorganism species suggest that adsorption is the first step in the formation of Au nanoparticles by bacteria, and the results enhance our ability to account for the behavior of Au in bacteria-bearing geologic systems.

  11. Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors

    Directory of Open Access Journals (Sweden)

    Lau Yu

    2008-09-01

    Full Text Available Abstract Background Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings. Results We selected lipopolysaccharide (LPS from Escherichia coli and lipoteichoic acid (LTA from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs, in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria. Conclusion In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

  12. Adhesion of bacterial pathogens to soil colloidal particles: influences of cell type, natural organic matter, and solution chemistry.

    Science.gov (United States)

    Zhao, Wenqiang; Walker, Sharon L; Huang, Qiaoyun; Cai, Peng

    2014-04-15

    Bacterial adhesion to granular soil particles is well studied; however, pathogen interactions with naturally occurring colloidal particles (organic matter (NOM), and solution chemistry. Specifically, batch adhesion experiments were conducted using NOM-present, NOM-stripped soil colloids, Streptococcus suis SC05 and Escherichia coli WH09 over a wide range of solution pH (4.0-9.0) and ionic strength (IS, 1-100 mM KCl). Cell characterization techniques, Freundlich isotherm, and Derjaguin-Landau-Verwey-Overbeek (DLVO) theory (sphere-sphere model) were utilized to quantitatively determine the interactions between cells and colloids. The adhesion coefficients (Kf) of S. suis SC05 to NOM-present and NOM-stripped soil colloids were significantly higher than E. coli WH09, respectively. Similarly, Kf values of S. suis SC05 and E. coli WH09 adhesion to NOM-stripped soil colloids were greater than those colloids with NOM-present, respectively, suggesting NOM inhibits bacterial adhesion. Cell adhesion to soil colloids declined with increasing pH and enhanced with rising IS (1-50 mM). Interaction energy calculations indicate these adhesion trends can be explained by DLVO-type forces, with S. suis SC05 and E. coli WH09 being weakly adhered in shallow secondary energy minima via polymer bridging and charge heterogeneity. S. suis SC05 adhesion decreased at higher IS 100 mM, which is attributed to the change of hydrophobic effect and steric repulsion resulted from the greater presence of extracellular polymeric substances (EPS) on S. suis SC05 surface as compared to E. coli WH09. Hence, pathogen adhesion to the colloidal material is determined by a combination of DLVO, charge heterogeneity, hydrophobic and polymer interactions as a function of solution chemistry.

  13. Timescales and Frequencies of Reversible and Irreversible Adhesion Events of Single Bacterial Cells.

    Science.gov (United States)

    Hoffman, Michelle D; Zucker, Lauren I; Brown, Pamela J B; Kysela, David T; Brun, Yves V; Jacobson, Stephen C

    2015-12-15

    In the environment, most bacteria form surface-attached cell communities called biofilms. The attachment of single cells to surfaces involves an initial reversible stage typically mediated by surface structures such as flagella and pili, followed by a permanent adhesion stage usually mediated by polysaccharide adhesives. Here, we determine the absolute and relative timescales and frequencies of reversible and irreversible adhesion of single cells of the bacterium Caulobacter crescentus to a glass surface in a microfluidic device. We used fluorescence microscopy of C. crescentus expressing green fluorescent protein to track the swimming behavior of individual cells prior to adhesion, monitor the cell at the surface, and determine whether the cell reversibly or irreversibly adhered to the surface. A fluorescently labeled lectin that binds specifically to polar polysaccharides, termed holdfast, discriminated irreversible adhesion events from reversible adhesion events where no holdfast formed. In wild-type cells, the holdfast production time for irreversible adhesion events initiated by surface contact (23 s) was 30-times faster than the holdfast production time that occurs through developmental regulation (13 min). Irreversible adhesion events in wild-type cells (3.3 events/min) are 15-times more frequent than in pilus-minus mutant cells (0.2 events/min), indicating the pili are critical structures in the transition from reversible to irreversible surface-stimulated adhesion. In reversible adhesion events, the dwell time of cells at the surface before departing was the same for wild-type cells (12 s) and pilus-minus mutant cells (13 s), suggesting the pili do not play a significant role in reversible adhesion. Moreover, reversible adhesion events in wild-type cells (6.8 events/min) occur twice as frequently as irreversible adhesion events (3.3 events/min), demonstrating that most cells contact the surface multiple times before transitioning from reversible to

  14. Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan.

    Science.gov (United States)

    Schanda, Paul; Triboulet, Sébastien; Laguri, Cédric; Bougault, Catherine M; Ayala, Isabel; Callon, Morgane; Arthur, Michel; Simorre, Jean-Pierre

    2014-12-24

    The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance, to date no structure of a protein in complex with an intact bacterial peptidoglycan has been resolved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spectroscopy to derive for the first time an atomic model of an l,d-transpeptidase from Bacillus subtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains-the catalytic domain as well as the proposed peptidoglycan recognition domain-are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the implication of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidoglycan complex paves the way for the design of new antibiotic drugs targeting l,d-transpeptidases. The strategy developed here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis.

  15. Short communication: bulk tank total bacterial count in dairy sheep: factors of variation and relationship with somatic cell count.

    Science.gov (United States)

    Gonzalo, C; Carriedo, J A; Beneitez, E; Juárez, M T; De La Fuente, L F; San Primitivo, F

    2006-02-01

    A total of 9,353 records for bulk tank total bacterial count (TBC) were obtained over 1 yr from 315 dairy ewe flocks belonging to the Sheep Improvement Consortium (CPO) in Castilla-León (Spain). Analysis of variance showed significant effects of flock, breed, month within flock, dry therapy, milking type and installation, and logSCC on logTBC. Flock and month within flock were important variation factors as they accounted for 22.0 and 22.1% of the variance, respectively. Considerable repeatability values were obtained for both random factors. Hand milking and bucket-milking machines elicited highest logTBC (5.31), whereas parlor systems with looped milkline (5.01) elicited the lowest logTBC. The implementation of dry therapy practice (5.12) showed significantly lower logTBC than when not used (5.25). Variability in logTBC among breeds ranged from 5.24 (Awassi) to 5.07 (Churra). However, clinical outbreaks of contagious agalactia did not increase TBC significantly. A statistically significant relationship was found between logTBC and logSCC, the correlation coefficient between the variables being r = 0.23. Programs for improving milk hygiene should be implemented for both total bacterial count and somatic cell count variables at the same time.

  16. Modeling of microfluidic microbial fuel cells using quantitative bacterial transport parameters

    Science.gov (United States)

    Mardanpour, Mohammad Mahdi; Yaghmaei, Soheila; Kalantar, Mohammad

    2017-02-01

    The objective of present study is to analyze the dynamic modeling of bioelectrochemical processes and improvement of the performance of previous models using quantitative data of bacterial transport parameters. The main deficiency of previous MFC models concerning spatial distribution of biocatalysts is an assumption of initial distribution of attached/suspended bacteria on electrode or in anolyte bulk which is the foundation for biofilm formation. In order to modify this imperfection, the quantification of chemotactic motility to understand the mechanisms of the suspended microorganisms' distribution in anolyte and/or their attachment to anode surface to extend the biofilm is implemented numerically. The spatial and temporal distributions of the bacteria, as well as the dynamic behavior of the anolyte and biofilm are simulated. The performance of the microfluidic MFC as a chemotaxis assay is assessed by analyzing the bacteria activity, substrate variation, bioelectricity production rate and the influences of external resistance on the biofilm and anolyte's features.

  17. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens

    2008-01-01

    of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs) by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C...... in vitro culture condition C. jejuni strains of both human and chicken origins can invade avian host cells with a pro-inflammatory response and that the virulence-associated genes of C. jejuni may play a role in this process....

  18. Bacterial biofilm formation versus mammalian cell growth on titanium-based mono- and bi-functional coating

    Directory of Open Access Journals (Sweden)

    G Subbiahdoss

    2010-05-01

    Full Text Available Biomaterials-associated-infections (BAI are serious complications in modern medicine. Although non-adhesive coatings, like polymer-brush coatings, have been shown to prevent bacterial adhesion, they do not support cell growth. Bi-functional coatings are supposed to prevent biofilm formation while supporting tissue integration. Here, bacterial and cellular responses to poly(ethylene glycol (PEG brush-coatings on titanium oxide presenting the integrin-active peptide RGD (arginine-glycine-aspartic acid (bioactive “PEG-RGD” were compared to mono-functional PEG brush-coatings (biopassive “PEG” and bare titanium oxide (TiO2 surfaces under flow. Staphylococcus epidermidis ATCC 35983 was deposited on the surfaces under a shear rate of 11 s-1 for 2 h followed by seeding of U2OS osteoblasts. Subsequently, both S. epidermidis and U2OS cells were grown simultaneously on the surfaces for 48 h under low shear (0.14 s-1. After 2 h, staphylococcal adhesion was reduced to 3.6±1.8 × 103 and 6.0±3.9 × 103 cm-2 on PEG and PEG-RGD coatings respectively, compared to 1.3±0.4 × 105 cm-2 for the TiO2 surface. When allowed to grow for 48 h, biofilms formed on all surfaces. However, biofilms detached from the PEG and PEG-RGD coatings when exposed to an elevated shear (5.6 s-1 U2OS cells neither adhered nor spread on PEG brush-coatings, regardless of the presence of biofilm. In contrast, in the presence of biofilm, U2OS cells adhered and spread on PEG-RGD coatings with a significantly higher surface coverage than on bare TiO2. The detachment of biofilm and the high cell surface coverage revealed the potential significance of PEG-RGD coatings in the context of the “race for the surface” between bacteria and mammalian cells.

  19. A rapid crosstalk of human gammadelta T cells and monocytes drives the acute inflammation in bacterial infections.

    Directory of Open Access Journals (Sweden)

    Matthias Eberl

    2009-02-01

    Full Text Available Vgamma9/Vdelta2 T cells are a minor subset of T cells in human blood and differ from other T cells by their immediate responsiveness to microbes. We previously demonstrated that the primary target for Vgamma9/Vdelta2 T cells is (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, an essential metabolite produced by a large range of pathogens. Here we wished to study the consequence of this unique responsiveness in microbial infection. The majority of peripheral Vgamma9/Vdelta2 T cells shares migration properties with circulating monocytes, which explains the presence of these two distinct blood cell types in the inflammatory infiltrate at sites of infection and suggests that they synergize in anti-microbial immune responses. Our present findings demonstrate a rapid and HMB-PP-dependent crosstalk between Vgamma9/Vdelta2 T cells and autologous monocytes that results in the immediate production of inflammatory mediators including the cytokines interleukin (IL-6, interferon (IFN-gamma, tumor necrosis factor (TNF-alpha, and oncostatin M (OSM; the chemokines CCL2, CXCL8, and CXCL10; and TNF-related apoptosis-inducing ligand (TRAIL. Moreover, under these co-culture conditions monocytes differentiate within 18 hours into inflammatory dendritic cells (DCs with antigen-presenting functions. Addition of further microbial stimuli (lipopolysaccharide, peptidoglycan induces CCR7 and enables these inflammatory DCs to trigger the generation of CD4(+ effector alphabeta T cells expressing IFN-gamma and/or IL-17. Importantly, our in vitro model replicates the responsiveness to microbes of effluent cells from peritoneal dialysis (PD patients and translates directly to episodes of acute PD-associated bacterial peritonitis, where Vgamma9/Vdelta2 T cell numbers and soluble inflammatory mediators are elevated in patients infected with HMB-PP-producing pathogens. Collectively, these findings suggest a direct link between invading pathogens, microbe

  20. Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles

    DEFF Research Database (Denmark)

    Chua, Song Lin; Liu, Yang; Yam, Joey Kuok Hoong

    2014-01-01

    Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. Increased levels of the intracellular messenger c-di-GMP determine the transition from planktonic to biofilm growth, while a reduction causes biofilm dispersal. It is generally assumed that cells dispersed from...... biofilms immediately go into the planktonic growth phase. Here we use single-nucleotide resolution transcriptomic analysis to show that the physiology of dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells. In dispersed cells, the expression...... of the small regulatory RNAs RsmY and RsmZ is downregulated, whereas secretion genes are induced. Dispersed cells are highly virulent against macrophages and Caenorhabditis elegans compared with planktonic cells. In addition, they are highly sensitive towards iron stress, and the combination of a biofilm...

  1. Microfluidic-Based Droplet and Cell Manipulations Using Artificial Bacterial Flagella

    Directory of Open Access Journals (Sweden)

    Yun Ding

    2016-02-01

    Full Text Available Herein, we assess the functionality of magnetic helical microswimmers as basic tools for the manipulation of soft materials, including microdroplets and single cells. Their ability to perform a range of unit operations is evaluated and the operational challenges associated with their use are established. In addition, we also report on interactions observed between the head of such helical swimmers and the boundaries of droplets and cells and discuss the possibilities of assembling an artificial swimming microorganism or a motorized cell.

  2. Bacterial scaffolds assemble novel higher-order complexes to reengineer eukaryotic cell processes.

    Science.gov (United States)

    Lesser, Cammie F; Leong, John M

    2011-07-05

    Many microbial pathogens use specialized secretion systems to inject proteins referred to as effectors directly into eukaryotic host cells. These effectors directly target various eukaryotic signaling pathways and cellular processes, often by mimicking the activity of host cell proteins. Effectors of pathogenic Escherichia coli and Salmonella typhimurium can also act as molecular scaffolds that not only recruit but also directly regulate the activity and localization of multiple eukaryotic proteins. By assembling and localizing disparate signaling pathways, the bacteria can reengineer host cell processes to generate novel processes not previously observed in eukaryotic cells.

  3. Non-canonical roles of tRNAs and tRNA mimics in bacterial cell biology.

    Science.gov (United States)

    Katz, Assaf; Elgamal, Sara; Rajkovic, Andrei; Ibba, Michael

    2016-08-01

    Transfer RNAs (tRNAs) are the macromolecules that transfer activated amino acids from aminoacyl-tRNA synthetases to the ribosome, where they are used for the mRNA guided synthesis of proteins. Transfer RNAs are ancient molecules, perhaps even predating the existence of the translation machinery. Albeit old, these molecules are tremendously conserved, a characteristic that is well illustrated by the fact that some bacterial tRNAs are efficient and specific substrates of eukaryotic aminoacyl-tRNA synthetases and ribosomes. Considering their ancient origin and high structural conservation, it is not surprising that tRNAs have been hijacked during evolution for functions outside of translation. These roles beyond translation include synthetic, regulatory and information functions within the cell. Here we provide an overview of the non-canonical roles of tRNAs and their mimics in bacteria, and discuss some of the common themes that arise when comparing these different functions.

  4. Bacterial cellulose-polyaniline nano-biocomposite: A porous media hydrogel bioanode enhancing the performance of microbial fuel cell

    Science.gov (United States)

    Mashkour, Mehrdad; Rahimnejad, Mostafa; Mashkour, Mahdi

    2016-09-01

    Microbial fuel cells (MFCs) are one of the possible renewable energy supplies which microorganisms play an active role in bio-oxidize reactions of a substrate such as glucose. Electrode materials and surface modifications are highly effective tools in enhancing MFCs' Performance. In this study, new composite anodes are fabricated. Bacterial cellulose (BC) is used as continuous phase and polyaniline (PANI) as dispersed one which is synthesized by in situ chemical oxidative polymerization on BC's fibers. With hydrogel nature of BC as a novel feature and polyaniline conductivity there meet the favorable conditions to obtain an active microbial biofilm on anode surface. Maximum power density of 117.76 mW/m2 in current density of 617 mA/m2 is achieved for BC/PANI anode. The amounts demonstrate a considerable enhancement compared with graphite plate (1 mW/m2 and 10 mA/m2).

  5. The oral immunogenicity of BioProtein, a bacterial single-cell protein, is affected by its particulate nature

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Larsen, L.C.; Frøkiær, Hanne

    2003-01-01

    The bacterial single-cell protein BioProtein (BP; Norferm Danmark, Odense, Denmark), produced by fermentation of natural gas with methanotrophic bacteria, is a potential protein source for man and animals. For human consumption, removal of the nucleic acid is necessary. Preliminary studies have...... shown that ingested BP induces a specific immune response. The objective of the present study was to characterize the type of response, its development over time and product-related causative factors. Mice were fed with diets containing 60 g nucleic acid-reduced BP/kg, 240 g nucleic acid-reduced BP...... and saliva. Ingested BP induced a steady specific mucosal and systemic immune response, characterized by a dose-dependent production of immunoglobulin and immunoglobulin A in blood and immunoglobulin A in saliva. Basic BP and nucleic acid-reduced BP induced identical responses. However, feeding mice BP...

  6. Overlapping two self-avoiding polymers in a closed cylindrical pore: Implications for chromosome segregation in a bacterial cell

    Science.gov (United States)

    Jung, Youngkyun; Ha, Bae-Yeun

    2010-11-01

    We study the spatial organization and segregation of two self-avoiding polymers trapped inside a closed cylindrical pore. Using molecular-dynamics simulations, we show how confinement shapes the chains, especially their mutual (entropic) force, chain miscibility, and segregation dynamics. Under strong confinement, the chains are shown to repel more strongly and thus segregate better if they are shorter and the confining space is more asymmetric, in contrast to the spherically confined case, where nonlinear chain topology is required for chain partitioning in equilibrium. When applied to bacterial chromosomes, our results imply that chromosome miscibility depends on how they are compacted and structured inside the cell (by proteins and supercoiling). Finally, longitudinal confinement is shown to have nontrivial effects on segregation dynamics by randomizing and thus slowing down the segregation process, which would otherwise be assisted with entropic forces.

  7. Prokaryotic ParA-ParB-parS system links bacterial chromosome segregation with the cell cycle.

    Science.gov (United States)

    Mierzejewska, Jolanta; Jagura-Burdzy, Grażyna

    2012-01-01

    While the essential role of episomal par loci in plasmid DNA partitioning has long been appreciated, the function of chromosomally encoded par loci is less clear. The chromosomal parA-parB genes are conserved throughout the bacterial kingdom and encode proteins homologous to those of the plasmidic Type I active partitioning systems. The third conserved element, the centromere-like sequence called parS, occurs in several copies in the chromosome. Recent studies show that the ParA-ParB-parS system is a key player of a mitosis-like process ensuring proper intracellular localization of certain chromosomal regions such as oriC domain and their active and directed segregation. Moreover, the chromosomal par systems link chromosome segregation with initiation of DNA replication and the cell cycle.

  8. Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.

    Science.gov (United States)

    Wong, Connie H Y; Jenne, Craig N; Petri, Björn; Chrobok, Navina L; Kubes, Paul

    2013-08-01

    Through the use of intravital imaging of the liver, we demonstrate a collaborative role for platelets with Kupffer cells (KCs) in eradicating blood-borne bacterial infection. Under basal conditions, platelets, via the platelet-adhesion receptor GPIb, formed transient 'touch-and-go' interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria such as Bacillus cereus and methicillin-resistant Staphylococcus aureus (MRSA) were rapidly caught by KCs and triggered platelets to switch from 'touch-and-go' adhesion to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GPIbα-deficient mice had more endothelial and KC damage than did their wild-type counterparts, which led to more fluid leakage, substantial polycythemia and rapid mortality. Our study identifies a previously unknown surveillance mechanism by which platelets survey macrophages that rapidly converts to a critical host response to blood-borne bacteria.

  9. Intrinsic disorder of the bacterial cell division protein ZipA: coil-to-brush conformational transition.

    Science.gov (United States)

    López-Montero, Iván; López-Navajas, Pilar; Mingorance, Jesús; Rivas, Germán; Vélez, Marisela; Vicente, Miguel; Monroy, Francisco

    2013-08-01

    The full-length ZipA protein from Escherichia coli, one of the essential elements of the cell division machinery, was studied in a surface model built as adsorbed monolayers. The interplay between lateral packing and molecular conformation was probed using a combined methodology based on the scaling analysis of the surface pressure isotherms and ellipsometry measurements of the monolayer thickness. The observed behavior is compatible with the one expected for an intrinsically disordered and highly flexible protein that is preferentially structured in a random coil conformation. At low grafting densities, ZipA coils organize in a mushroom-like regime, whereas a coil-to-brush transition occurs on increasing lateral packing. The structural results suggest a functional scenario in which ZipA acts as a flexible tether anchoring bacterial proto-ring elements to the membrane during the earlier stages of division.

  10. Bacterial Vaginosis

    Science.gov (United States)

    ... Issues > Conditions > Sexually Transmitted > Bacterial Vaginosis Health Issues Listen Español Text Size Email Print Share Bacterial Vaginosis Page Content Bacterial vaginosis (BV) is the most common vaginal infection in sexually active teenaged girls . It appears to be caused by ...

  11. Bacterial clearance reverses a skewed T-cell repertoire induced by Salmonella infection.

    Science.gov (United States)

    Leyva-Rangel, Jessica P; de Los Angeles Hernández-Cueto, Maria; Galan-Enriquez, Carlos-Samuel; López-Medina, Marcela; Ortiz-Navarrete, Vianney

    2015-09-01

    Salmonella typhimurium invades the spleen, liver, and peripheral lymph nodes and has recently been detected in the bone marrow and thymus, resulting in a reduced thymic size and a decline in the total number of thymic cells. A specific deletion of the double-positive cell subset has been characterized, yet the export of mature T cells to the periphery remains normal. We analyzed Salmonella pathogenesis regarding thymic structure and the T-cell maturation process. We demonstrate that, despite alterations in the thymic structure, T-cell development is maintained during Salmonella infection, allowing the selection of single-positive T-cell clones expressing particular T-cell receptor beta chains (TCR-Vβ). Moreover, the treatment of infected mice with an antibiotic restored the normal thymic architecture and thymocyte subset distribution. Additionally, the frequency of TCR-Vβ usage after treatment was comparable to that in non-infected mice. However, bacteria were still recovered from the thymus after 1 month of treatment. Our data reveal that a skewed T-cell developmental process is present in the Salmonella-infected thymus that alters the TCR-Vβ usage frequency. Likewise, the post-treatment persistence of Salmonella reveals a novel function of the thymus as a potential reservoir for this infectious agent.

  12. B and T cell crosstalk in anti-bacterial immune responses

    NARCIS (Netherlands)

    J. de Wit

    2012-01-01

    This thesis shows that phagocytosis of Salmonella by B cells may generate a survival niche and transport vehicle for Salmonella, but that simultaneously Salmonella-infected B cells induce an optimal anti-Salmonella response through activation of multiple arms of the adaptive immune response. The the

  13. Autonomous Bacterial Localization and Gene Expression Based on Nearby Cell Receptor Density

    Science.gov (United States)

    2013-01-22

    Upon detection of B1–5 mM AI-2, these cells express T7 polymerase that amplifies the native lsr operon response by overexpressing DsRed (see...2 for initiating gene expression (lsr operon ). (B) Indicated densities of PCI-15B or HEK293 cells were seeded to wells followed by mouse anti-EGFR

  14. Anti-Inflammatory Effects of Vitamin D on Human Immune Cells in the Context of Bacterial Infection

    Science.gov (United States)

    Hoe, Edwin; Nathanielsz, Jordan; Toh, Zheng Quan; Spry, Leena; Marimla, Rachel; Balloch, Anne; Mulholland, Kim; Licciardi, Paul V.

    2016-01-01

    Vitamin D induces a diverse range of biological effects, including important functions in bone health, calcium homeostasis and, more recently, on immune function. The role of vitamin D during infection is of particular interest given data from epidemiological studies suggesting that vitamin D deficiency is associated with an increased risk of infection. Vitamin D has diverse immunomodulatory functions, although its role during bacterial infection remains unclear. In this study, we examined the effects of 1,25(OH)2D3, the active metabolite of vitamin D, on peripheral blood mononuclear cells (PBMCs) and purified immune cell subsets isolated from healthy adults following stimulation with the bacterial ligands heat-killed pneumococcal serotype 19F (HK19F) and lipopolysaccharide (LPS). We found that 1,25(OH)2D3 significantly reduced pro-inflammatory cytokines TNF-α, IFN-γ, and IL-1β as well as the chemokine IL-8 for both ligands (three- to 53-fold), while anti-inflammatory IL-10 was increased (two-fold, p = 0.016) in HK19F-stimulated monocytes. Levels of HK19F-specific IFN-γ were significantly higher (11.7-fold, p = 0.038) in vitamin D-insufficient adults (50 nmol/L). Vitamin D also shifted the pro-inflammatory/anti-inflammatory balance towards an anti-inflammatory phenotype and increased the CD14 expression on monocytes (p = 0.008) in response to LPS but not HK19F stimulation. These results suggest that 1,25(OH)2D3 may be an important regulator of the inflammatory response and supports further in vivo and clinical studies to confirm the potential benefits of vitamin D in this context. PMID:27973447

  15. A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell

    Science.gov (United States)

    Xie, Sunney; Choi, Paul; Cai, Long

    2009-03-01

    By monitoring fluorescently labeled lactose permease with single-molecule sensitivity, we investigated the molecular mechanism of how an Escherichia coli cell with the lac operon switches from one phenotype to another. At intermediate inducer concentrations, a population of genetically identical cells exhibits two phenotypes: induced cells with highly fluorescent membranes and uninduced cells with a small number of membrane-bound permeases. We found that this basal-level expression results from partial dissociation of the tetrameric lactose repressor from one of its operators on looped DNA. In contrast, infrequent events of complete dissociation of the repressor from DNA result in large bursts of permease expression that trigger induction of the lac operon. Hence, a stochastic single-molecule event determines a cell's phenotype.

  16. Investigation of engineered bacterial adhesins for opportunity to interface cells with abiotic materials

    Science.gov (United States)

    Terrell, Jessica L.; Dong, Hong; Holthoff, Ellen L.; Small, Meagan C.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

    2016-05-01

    The convenience of cellular genetic engineering has afforded the power to build `smart' synthetic biological tools with novel applications. Here, we have explored opportunities to hybridize engineered cells with inorganic materials toward the development of 'living' device-compatible systems. Cellular structural biology is engineerable based on the ability to rewrite genetic code to generate recombinant, foreign, or even unnatural proteins. With this capability on the biological end, it should be possible to achieve superior abio-compatibility with the inorganic materials that compose current microfabricated technology. This work investigated the hair-like appendages of Escherichia coli known as Type 1 fimbriae that enable natural adhesion to glycosylated substrates. Sequence alterations within the fimbrial gene cluster were found to be well-tolerated, evidenced by tagging the fimbriae with peptide-based probes. As a further development, fimbriae tips could be reconfigured to, in turn, alter cell binding. In particular, the fimbriae were fused with a genetically optimized peptide-for-inorganics to enable metal binding. This work established methodologies to systematically survey cell adhesion properties across a suite of fimbriae-modified cell types as well as to direct patterned cell adhesion. Cell types were further customized for added complexity including turning on secondary gene expression and binding to gold surfaces. The former demonstrates potential for programmable gene switches and the latter for interfacing biology with inorganic materials. In general, the incorporation of 'programmed' cells into devices can be used to provide the feature of dynamic and automated cell response. The outcomes of this study are foundational toward the critical feature of deliberate positioning of cells as configurable biocomponentry. Overall, cellular integration into bioMEMs will yield advanced sensing and actuation.

  17. Efficacy of coating activated carbon with milk proteins to prevent binding of bacterial cells from foods for PCR detection.

    Science.gov (United States)

    Opet, Nathan J; Levin, Robert E

    2013-08-01

    Foods contaminated with pathogens are common sources of illness. Currently, the most common and sensitive rapid detection method involves the PCR. However, food matrices are complex and contain inhibitors that limit the sensitivity of the PCR. The use of coated activated carbon can effectively facilitate the removal of PCR inhibitors without binding targeted bacterial cells from food samples. With the use of activated carbon coated with milk proteins, a cell recovery at pH 7.0 of 95.7%±2.0% was obtained, compared to control uncoated activated carbon, which yielded a cell recovery of only 1.1%±0.8%. In addition, the milk protein coated activated carbon was able to absorb similar amounts of soluble compounds as uncoated activated carbon, with the exception of bovine hemoglobin. This suggests that the use of milk proteins to coat activated carbon may therefore serve as a suitable replacement for bentonite in the coating of activated carbon, which has previously been used for the removal of PCR inhibitors from food.

  18. Flow Cytometry Detection of Bacterial Cell Entrapment within the Chitosan Hydrogel and Antibacterial Property of Extracted Chitosan

    Directory of Open Access Journals (Sweden)

    Nafise Sadat Majidi

    2016-09-01

    Full Text Available Background:   Chitosan is unbranched polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine. Chitosan, derived from shrimp shell, has broad antimicrobial properties against Gram-negative, Gram-positive bacteria and fungi. Methods:  Chitosan was extracted from shrimp shell and studied for cell entrapment and anti-bacterial properties. The hydrogel chitosan was used as the beads for cell entrapment and chitosan beads were designed to deliver cells and nutrients. These data confirmed with flow cytometric analyses.                 Results:   Experimental results exhibited that internal diffusion through the chitosan matrix was the main mechanism for whole gelation by TPP (Tri-polyphosphate. The minimum inhibitory concentration (MIC for chitosan against Staphylococcus aureus and Escherichia coli was 16 and 32 μg/ml respectively. Conclusion:  Despite the antimicrobial properties of chitosan, trapped bacteria in the gel network were alive and were chelated indicating that their access to the outside was limited.

  19. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions

    DEFF Research Database (Denmark)

    Schwarz, Sandra; West, T Eoin; Boyer, Frédéric

    2010-01-01

    . From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas...... fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly...

  20. Towards identifying host cell-type specific response patterns to bacterial endosymbiosis

    DEFF Research Database (Denmark)

    Gavrilovic, Srdjan

    of view, available techniques have relied heavily on whole organ analyses that disregard specificities of individual cell types. To address this issue we aimed to develop a technology for comparative global analysis of mature mRNA and small RNA populations at the cell type specific level in the model...... plant Lotus japonicus. A powerful approach referred to here as Defined Expression and RNA Affinity co-Purification (DERAP) was developed to study gene expression and small RNA populations in the host roots during early phases of signal exchange at the cell-type level. As a basis for DERAP analysis...

  1. CD8(+) T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity.

    Science.gov (United States)

    Shen, Haiqian; Gonzalez-Juarbe, Norberto; Blanchette, Krystle; Crimmins, Gregory; Bergman, Molly A; Isberg, Ralph R; Orihuela, Carlos J; Dube, Peter H

    2016-09-01

    CD8(+) T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8(+) T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8(+) T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8(+) T cells are generated and provide limited protection against challenge with virulent OVA(+)Y. pseudotuberculosis. Perforin expression by OVA-specific CD8(+) T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8(+) T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8(+) T cell status. These studies indicate that CD8(+) T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death.

  2. Detection of antibodies to bacterial cell wall peptidoglycan in human sera. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Heymer, B.; Schleifer, K.H.; Read, S.; Zabriskie, J.B.; Krause, R.M.

    1976-07-01

    A radioimmunoassay has been developed for the measurement of antibodies to peptidoglycan in human sera including patients with rheumatic feaver and juvenile rheumatoid arthritis. The assay is based on the percentage of binding of the hapten /sup 125/I-L-Ala-..gamma..-D-Glu-L-Lys-D-Ala-D-Ala, the major peptide determinant of peptidoglycan. Because of differences in the avidity of the antibodies in different sera, the amount of antibody was expressed as pentapeptide hapten-binding capacity (pentapeptide-HBC in ng/ml of serum). Fourteen out of 105 normal blood donors had a pentapeptide-HBC value greater than or equal to 75 ng/ml serum. Values in healthy children 5 to 18 years of age were less than or equal to 50 ng/ml. Sixty-eight percent of the individuals with rheumatic fever had values greater than or equal to 75 ng/ml, an indication that streptococcal infections can stimulate an immune response to peptidoglycan. Thirty-five percent of the patients with juvenile rheumatoid arthritis had values greater than or equal to 75 ng/ml. Such a finding points to a possible association between bacterial infections and juvenile rheumatoid arthritis.

  3. Laboratory studies with cloud-derived Bacterial Cells acting as Ice Nuclei in the Immersion and Deposition Mode

    Science.gov (United States)

    Oehm, C.; Chou, C.; Amato, P.; Attard, E.; Delort, A.-M.; Morris, C.; Kiselev, A.; Stetzer, O.; Möhler, O.; Leisner, T.

    2012-04-01

    Atmospheric aerosol particles play an important role in cloud microphysics. Aerosols of biological origin are a subgroup, and some of them are able to act as heterogeneous ice nuclei and thus influence cloud life cycles and the climate. Some bacteria species have been found to act as ice nuclei at relatively high temperatures up to -2 degree Celsius and are therefore of particular importance as "high temperature" ice nuclei. Recently, ice nucleation experiments with bacterial cells from different sources were performed at the aerosol and cloud simulation chamber AIDA at the Karlsruhe Institute of Technology. At the AIDA facility, microphysical cloud processes can be simulated and investigated in laboratory at realistic atmospheric cloud conditions. Different ice nucleation active (INA) bacteria strains were isolated from cloud water, glacier melt water and phyllosphere and examined in AIDA experiments. The living cells were suspended in nanopure or artificial cloud water and injected into the cloud chamber through a dispersion nozzle. The injected droplets evaporated in the chamber and the bacterial cells were transformed into the aerosol phase. After the spraying, the cloud formation was started by expansion cooling. Experiments were performed in the temperature range from -2 down to -20 degree Celsius. Detailed measurements of the number concentration and size distribution of the aerosol particles as well as of the droplets and ice particles were carried out during the AIDA experiments. A minor fraction of the bacteria cells was observed to act as ice nuclei in the immersion nucleation mode at higher temperatures as well as in the deposition nucleation mode at lower temperatures. The ice activity started at -6 degree Celsius. The most efficient INA bacteria species were Pseudomonas syringae 32b74 and Pseudomonas fluorescens Antarctica1. The ice active number fraction with respect to the cells varied from 0,01 to 0,1, and it does not change at different

  4. Structure of ristocetin A in complex with a bacterial cell-wall mimetic

    OpenAIRE

    Nahoum, Virginie; Spector, Sherri; Loll, Patrick J.

    2009-01-01

    The crystal structure of the complex between ristocetin A and the cell-wall peptide mimetic N-acetyl-lysine-d-alanine-d-alanine has been solved. Structural details explaining the anticooperativity of the antibiotic have been identified.

  5. SIMSISH technique does not alter the apparent isotopic composition of bacterial cells.

    Directory of Open Access Journals (Sweden)

    Olivier Chapleur

    Full Text Available In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH and nanoscale secondary ion mass spectrometry (nanoSIMS imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine - iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific (13C uptake during labelled methanol anaerobic degradation.

  6. Different phenotypes of CD8+ T cells associated with bacterial load in active tuberculosis.

    Science.gov (United States)

    Silva, Bruna Daniella de Souza; Trentini, Monalisa Martins; da Costa, Adeliane Castro; Kipnis, Andre; Junqueira-Kipnis, Ana Paula

    2014-07-01

    Tuberculosis is an infectious disease that affects millions of people worldwide with an annual mortality rate of 1.3 million. The mechanisms contributing to the loss of balance of immune responses and progression to active tuberculosis disease are unknown. Although CD4+ and CD8+ T cells and the cytokines they produce are crucial for protection against tuberculosis they have different roles in tuberculosis immunology. The function of CD4+ T cells has been extensively studied; however, less is known about the phenotype and function of CD8+ T cells. This study evaluated the specific expression of IFN-γ, IL-17, IL-10, and TGF-β and ex vivo expression of perforin and granzyme-B by CD8+ T cells from active tuberculosis individuals compared with latent infected individuals and non-latent infected individuals. Tuberculosis responses were correlated with the baciloscopy score. We observed that the presence of IL-10 and TGF-β expression and down-expression of granzyme-B in CD8+ T cells correlated with increased sputum bacillary load in active tuberculosis individuals. These findings provide new insights into the role of CD8+ T cells in Mycobacterium tuberculosis disease.

  7. Whole genome amplification and de novo assembly of single bacterial cells.

    Directory of Open Access Journals (Sweden)

    Sébastien Rodrigue

    Full Text Available BACKGROUND: Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA and complete genome sequencing of individual cells. METHODOLOGY/PRINCIPAL FINDINGS: We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA, and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs. CONCLUSIONS/SIGNIFICANCE: The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.

  8. Unique interplay between sugar and lipid in determining the antigenic potency of bacterial antigens for NKT cells.

    Directory of Open Access Journals (Sweden)

    Enrico Girardi

    2011-11-01

    Full Text Available Invariant natural killer T (iNKT cells are an evolutionary conserved T cell population characterized by features of both the innate and adaptive immune response. Studies have shown that iNKT cells are required for protective responses to Gram-positive pathogens such as Streptococcus pneumoniae, and that these cells recognize bacterial diacylglycerol antigens presented by CD1d, a non-classical antigen-presenting molecule. The combination of a lipid backbone containing an unusual fatty acid, vaccenic acid, as well as a glucose sugar that is weaker or not stimulatory when linked to other lipids, is required for iNKT cell stimulation by these antigens. Here we have carried out structural and biophysical studies that illuminate the reasons for the stringent requirement for this unique combination. The data indicate that vaccenic acid bound to the CD1d groove orients the protruding glucose sugar for TCR recognition, and it allows for an additional hydrogen bond of the glucose with CD1d when in complex with the TCR. Furthermore, TCR binding causes an induced fit in both the sugar and CD1d, and we have identified the CD1d amino acids important for iNKT TCR recognition and the stability of the ternary complex. The studies show also how hydrogen bonds formed by the glucose sugar can account for the distinct binding kinetics of the TCR for this CD1d-glycolipid complex. Therefore, our studies illuminate the mechanism of glycolipid recognition for antigens from important pathogens.

  9. In Situ Confocal Raman Microscopy of Hydrated Early Stages of Bacterial Biofilm Formation on Various Surfaces in a Flow Cell.

    Science.gov (United States)

    Smith-Palmer, Truis; Lin, Sicheng; Oguejiofor, Ikenna; Leng, Tianyang; Pustam, Amanda; Yang, Jin; Graham, Lori L; Wyeth, Russell C; Bishop, Cory D; DeMont, M Edwin; Pink, David

    2016-02-01

    Bacterial biofilms are precursors to biofouling by other microorganisms. Understanding their initiation may allow us to design better ways to inhibit them, and thus to inhibit subsequent biofouling. In this study, the ability of confocal Raman microscopy to follow the initiation of biofouling by a marine bacterium, Pseudoalteromonas sp. NCIMB 2021 (NCIMB 2021), in a flow cell, using optical and confocal Raman microscopy, was investigated. The base of the flow cell comprised a cover glass. The cell was inoculated and the bacteria attached to, and grew on, the cover glass. Bright field images and Raman spectra were collected directly from the hydrated biofilms over several days. Although macroscopically the laser had no effect on the biofilm, within the first 24 h cells migrated away from the position of the laser beam. In the absence of flow, a buildup of extracellular substances occurred at the base of the biofilm. When different coatings were applied to cover glasses before they were assembled into the flow cells, the growth rate, structure, and composition of the resulting biofilm was affected. In particular, the ratio of Resonance Raman peaks from cytochrome c (CC) in the extracellular polymeric substances, to the Raman phenylalanine (Phe) peak from protein in the bacteria, depended on both the nature of the surface and the age of the biofilm. The ratios were highest for 24 h colonies on a hydrophobic surface. Absorption of a surfactant with an ethyleneoxy chain into the hydrophobic coating created a surface similar to that given with a simple PEG coating, where bacteria grew in colonies away from the surface rather than along the surface, and CC:Phe ratios were initially low but increased at least fivefold in the first 48 h.

  10. Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results.

    Directory of Open Access Journals (Sweden)

    Natalie Bordag

    Full Text Available The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli as well as mammalian cells chinese hamster ovary (CHO and mouse myeloma cells (NS0.The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

  11. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    Directory of Open Access Journals (Sweden)

    Bang Dang D

    2008-06-01

    Full Text Available Abstract Background Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation. Results C. jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under in vitro culture condition C. jejuni strains of both human and chicken origins can invade avian host cells with a pro-inflammatory response and that the virulence-associated genes of C. jejuni may play a role in this process.

  12. Tet38 Efflux Pump Affects Staphylococcus aureus Internalization by Epithelial Cells through Interaction with CD36 and Contributes to Bacterial Escape from Acidic and Nonacidic Phagolysosomes.

    Science.gov (United States)

    Truong-Bolduc, Q C; Khan, N S; Vyas, J M; Hooper, D C

    2017-02-01

    We previously reported that the Tet38 efflux pump is involved in internalization of Staphylococcus aureus by A549 lung epithelial cells. A lack of tet38 reduced bacterial uptake by A549 cells to 36% of that of the parental strain RN6390. Using invasion assays coupled with confocal microscopy imaging, we studied the host cell receptor(s) responsible for bacterial uptake via interaction with Tet38. We also assessed the ability of S. aureus to survive following alkalinization of the phagolysosomes by chloroquine. Antibody to the scavenger receptor CD36 reduced the internalization of S. aureus RN6390 by A549 cells, but the dependence on CD36 was reduced in QT7 tet38, suggesting that an interaction between Tet38 and CD36 contributed to S. aureus internalization. Following fusion of the S. aureus-associated endosomes with lysosomes, alkalinization of the acidic environment with chloroquine led to a rapid increase in the number of S. aureus RN6390 bacteria in the cytosol, followed by a decrease shortly thereafter. This effect of chloroquine was not seen in the absence of intact Tet38 in mutant QT7. These data taken together suggest that Tet38 plays a role both in bacterial internalization via interaction with CD36 and in bacterial escape from the phagolysosomes.

  13. Saponin, an inhibitory agent of carbon dioxide production by white cells : its use in the microbiologic examination of blood components in an automated bacterial culture system

    NARCIS (Netherlands)

    van Doorne, H; van der Tuuk Adriani, WPA; van de Ven, LI; Bosch, EH; de Natris, T; Sibinga, CTS

    1998-01-01

    BACKGROUND: Blood components with a white cell count >100 x 10(9) per L may cause false-positive results when the BacT/Alert system is used for the microbiologic examination. The effects of different concentrations of saponin on bacterial growth and on carbon dioxide production by blood fractions wi

  14. Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Chan, J W; Winhold, H; Corzett, M H; Ulloa, J M; Cosman, M; Balhorn, R; Huser, T

    2007-01-09

    Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG(1-120)) Raman spectra were acquired of individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of three hours. Data was also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in

  15. Gene dosage imbalance during DNA replication controls bacterial cell-fate decision

    Science.gov (United States)

    Igoshin, Oleg

    Genes encoding proteins in a common regulatory network are frequently located close to one another on the chromosome to facilitate co-regulation or couple gene expression to growth rate. Contrasting with these observations, here we demonstrate a functional role for the arrangement of Bacillus subtilis sporulation network genes on opposite sides of the chromosome. We show that the arrangement of two sporulation network genes, one located close to the origin, the other close to the terminus leads to a transient gene dosage imbalance during chromosome replication. This imbalance is detected by the sporulation network to produce cell-cycle coordinated pulses of the sporulation master regulator Spo0A~P. This pulsed response allows cells to decide between sporulation and continued vegetative growth during each cell-cycle spent in starvation. Furthermore, changes in DNA replication and cell-cycle parameters with decreased growth rate in starvation conditions enable cells to indirectly detect starvation without the need for evaluating specific metabolites. The simplicity of the uncovered coordination mechanism and starvation sensing suggests that it may be widely applicable in a variety of gene regulatory and stress-response settings. This work is supported by National Science Foundation Grants MCB-1244135, EAGER-1450867, MCB-1244423, NIH NIGMS Grant R01 GM088428 and HHMI International Student Fellowship.

  16. Single-cell level based approach to investigate bacterial metabolism during batch industrial fermentation

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Larsen, Poul; Eriksen, Niels T.

    Escherichia coli fermentations have been studied for decades, but most results are based on average measurements of the whole populations of cells, whilst averaged data can mask the distribution of activities at the sub-population or single-cell level. A population of genetically identical cells...... can exhibit different phenotypes under specific environmental conditions that show significant differences in physiological parameters from the population average. However, studies concerning segregation of populations into metabolically diversified subpopulations are scarce. Acetate is a product...... of Escherichia coli overflow metabolism when the bacteria are grown under aerobic conditions and glucose is present in excessive concentrations. Acetate accumulation is of the utmost importance in batch fermentation processes as it is an undesirable byproduct that negatively affects growth, physiology...

  17. THE TRANSMEMBRANE SIGNAL TRANSDUCTION IN HEp-2 CELLS INDUCED BY BACTERIAL ADHERENCE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@ In order to understand the role of transmembrane signal transduction of host cells in the early steps of infection,the adherence of E. coli to HEp-2 cells and the change of activity of phospholipase C-γ (PLC-γ) induced by the adherence were investigated.The adherence of enteropathogenic E.coli (EPEC), strain E.7, induced a significant increase of inositol-triphosphat (IP-3) level in HEp-2 cells. The adherence of the bacteria and the increase of IP-3 was kinetically correlated. Whereas the increase of IP3 level induced by the adherence of the control strain EPEC (H511), a non-piliated strain, was much meager than that by E7, a piliated strain. The results highlighted an important role of transmembrane signals like IP-3 in the pathogenesis of EPEC.

  18. Scaling laws governing stochastic growth and division of single bacterial cells

    CERN Document Server

    Iyer-Biswas, Srividya; Henry, Jonathan T; Lo, Klevin; Burov, Stanislav; Lin, Yihan; Crooks, Gavin E; Crosson, Sean; Dinner, Aaron R; Scherer, Norbert F

    2014-01-01

    Uncovering the quantitative laws that govern the growth and division of single cells remains a major challenge. Using a unique combination of technologies that yields unprecedented statistical precision, we find that the sizes of individual Caulobacter crescentus cells increase exponentially in time. We also establish that they divide upon reaching a critical multiple ($\\approx$1.8) of their initial sizes, rather than an absolute size. We show that when the temperature is varied, the growth and division timescales scale proportionally with each other over the physiological temperature range. Strikingly, the cell-size and division-time distributions can both be rescaled by their mean values such that the condition-specific distributions collapse to universal curves. We account for these observations with a minimal stochastic model that is based on an autocatalytic cycle. It predicts the scalings, as well as specific functional forms for the universal curves. Our experimental and theoretical analysis reveals a ...

  19. A polymerization–depolymerization model for generation of contractile force during bacterial cell division

    Indian Academy of Sciences (India)

    Biplab Ghosh; Anirban Sain

    2008-08-01

    During the last phase of cell division in bacteria, a polymeric ring forms at the division site. The ring, made of intracellular proteins, anchors to the cell wall and starts to contract. That initiates a dividing septum to close in, like the shutter of a camera, eventually guillotining the cell into two daughters. All through, the ring remains at the leading edge of the septum and seems to power its closure. It is not understood why does the ring contract. We propose a theoretical model to explain this. It is worth mentioning that a similar contraction phenomenon occurs for the actin ring in eukaryotes, but there it is due to motor proteins, which however, are absent in bacteria.

  20. Overloaded and stressed: whole-cell considerations for bacterial synthetic biology.

    Science.gov (United States)

    Borkowski, Olivier; Ceroni, Francesca; Stan, Guy-Bart; Ellis, Tom

    2016-10-01

    The predictability and robustness of engineered bacteria depend on the many interactions between synthetic constructs and their host cells. Expression from synthetic constructs is an unnatural load for the host that typically reduces growth, triggers stresses and leads to decrease in performance or failure of engineered cells. Work in systems and synthetic biology has now begun to address this through new tools, methods and strategies that characterise and exploit host-construct interactions in bacteria. Focusing on work in E. coli, we review here a selection of the recent developments in this area, highlighting the emerging issues and describing the new solutions that are now making the synthetic biology community consider the cell just as much as they consider the construct.

  1. Expression of innate immune complement regulators on brain epithelial cells during human bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Gasque Philippe

    2006-09-01

    Full Text Available Abstract Background In meningitis, the cerebrospinal fluid contains high levels of innate immune molecules (e.g. complement which are essential to ward off the infectious challenge and to promote the infiltration of phagocytes (neutrophils, monocytes. However, epithelial cells of either the ependymal layer, one of the established niche for adult neural stem cells, or of the choroid plexus may be extremely vulnerable to bystander attack by cytotoxic and cytolytic complement components. Methods In this study, we assessed the capacity of brain epithelial cells to express membrane-bound complement regulators (ie, CD35, CD46, CD55 and CD59 in vitro and in situ by immunostaining of control and meningitis human brain tissue sections. Results Double immunofluorescence experiments for ependymal cell markers (GFAP, S100, ZO-1, E-cadherin and complement regulators indicated that the human ependymal cell line model was strongly positive for CD55, CD59 compared to weak stainings for CD46 and CD35. In tissues, we found that CD55 was weakly expressed in control choroid plexus and ependyma but was abundantly expressed in meningitis. Anti-CD59 stained both epithelia in apical location while increased CD59 staining was solely demonstrated in inflamed choroid plexus. CD46 and CD35 were not detected in control tissue sections. Conversely, in meningitis, the ependyma, subependyma and choroid plexus epithelia were strongly stained for CD46 and CD35. Conclusion This study delineates for the first time the capacity of brain ependymal and epithelial cells to respond to and possibly sustain the innate complement-mediated inflammatory insult.

  2. Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Julien Nourikyan

    2015-09-01

    Full Text Available Bacterial capsular polysaccharides (CPS are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with

  3. Modeling the induced mutation process in bacterial cells with defects in excision repair system

    Science.gov (United States)

    Bugay, A. N.; Vasilyeva, M. A.; Krasavin, E. A.; Parkhomenko, A. Yu.

    2015-12-01

    A mathematical model of the UV-induced mutation process in Escherichia coli cells with defects in the uvrA and polA genes has been developed. The model describes in detail the reaction kinetics for the excision repair system. The number of mismatches as a result of translesion synthesis is calculated for both wild-type and mutant cells. The effect of temporal modulation of the number of single-stranded DNA during postreplication repair has been predicted. A comparison of effectiveness of different repair systems has been conducted.

  4. Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes

    Directory of Open Access Journals (Sweden)

    Romeo Tony

    2009-01-01

    Full Text Available Abstract Background The phylum Verrucomicrobia is a divergent phylum within domain Bacteria including members of the microbial communities of soil and fresh and marine waters; recently extremely acidophilic members from hot springs have been found to oxidize methane. At least one genus, Prosthecobacter, includes species with genes homologous to those encoding eukaryotic tubulins. A significant superphylum relationship of Verrucomicrobia with members of phylum Planctomycetes possessing a unique compartmentalized cell plan, and members of the phylum Chlamydiae including human pathogens with a complex intracellular life cycle, has been proposed. Based on the postulated superphylum relationship, we hypothesized that members of the two separate phyla Planctomycetes and Verrucomicrobia might share a similar ultrastructure plan differing from classical prokaryote organization. Results The ultrastructure of cells of four members of phylum Verrucomicrobia – Verrucomicrobium spinosum, Prosthecobacter dejongeii, Chthoniobacter flavus, and strain Ellin514 – was examined using electron microscopy incorporating high-pressure freezing and cryosubstitution. These four members of phylum Verrucomicrobia, representing 3 class-level subdivisions within the phylum, were found to possess a compartmentalized cell plan analogous to that found in phylum Planctomycetes. Like all planctomycetes investigated, they possess a major pirellulosome compartment containing a condensed nucleoid and ribosomes surrounded by an intracytoplasmic membrane (ICM, as well as a ribosome-free paryphoplasm compartment between the ICM and cytoplasmic membrane. Conclusion A unique compartmentalized cell plan so far found among Domain Bacteria only within phylum Planctomycetes, and challenging our concept of prokaryote cell plans, has now been found in a second phylum of the Domain Bacteria, in members of phylum Verrucomicrobia. The planctomycete cell plan thus occurs in at least two

  5. How do bacterial cells ensure that metalloproteins get the correct metal?

    Science.gov (United States)

    Waldron, Kevin J; Robinson, Nigel J

    2009-01-01

    Protein metal-coordination sites are richly varied and exquisitely attuned to their inorganic partners, yet many metalloproteins still select the wrong metals when presented with mixtures of elements. Cells have evolved elaborate mechanisms to scavenge for sufficient metal atoms to meet their needs and to adjust their needs to match supply. Metal sensors, transporters and stores have often been discovered as metal-resistance determinants, but it is emerging that they perform a broader role in microbial physiology: they allow cells to overcome inadequate protein metal affinities to populate large numbers of metalloproteins with the right metals.

  6. FACTORS LIMITING BACTERIAL GROWTH : III. CELL SIZE AND "PHYSIOLOGIC YOUTH" IN BACTERIUM COLI CULTURES.

    Science.gov (United States)

    Hershey, A D; Bronfenbrenner, J

    1938-07-20

    1. Measurements of the rate of oxygen uptake per cell in transplants of Bacterium coli from cultures of this organism in different phases of growth have given results in essential agreement with the observations of others. 2. Correlations of viable count, centrifugable nitrogen, and turbidity, with oxygen consumption, indicate that the increased metabolism during the early portion of the growth period is quantitatively referable to increased average size of cells. 3. Indirect evidence has suggested that the initial rate of growth of transplants is not related to the phase of growth of the parent culture.

  7. Characterization of bacterial and archaeal communities in air-cathode microbial fuel cells, open circuit and sealed-off reactors

    KAUST Repository

    Chehab, Noura A.

    2013-06-18

    A large percentage of organic fuel consumed in a microbial fuel cell (MFC) is lost as a result of oxygen transfer through the cathode. In order to understand how this oxygen transfer affects the microbial community structure, reactors were operated in duplicate using three configurations: closed circuit (CC; with current generation), open circuit (OC; no current generation), and sealed off cathodes (SO; no current, with a solid plate placed across the cathode). Most (98 %) of the chemical oxygen demand (COD) was removed during power production in the CC reactor (maximum of 640 ± 10 mW/m 2), with a low percent of substrate converted to current (coulombic efficiency of 26.5 ± 2.1 %). Sealing the cathode reduced COD removal to 7 %, but with an open cathode, there was nearly as much COD removal by the OC reactor (94.5 %) as the CC reactor. Oxygen transfer into the reactor substantially affected the composition of the microbial communities. Based on analysis of the biofilms using 16S rRNA gene pyrosequencing, microbes most similar to Geobacter were predominant on the anodes in the CC MFC (72 % of sequences), but the most abundant bacteria were Azoarcus (42 to 47 %) in the OC reactor, and Dechloromonas (17 %) in the SO reactor. Hydrogenotrophic methanogens were most predominant, with sequences most similar to Methanobacterium in the CC and SO reactor, and Methanocorpusculum in the OC reactors. These results show that oxygen leakage through the cathode substantially alters the bacterial anode communities, and that hydrogenotrophic methanogens predominate despite high concentrations of acetate. The predominant methanogens in the CC reactor most closely resembled those in the SO reactor, demonstrating that oxygen leakage alters methanogenic as well as general bacterial communities. © 2013 Springer-Verlag Berlin Heidelberg.

  8. Activation of toll-like receptors and dendritic cells by a broad range of bacterial molecules

    NARCIS (Netherlands)

    Boele, L.C.L.; Bajramovic, J.J.; Vries, A.M.M.B.C. de; Voskamp-Visser, I.A.I.; Kaman, W.E.; Kleij, D. van der

    2009-01-01

    Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, the

  9. Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

    Directory of Open Access Journals (Sweden)

    Chiou-Yueh Yeh

    Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

  10. The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes

    DEFF Research Database (Denmark)

    Chindera, Kantaraja; Mahato, Manohar; Sharma, Ashwani Kumar;

    2016-01-01

    To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption...

  11. An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments

    DEFF Research Database (Denmark)

    Yu, Ran; Graf, Joerg; Smets, Barth F.

    2008-01-01

    Two cell recovery methods for IOB enrichments were evaluated for DNA extraction and further PCR-based 16S rRNA gene clone library creation. One was a published method consisting of heating plus oxalic acid treatment and the other one was a new method based on enzymatic agarose digestion (using β-...

  12. In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection

    NARCIS (Netherlands)

    Bartfeld, Sina; Bayram, Tülay; van de Wetering, Marc; Huch, Meritxell; Begthel, Harry; Kujala, Pekka; Vries, Robert; Peters, Peter J; Clevers, Hans

    2015-01-01

    BACKGROUND & AIMS: We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technolo

  13. The membrane steps of bacterial cell wall synthesis as antibiotic targets

    NARCIS (Netherlands)

    Liu, Yao; Breukink, Eefjan

    2016-01-01

    Peptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied ext

  14. Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida).

    Science.gov (United States)

    Hirao, A; Ehlers, R-U

    2009-08-01

    The rhabditid nematodes Steinernema carpocapsae and Steinernema feltiae are used in biological control of insect pests. Mass production is done in liquid culture media pre-incubated with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, before nematode dauer juveniles (DJs) are inoculated. As a response to food signals produced by the bacterial symbionts, the DJs exit from the developmentally arrested dauer stage (they recover development) and grow to adults, which produce DJ offspring. Variable DJ recovery after inoculation often causes process failure due to non-synchronous population development and low numbers of adult nematodes. This contribution investigated the influence of the bacterial cell density on DJ recovery and development to adults. At higher density of 10(10) bacterial cells ml(-1), a higher percentage of DJ recovery was induced, and adults occurred earlier in both Steinernema spp. than at lower density of 10(9) and 10(8) cells ml(-1). Xenorhabdus symbionts produce phase variants. Recovery in bacteria-free supernatants was lower than in supernatants containing bacterial cells for both primary and secondary phase Xenorhabdus spp. and lower in secondary than in primary phase supernatants or cell suspensions. In general, recovery was lower for Steinernema feltiae and the time at which 50% of the population had recovered after exposure to the food signal was longer (RT(50) = 17.1 h) than for Steinernema carpocapsae (RT(50) = 6.6 h). Whereas >90% S. carpocapsae DJs recovered in hemolymph serum of the lepidopteran insect Galleria mellonella, recovery of S. feltiae only reached 31%. Penetration into a host insect prior to exposure to the insect's food signal did not enhance DJ recovery. Consequences for liquid culture mass production of the nematodes and differences between species of the genera Steinernema and Heterorhabditis are discussed.

  15. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation.

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-04-13

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.

  16. Robotic milking and milk quality: effects on bacterial counts, somatic cell counts, freezing point and free fatty acids

    Directory of Open Access Journals (Sweden)

    Yvonne van der Vorst

    2010-01-01

    Full Text Available Changes in milk quality after the introduction of automatic milking systems (AM-systems on dairy farms in TheNetherlands, Germany and Denmark were examined and the data were compared with milk quality results of farms withconventional milking technology. After introduction, a small, but significant increase in total bacterial count, somatic cellcount, freezing point and free fatty acids was observed. The highest levels for total plate count and cell count are foundin the first six months after introduction. After this period the milk quality slightly improves to a more stable level.Risk factors related with milk quality concern general farm characteristics, animal health, AM-system, cleaning and cooling,housing, management skills of the farmer and the hygiene on the farm. Total plate count was significantly relatedto milk yield of the herd, cleaning of the area around the AM-system and the overall hygiene on the farm. Bulk milksomatic cell count appeared to be significantly related to milk yield of the herd and the number of milkings before replacementof the liners. An increased milking frequency is not the only explanation of increased free fatty acid levels. Technicalfactors related to free fatty acids mainly concerned the air inlet in the teat cups, bubbling (excessive air inlet and a toolong post run time of the milk pump. However, several questions regarding the causes of increased free fatty acid levelsremained unclear.

  17. Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation

    Science.gov (United States)

    Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

    2016-01-01

    Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1–45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance. PMID:27089320

  18. Artificial Intelligence vs. Statistical Modeling and Optimization of Continuous Bead Milling Process for Bacterial Cell Lysis.

    Science.gov (United States)

    Haque, Shafiul; Khan, Saif; Wahid, Mohd; Dar, Sajad A; Soni, Nipunjot; Mandal, Raju K; Singh, Vineeta; Tiwari, Dileep; Lohani, Mohtashim; Areeshi, Mohammed Y; Govender, Thavendran; Kruger, Hendrik G; Jawed, Arshad

    2016-01-01

    For a commercially viable recombinant intracellular protein production process, efficient cell lysis and protein release is a major bottleneck. The recovery of recombinant protein, cholesterol oxidase (COD) was studied in a continuous bead milling process. A full factorial response surface methodology (RSM) design was employed and compared to artificial neural networks coupled with genetic algorithm (ANN-GA). Significant process variables, cell slurry feed rate (A), bead load (B), cell load (C), and run time (D), were investigated and optimized for maximizing COD recovery. RSM predicted an optimum of feed rate of 310.73 mL/h, bead loading of 79.9% (v/v), cell loading OD600nm of 74, and run time of 29.9 min with a recovery of ~3.2 g/L. ANN-GA predicted a maximum COD recovery of ~3.5 g/L at an optimum feed rate (mL/h): 258.08, bead loading (%, v/v): 80%, cell loading (OD600nm): 73.99, and run time of 32 min. An overall 3.7-fold increase in productivity is obtained when compared to a batch process. Optimization and comparison of statistical vs. artificial intelligence techniques in continuous bead milling process has been attempted for the very first time in our study. We were able to successfully represent the complex non-linear multivariable dependence of enzyme recovery on bead milling parameters. The quadratic second order response functions are not flexible enough to represent such complex non-linear dependence. ANN being a summation function of multiple layers are capable to represent complex non-linear dependence of variables in this case; enzyme recovery as a function of bead milling parameters. Since GA can even optimize discontinuous functions present study cites a perfect example of using machine learning (ANN) in combination with evolutionary optimization (GA) for representing undefined biological functions which is the case for common industrial processes involving biological moieties.

  19. Water reservoir maintained by cell growth fuels the spreading of a bacterial swarm.

    Science.gov (United States)

    Wu, Yilin; Berg, Howard C

    2012-03-13

    Flagellated bacteria can swim across moist surfaces within a thin layer of fluid, a means for surface colonization known as swarming. This fluid spreads with the swarm, but how it does so is unclear. We used micron-sized air bubbles to study the motion of this fluid within swarms of Escherichia coli. The bubbles moved diffusively, with drift. Bubbles starting at the swarm edge drifted inward for the first 5 s and then moved outward. Bubbles starting 30 μm from the swarm edge moved inward for the first 20 s, wandered around in place for the next 40 s, and then moved outward. Bubbles starting at 200 or 300 μm from the edge moved outward or wandered around in place, respectively. So the general trend was inward near the outer edge of the swarm and outward farther inside, with flows converging on a region about 100 μm from the swarm edge. We measured cellular metabolic activities with cells expressing a short-lived GFP and cell densities with cells labeled with a membrane fluorescent dye. The fluorescence plots were similar, with peaks about 80 μm from the swarm edge and slopes that mimicked the particle drift rates. These plots suggest that net fluid flow is driven by cell growth. Fluid depth is largest in the multilayered region between approximately 30 and 200 μm from the swarm edge, where fluid agitation is more vigorous. This water reservoir travels with the swarm, fueling its spreading. Intercellular communication is not required; cells need only grow.

  20. Mammalian cell entry genes in Streptomyces may provide clues to the evolution of bacterial virulence

    Science.gov (United States)

    Clark, Laura C.; Seipke, Ryan F.; Prieto, Pilar; Willemse, Joost; van Wezel, Gilles P.; Hutchings, Matthew I.; Hoskisson, Paul A.

    2013-01-01

    Understanding the evolution of virulence is key to appreciating the role specific loci play in pathogenicity. Streptomyces species are generally non-pathogenic soil saprophytes, yet within their genome we can find homologues of virulence loci. One example of this is the mammalian cell entry (mce) locus, which has been characterised in Mycobacterium tuberculosis. To investigate the role in Streptomyces we deleted the mce locus and studied its impact on cell survival, morphology and interaction with other soil organisms. Disruption of the mce cluster resulted in virulence towards amoebae (Acanthamoeba polyphaga) and reduced colonization of plant (Arabidopsis) models, indicating these genes may play an important role in Streptomyces survival in the environment. Our data suggest that loss of mce in Streptomyces spp. may have profound effects on survival in a competitive soil environment, and provides insight in to the evolution and selection of these genes as virulence factors in related pathogenic organisms. PMID:23346366

  1. Structural insight into the transglycosylation step of bacterial cell-wall biosynthesis.

    Science.gov (United States)

    Lovering, Andrew L; de Castro, Liza H; Lim, Daniel; Strynadka, Natalie C J

    2007-03-09

    Peptidoglycan glycosyltransferases (GTs) catalyze the polymerization step of cell-wall biosynthesis, are membrane-bound, and are highly conserved across all bacteria. Long considered the "holy grail" of antibiotic research, they represent an essential and easily accessible drug target for antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus. We have determined the 2.8 angstrom structure of a bifunctional cell-wall cross-linking enzyme, including its transpeptidase and GT domains, both unliganded and complexed with the substrate analog moenomycin. The peptidoglycan GTs adopt a fold distinct from those of other GT classes. The structures give insight into critical features of the catalytic mechanism and key interactions required for enzyme inhibition.

  2. The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

    Science.gov (United States)

    Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

    2015-04-21

    A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

  3. Bacterial viruses enable their host to acquire antibiotic resistance genes from neighbouring cells

    DEFF Research Database (Denmark)

    Haaber, Jakob Krause; Leisner, Jørgen; Cohn, Marianne Thorup

    2016-01-01

    Prophages are quiescent viruses located in the chromosomes of bacteria. In the human pathogen, Staphylococcus aureus, prophages are omnipresent and are believed to be responsible for the spread of some antibiotic resistance genes. Here we demonstrate that release of phages from a subpopulation of S....... aureus cells enables the intact, prophage-containing population to acquire beneficial genes from competing, phage-susceptible strains present in the same environment. Phage infection kills competitor cells and bits of their DNA are occasionally captured in viral transducing particles. Return...... of such particles to the prophage-containing population can drive the transfer of genes encoding potentially useful traits such as antibiotic resistance. This process, which can be viewed as ‘auto-transduction’, allows S. aureus to efficiently acquire antibiotic resistance both in vitro and in an in vivo virulence...

  4. Biosorption of heavy metal by thermotolerant polymerproducing bacterial cells and the bioflocculant

    Directory of Open Access Journals (Sweden)

    Saithong Kaewchai

    2002-07-01

    Full Text Available Three strains of thermotolerant polymer-producing bacteria; Bacillus subtilis WD 90, Bacillus subtilis SM 29, and Enterobacter agglomerans SM 38 as well as their biofloculants were used to investigate on the adsorption of heavy metal, nickel and cadmium. The effects of pH and concentrations of heavy metal were investigated. The optimum pH for nickel and cadmium adsorption by the dried cells of E. agglomerans SM 38 were found to be 7.0 (25.5% removal and 8.0 (32% removal, respectively. For B. subtilis WD 90 and B. subtilis SM 29, the optimum pH at 8.0 exhibited the nickel removal of 27% and 25%, respectively, and cadmium removal of 28% and 28.5%, respectively. The heavy metal adsorption by the dried cells and wet cells of E. agglomerans SM 38 were slightly increased with increasing initial concentrations of nickel and cadmium up to 60 and 30 ppm, respectively. The bioflocculant of B. subtilis WD 90 and B. subtilis SM 29 showed the highest nickel removal of 90.7% and 87.0% respectively, while the cadmium removal was 90.9 and 91.4%, respectively. The optimum pH for adsorption of both nickel and cadmium by the bioflocculant of E. agglomerans SM 38 was 7.0 with the removal of 92.8 and 84.2%, respectively. The optimum nickel concentration for adsorption by the bioflocculant of E. agglomerans SM 38 was 10 ppm, with the removal of 92.5%, and rather stable up to 60 ppm. The optimum cadmium concentration for adsorption by the bioflocculant of B. subtilis SM 29 was 60 ppm at pH 8.0 with the removal of 85.7%. Therefore, the bioflocculant of the three isolates gave higher heavy metal adsorption than the cells.

  5. Community Composition of Bacterial Biofilms Formed on Simple Soil Based Bioelectrochemical Cell Anodes and Cathodes

    Science.gov (United States)

    2012-04-01

    3 Table 2. Relative molar percentages and absolute abundances of prokaryotic and eukaryotic fatty acid (FA...density for the three soils varied from 5 to 8 × 108 cells per gram of soil. 3.1 Anode biofilms Although eukaryotic biomarkers were detected on the...percentages and absolute abundances of prokaryotic and eukaryotic fatty acid (FA) biomarkers detected in the soils, top middle and bottom fractions, on the

  6. A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence

    Science.gov (United States)

    Bertolo, Lisa; Monteiro, Mario A.; Agellon, Al; Viswanathan, V. K.; Vedantam, Gayatri

    2016-01-01

    Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence. PMID:27741317

  7. Rv3351c, a Mycobacterium tuberculosis gene that affects bacterial growth and alveolar epithelial cell viability.

    Science.gov (United States)

    Pavlicek, Rebecca L; Fine-Coulson, Kari; Gupta, Tuhina; Quinn, Frederick D; Posey, James E; Willby, Melisa; Castro-Garza, Jorge; Karls, Russell K

    2015-12-01

    Despite the interactions known to occur between various lower respiratory tract pathogens and alveolar epithelial cells (AECs), few reports examine factors influencing the interplay between Mycobacterium tuberculosis bacilli and AECs during infection. Importantly, in vitro studies have demonstrated that the M. tuberculosis hbha and esxA gene products HBHA and ESAT6 directly or indirectly influence AEC survival. In this report, we identify Rv3351c as another M. tuberculosis gene that impacts the fate of both the pathogen and AEC host. Intracellular replication of an Rv3351c mutant in the human AEC type II pneumocyte cell line A549 was markedly reduced relative to the complemented mutant and parent strain. Deletion of Rv3351c diminished the release of lactate dehydrogenase and decreased uptake of trypan blue vital stain by host cells infected with M. tuberculosis bacilli, suggesting attenuated cytotoxic effects. Interestingly, an isogenic hbha mutant displayed reductions in AEC killing similar to those observed for the Rv3351c mutant. This opens the possibility that multiple M. tuberculosis gene products interact with AECs. We also observed that Rv3351c aids intracellular replication and survival of M. tuberculosis in macrophages. This places Rv3351c in the same standing as HBHA and ESAT6, which are important factors in AECs and macrophages. Defining the mechanism(s) by which Rv3351c functions to aid pathogen survival within the host may lead to new drug or vaccine targets.

  8. Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins.

    Science.gov (United States)

    Schofield, Whitman B; Lim, Hoong Chuin; Jacobs-Wagner, Christine

    2010-09-15

    What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back-and-forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole-organizing protein PopZ, compensate for the defect in segregation regulation in ΔtipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self-organizing oscillator to create motion suitable for chromosome segregation.

  9. Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane.

    Science.gov (United States)

    Adams, David William; Wu, Ling Juan; Errington, Jeff

    2015-02-12

    To proliferate efficiently, cells must co-ordinate division with chromosome segregation. In Bacillus subtilis, the nucleoid occlusion protein Noc binds to specific DNA sequences (NBSs) scattered around the chromosome and helps to protect genomic integrity by coupling the initiation of division to the progression of chromosome replication and segregation. However, how it inhibits division has remained unclear. Here, we demonstrate that Noc associates with the cell membrane via an N-terminal amphipathic helix, which is necessary for function. Importantly, the membrane-binding affinity of this helix is weak and requires the assembly of nucleoprotein complexes, thus establishing a mechanism for DNA-dependent activation of Noc. Furthermore, division inhibition by Noc requires recruitment of NBS DNA to the cell membrane and is dependent on its ability to bind DNA and membrane simultaneously. Indeed, Noc production in a heterologous system is sufficient for recruitment of chromosomal DNA to the membrane. Our results suggest a simple model in which the formation of large membrane-associated nucleoprotein complexes physically occludes assembly of the division machinery.

  10. Bacterial pathogens induce abscess formation by CD4(+) T-cell activation via the CD28-B7-2 costimulatory pathway.

    Science.gov (United States)

    Tzianabos, A O; Chandraker, A; Kalka-Moll, W; Stingele, F; Dong, V M; Finberg, R W; Peach, R; Sayegh, M H

    2000-12-01

    Abscesses are a classic host response to infection by many pathogenic bacteria. The immunopathogenesis of this tissue response to infection has not been fully elucidated. Previous studies have suggested that T cells are involved in the pathologic process, but the role of these cells remains unclear. To delineate the mechanism by which T cells mediate abscess formation associated with intra-abdominal sepsis, the role of T-cell activation and the contribution of antigen-presenting cells via CD28-B7 costimulation were investigated. T cells activated in vitro by zwitterionic bacterial polysaccharides (Zps) known to induce abscess formation required CD28-B7 costimulation and, when adoptively transferred to the peritoneal cavity of naïve rats, promoted abscess formation. Blockade of T-cell activation via the CD28-B7 pathway in animals with CTLA4Ig prevented abscess formation following challenge with different bacterial pathogens, including Staphylococcus aureus, Bacteroides fragilis, and a combination of Enterococcus faecium and Bacteroides distasonis. In contrast, these animals had an increased abscess rate following in vivo T-cell activation via CD28 signaling. Abscess formation in vivo and T-cell activation in vitro required costimulation by B7-2 but not B7-1. These results demonstrate that abscess formation by pathogenic bacteria is under the control of a common effector mechanism that requires T-cell activation via the CD28-B7-2 pathway.

  11. Pleural fluid soluble triggering receptor expressed on myeloid cells-1 as a marker of bacterial infection: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Jiang Hong-Ni

    2011-10-01

    Full Text Available Abstract Background Pleural infection is a common clinical problem. Its successful treatment depends on rapid diagnosis and early initiation of antibiotics. The measurement of soluble triggering receptor expressed in myeloid cells-1 (sTREM-1 level in pleural effusions has proven to be a valuable diagnostic tool for differentiating bacterial effusions from effusions of other etiologies. Herein, we performed a meta-analysis to assess the accuracy of pleural fluid sTREM-1 in the diagnosis of bacterial infection. Methods We searched Web of Knowledge and Medline from 1990 through March 2011 for studies reporting diagnostic accuracy data regarding the use of sTREM-1 in the diagnosis of bacterial pleural effusions. Pooled sensitivity and specificity and summary measures of accuracy and Q* were calculated. Results Overall, the sensitivity of sTREM-1was 78% (95% CI: 72%-83%; the specificity was 84% (95% CI: 80%-87%; the positive likelihood ratio was 6.0 (95% CI: 3.3-10.7; and the negative likelihood ratio was 0.22 (95% CI: 0.12-0.40. The area under the SROC curve for sTREM-1 was 0.92. Statistical heterogeneity and inconsistency were found for sensitivity (p = 0.015, χ2 = 15.73, I2 = 61.9%, specificity (p = 0.000, χ2 = 29.90, I2 = 79.9%, positive likelihood ratio (p = 0.000, χ2 = 33.09, I2 = 81.9%, negative likelihood ratio (p = 0.008, χ2 = 17.25, I2 = 65.2%, and diagnostic odds ratio (p = 0.000, χ2 = 28.49, I2 = 78.9%. A meta-regression analysis performed showed that the Quality Assessment of Diagnostic Accuracy Studies score (p = 0.3245; RDOR, 4.34; 95% CI, 0.11 to 164.01, the Standards for Reporting of Diagnostic Accuracy score (p = 0.3331; RDOR, 1.70; 95% CI, 0.44 to 6.52, lack of blinding (p = 0.7439; RDOR, 0.60; 95% CI, 0.01 to 33.80, and whether the studies were prospective or retrospective studies (p = 0.2068; RDOR, 7.44; 95% CI, 0.18 to 301.17 did not affect the test accuracy. A funnel plot for publication bias suggested a remarkable trend

  12. Antibacterial compounds of Canadian honeys target bacterial cell wall inducing phenotype changes, growth inhibition and cell lysis that resemble action of β-lactam antibiotics.

    Directory of Open Access Journals (Sweden)

    Katrina Brudzynski

    Full Text Available Honeys show a desirable broad spectrum activity against Gram-positive and negative bacteria making antibacterial activity an intrinsic property of honey and a desirable source for new drug development. The cellular targets and underlying mechanism of action of honey antibacterial compounds remain largely unknown. To facilitate the target discovery, we employed a method of phenotypic profiling by directly comparing morphological changes in Escherichia coli induced by honeys to that of ampicillin, the cell wall-active β-lactam of known mechanism of action. Firstly, we demonstrated the purity of tested honeys from potential β-lactam contaminations using quantitative LC-ESI-MS. Exposure of log-phase E. coli to honey or ampicillin resulted in time- and concentration-dependent changes in bacterial cell shape with the appearance of filamentous phenotypes at sub-inhibitory concentrations and spheroplasts at the MBC. Cell wall destruction by both agents, clearly visible on microscopic micrographs, was accompanied by increased permeability of the lipopolysaccharide outer membrane as indicated by fluorescence-activated cell sorting (FACS. More than 90% E. coli exposed to honey or ampicillin became permeable to propidium iodide. Consistently with the FACS results, both honey-treated and ampicillin-treated E. coli cells released lipopolysaccharide endotoxins at comparable levels, which were significantly higher than controls (p<0.0001. E. coli cells transformed with the ampicillin-resistance gene (β-lactamase remained sensitive to honey, displayed the same level of cytotoxicity, cell shape changes and endotoxin release as ampicillin-sensitive cells. As expected, β-lactamase protected the host cell from antibacterial action of ampicillin. Thus, both honey and ampicillin induced similar structural changes to the cell wall and LPS and that this ability underlies antibacterial activities of both agents. Since the cell wall is critical for cell growth and

  13. Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.

    Directory of Open Access Journals (Sweden)

    Maura De Simone

    Full Text Available Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s may have a role in the reduction of cell-mediated immunity playing a critical role in

  14. Host genetic background influences the response to the opportunistic Pseudomonas aeruginosa infection altering cell-mediated immunity and bacterial replication.

    Science.gov (United States)

    De Simone, Maura; Spagnuolo, Lorenza; Lorè, Nicola Ivan; Rossi, Giacomo; Cigana, Cristina; De Fino, Ida; Iraqi, Fuad A; Bragonzi, Alessandra

    2014-01-01

    Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P

  15. Halomonas desiderata as a bacterial model to predict the possible biological nitrate reduction in concrete cells of nuclear waste disposals.

    Science.gov (United States)

    Alquier, Marjorie; Kassim, Caroline; Bertron, Alexandra; Sablayrolles, Caroline; Rafrafi, Yan; Albrecht, Achim; Erable, Benjamin

    2014-01-01

    After closure of a waste disposal cell in a repository for radioactive waste, resaturation is likely to cause the release of soluble species contained in cement and bituminous matrices, such as ionic species (nitrates, sulfates, calcium and alkaline ions, etc.), organic matter (mainly organic acids), or gases (from steel containers and reinforced concrete structures as well as from radiolysis within the waste packages). However, in the presence of nitrates in the near-field of waste, the waste cell can initiate oxidative conditions leading to enhanced mobility of redox-sensitive radionuclides (RN). In biotic conditions and in the presence of organic matter and/or hydrogen as electron donors, nitrates may be microbiologically reduced, allowing a return to reducing conditions that promote the safety of storage. Our work aims to analyze the possible microbial reactivity of nitrates at the bitumen - concrete interface in conditions as close as possible to radioactive waste storage conditions in order (i) to evaluate the nitrate reaction kinetics; (ii) to identify the by-products (NO2(-), NH4(+), N2, N2O, etc.); and (iii) to discriminate between the roles of planktonic bacteria and those adhering as a biofilm structure in the denitrifying activity. Leaching experiments on solid matrices (bitumen and cement pastes) were first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface, e.g. highly alkaline pH conditions (10 < pH < 11) imposed by the cement matrix. The screening of a range of anaerobic denitrifying bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these particular conditions of pH. The denitrifying activity of H. desiderata was quantified in a batch bioreactor in the presence of solid matrices and/or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement

  16. Comparison of availability of copper(II) complexes with organic ligands to bacterial cells and to chitin

    Energy Technology Data Exchange (ETDEWEB)

    Vasconcelos, M.T.S.D.; Azenha, M.A.O. [Laquipai, Porto (Portugal). Faculdade de Ciencias do Porto; Cabral, J.P.S. [Inst. de Botanica e Centro de Citologia Experimental U.P., Porto (Portugal)

    1997-10-01

    Bacterial cells or chitin were exposed to solutions with 100 {micro}M total but only 5 {micro}M free copper, due to the presence of a proper concentration of proline, lysine, cysteine, or ethylenediamine tetraacetate (EDTA). The influence of the nature and concentration of the particles and soluble ligands, on the sorption and on the desorption of the copper, at pH 6.50 and 25.0 C, was investigated. The metal sorbed by the particles and that left in the solution were measured by atomic absorption spectrometry, after different periods of contact between particles and solution. The interpretation of the results was based on the copper(II) speciation calculated through equilibrium approaches applied to homogeneous or heterogeneous systems. A significant fraction of copper bound to the organic ligands was displaced to the bacteria or chitin, and the extent of chemical reaction depended on the nature of both the soluble (or leaving) ligands and sites on the particle surface (or entering ligands), as expected by the equilibrium theory. But with chitin, the uptake of copper in the presence of cysteine or EDTA was higher than expected, which may be due to the adsorption of the soluble copper complexes on the particle surface. In consequence of a competition between soluble and particulate ligands (cells or chitin), the free copper(II) concentration decreased in the solution, even in the presence of very strong chelators. The results indicate that copper availability is not a simple function of the initial free copper concentration in the solution. Desorption of the previously fixed copper, originated by free soluble ligands indicated that the sorption of copper was quasireversible for both particles, though a larger dismissal of the equilibrium position occurred for the cells, probably due to their biological activity.

  17. Development and Use of Anucleate Bacterial Cells to Assay the in vitro Activity of Pollutants

    Science.gov (United States)

    1982-12-07

    1052-1064. 2. Reeve, J. N., R. A. Weigand, and J. W. Greenawalt. 1971. Presence of bacteriophace-like inhz.bitory particles in Escherichi a coli . J...H3; 1am193;LG3 infected E. coli cells DNase digestion. Bring to 0.3M NIH4Cl S Slow speed centrifugation High speed centrifugation + Pass supernatant...7 ’I -.&. - - ~ ,-..4 R IP-PAC;E: 3 S-1lethxonine Labullcd P. coli B3 Extract ,44r~~.a~~c2d 3a3b 3P. 3d 0 40- FIGURE 3 la = Uninfected E. coli

  18. Hierarchical micro/nanostructured titanium with balanced actions to bacterial and mammalian cells for dental implants

    Directory of Open Access Journals (Sweden)

    Zhu Y

    2015-10-01

    Full Text Available Yu Zhu,1,* Huiliang Cao,2,* Shichong Qiao,1,* Manle Wang,2,3 Yingxin Gu,1 Huiwen Luo,1 Fanhao Meng,2 Xuanyong Liu,2 Hongchang Lai1 1Department of Oral and Maxillofacial Implantology, Shanghai Key Laboratory of Stomatology, Shanghai Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University, 2State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 3School of Materials Engineering, Shanghai University of Engineering Science, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: A versatile strategy to endow dental implants with long-term antibacterial ability without compromising the cytocompatibility is highly desirable to combat implant-related infection. Silver nanoparticles (Ag NPs have been utilized as a highly effective and broad-spectrum antibacterial agent for surface modification of biomedical devices. However, the high mobility and subsequent hazardous effects of the particles on mammalian cells may limit its practical applications. Thus, Ag NPs were immobilized on the surface of sand-blasted, large grit, and acid-etched (SLA titanium by manipulating the atomic-scale heating effect of silver plasma immersion ion implantation. The silver plasma immersion ion implantation-treated SLA surface gave rise to both good antibacterial activity and excellent compatibility with mammalian cells. The antibacterial activity rendered by the immobilized Ag NPs was assessed using Fusobacterium nucleatum and Staphylococcus aureus, commonly suspected pathogens for peri-implant disease. The immobilized Ag NPs offered a good defense against multiple cycles of bacteria attack in both F. nucleatum and S. aureus, and the mechanism was independent of silver release. F. nucleatum showed a higher susceptibility to Ag NPs than S. aureus, which might be explained by the presence of different wall structures. Moreover, the

  19. Antigen 43-mediated autotransporter display, a versatile bacterial cell surface presentation system

    DEFF Research Database (Denmark)

    Kjærgaard, Kristian; Hasman, Henrik; Schembri, Mark;

    2002-01-01

    Antigen 43 (Ag43), a self-recognizing outer membrane protein of Escherichia coli, has been converted into an efficient and versatile tool for surface display of foreign protein segments. Ag43 is an autotransporter protein characterized by the feature that all information required for transport...... to the outer membrane and secretion through the cell envelope is contained within the protein itself. Ag43 consists of two subunits (alpha and beta), where the beta-subunit forms an integral outer membrane translocator to which the alpha-subunit is noncovalently attached. The simplicity of the Ag43 system...

  20. Experimental manipulation of sponge/bacterial symbiont community composition with antibiotics: sponge cell aggregates as a unique tool to study animal/microorganism symbiosis.

    Science.gov (United States)

    Richardson, Crystal; Hill, Malcolm; Marks, Carolyn; Runyen-Janecky, Laura; Hill, April

    2012-08-01

    Marine sponges can harbor dense and diverse bacterial communities, yet we have a limited understanding of important aspects of this symbiosis. We developed an experimental methodology that permits manipulating the composition of the microbial community. Specifically, we evaluated sponge cell aggregates (SCA) from Clathria prolifera that had been treated with different classes of antibiotics to determine whether this system might offer novel experimental approaches to the study of sponge/bacterial symbioses. Microscopic analysis of the SCA demonstrated that two distinct morphological types of microbiota existed on the external surface vs. the internal regions of the SCA. Denaturing gradient gel electrophoresis and sequence analysis of 16S rRNA gene clone libraries indicated that we were unable to create entirely aposymbiotic SCA but that different classes of antibiotics produced distinctive shifts in the SCA-associated bacterial community. After exposure to antibiotics, some bacterial species were 'revealed', thus uncovering novel components of the sponge-associated community. The antibiotic treatments used here had little discernible effect on the formation of SCA or subsequent development of the adult. The experimental approach we describe offers empirical options for studying the role symbionts play in sponge growth and development and for ascertaining relationships among bacterial species in communities residing in sponges.

  1. Evolutionary Cell Biology of Division Mode in the Bacterial Planctomycetes-Verrucomicrobia- Chlamydiae Superphylum

    Science.gov (United States)

    Rivas-Marín, Elena; Canosa, Inés; Devos, Damien P.

    2016-01-01

    Bacteria from the Planctomycetes, Verrucomicrobia, and Chlamydiae (PVC) superphylum are exceptions to the otherwise dominant mode of division by binary fission, which is based on the interaction between the FtsZ protein and the peptidoglycan (PG) biosynthesis machinery. Some PVC bacteria are deprived of the FtsZ protein and were also thought to lack PG. How these bacteria divide is still one of the major mysteries of microbiology. The presence of PG has recently been revealed in Planctomycetes and Chlamydiae, and proteins related to PG synthesis have been shown to be implicated in the division process in Chlamydiae, providing important insights into PVC mechanisms of division. Here, we review the historical lack of observation of PG in PVC bacteria, its recent detection in two phyla and its involvement in chlamydial cell division. Based on the detection of PG-related proteins in PVC proteomes, we consider the possible evolution of the diverse division mechanisms in these bacteria. We conclude by summarizing what is known and what remains to be understood about the evolutionary cell biology of PVC division modes. PMID:28018303

  2. Evolutionary cell biology of division mode in the bacterial Planctomycetes-Verrucomicrobia-Chlamydiae superphylum

    Directory of Open Access Journals (Sweden)

    Elena Rivas-Marín

    2016-12-01

    Full Text Available Bacteria from the Planctomycetes, Verrucomicrobia and Chlamydiae (PVC superphylum are exceptions to the otherwise dominant mode of division by binary fission, which is based on the interaction between the FtsZ protein and the peptidoglycan (PG biosynthesis machinery. Some PVC bacteria are deprived of the FtsZ protein and were also thought to lack PG. How these bacteria divide is still one of the major mysteries of microbiology. The presence of PG has recently been revealed in Planctomycetes and Chlamydiae, and proteins related to PG synthesis have been shown to be implicated in the division process in Chlamydiae, providing important insights into PVC mechanisms of division. Here, we review the historical lack of observation of PG in PVC bacteria, its recent detection in two phyla and its involvement in chlamydial cell division. Based on the detection of PG-related proteins in PVC proteomes, we consider the possible evolution of the diverse division mechanisms in these bacteria. We conclude by summarizing what is known and what remains to be understood about the evolutionary cell biology of PVC division modes.

  3. Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway.

    Science.gov (United States)

    Li, Xiu-Mei; Luo, Xue-Gang; Si, Chuan-Ling; Wang, Nan; Zhou, Hao; He, Jun-Fang; Zhang, Tong-Cun

    2015-01-01

    Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-β-(2″-acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-β-(2″-acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-β-(2″-acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs.

  4. Amperometric L-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Bo [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China); Zhang, Shu [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Lang, Qiaolin [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); Song, Jianxia; Han, Lihui [Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, Ocean University of China, 238 Songling Road, Qingdao 266100 (China); Liu, Aihua, E-mail: liuah@qibebt.ac.cn [Laboratory for Biosensing, Key Laboratory of Biofuels, and Shandong Provinicial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101 (China); University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049 (China)

    2015-07-16

    Highlights: • E. coli surface-dispalyed Gldh exhibiting excellent enzyme activity and stability. • Sensitive amperometric biosensor for glutamate using Gldh-bacteria and MWNTs. • The glutamate biosensor exhibited high specificity and stability. - Abstract: A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP{sup +}-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE). The MWNTs could not only significantly lower the oxidation overpotential towards NAPDH, which was the product of NADP{sup +} involving in the oxidation of glutamate by Gldh, but also enhanced the current response. Under the optimized experimental conditions, the current–time curve of the Nafion/Gldh-bacteria/PEI-MWNTs/GCE was performed at +0.52 V (vs. SCE) by amperometry varying glutamate concentration. The current response was linear with glutamate concentration in two ranges (10 μM–1 mM and 2–10 mM). The low limit of detection was estimated to be 2 μM glutamate (S/N = 3). Moreover, the proposed biosensor is stable, specific, reproducible and simple, which can be applied to real samples detection.

  5. Visualization and quantification of archaeal and bacterial metabolically active cells in soil using fluorescence in situ hybridization method

    Science.gov (United States)

    Semenov, Mikhail; Manucharova, Natalia; Stepanov, Alexey

    2015-04-01

    The method of in situ hybridization using fluorescent labeled 16S rRNA-targeted oligonucleotide probes (FISH - fluorescence in situ hybridization) combines identification and quantification of groups of microorganisms at different phylogenetic levels, from domain to species. The FISH method enables to study the soil microbial community in situ, avoiding plating on nutrient media, and allows to identify and quantify living, metabolically active cells of Bacteria and Archaea. The full procedure consists of the following steps: desorption of the cells from the soil particles, fixation of cells, coating a fixed sample on the glass slide, hybridization with the specific probes and, finally, microscopic observation and cell counting. For the FISH analysis of Bacteria and Archaea, the paraformaldehyde-fixed samples were hybridized with Cy3-labeled Archaea-specific probe(Arc915) and 6-carboxyfluorescein (FAM)-labeled Bacteria-specific probe(EUB338). When a molecular probe is incorporated into a cell, it can hybridize solely with a complementary rRNA sequence. The hybridization can be visualized under the fluorescent microscope and counted. The application of FISH will be demonstrated by the abundance of metabolically active cells of Archaea and Bacteria depending on soil properties, depth and land use. The research was carried out at field and natural ecosystems of European part of Russia. Samples were collected within the soil profiles (3-6 horizons) of Chernozem and Kastanozem with distinct land use. Quantification of metabolically active cells in virgin and arable Chernozem revealed that the abundance of Archaea in topsoil of virgin Chernozem was doubled as compared with arable soil, but it leveled off in the deeper horizons. Plowing of Chernozem decreased an amount of archaeal and bacterial active cells simultaneously, however, Bacteria were more resistant to agrogenic impact than Archaea. In Kastanozem, a significant change in the abundance of metabolically active

  6. The detection of genomic DNA from bacterial cells using iron oxide nanoparticles synthesized by a hydrothermal process

    Science.gov (United States)

    Kim, Young-Sung; Kim, Ki-Chul; Hong, Tae-Whan

    2010-04-01

    We used iron oxide nanoparticles in order to extract purified DNA from bacterial cells. Magnetite (Fe3O4) and maghemite (γ-Fe2O3) are synthesized with FeSO4·7H2O via a hydrothermal process and used as a medium to detect DNA. Various characterizations were performed including X-ray powder diffraction (XRD), high resolution transmission electron microscopy (HRTEM), field emission scanning electron microscopy, vibrating sample magnetometry, and Mössbauer spectroscopy. According to the XRD results, the XRD peaks of the synthesized magnetite and maghemite nanoparticles corresponded well with JCPDS standard data, respectively. The particle size of the iron oxide nanoparticles was about 20 nm, and the particle shape was almost spherical, which was confirmed by observation of the HRTEM image. The magnetite nanoparticles have a face-centeredcubic inverse spinel structure with a space group Fd bar 3 m, as confirmed by HRTEM and Mössbauer spectroscopy analyses. An agarose gel eletrophoresis analysis was performed to confirm the extraction ability of DNA using these iron oxide nanoparticles, revealing stronger reaction of the maghemite nanoparticles than the magnetite nanoparticles.

  7. The Use of MALDI-TOF-MS and In Silico Studies for Determination of Antimicrobial Peptides' Affinity to Bacterial Cells

    Science.gov (United States)

    Mandal, Santi M.; Migliolo, Ludovico; Franco, Octavio L.

    2012-11-01

    Several methods have been proposed for determining the binding affinity of antimicrobial peptides (AMPs) to bacterial cells. Here the utilization of MALDI-TOF-MS was proposed as a reliable and efficient method for high throughput AMP screening. The major advantage of the technique consists of finding AMPs that are selective and specific to a wide range of Gram-negative and -positive bacteria, providing a simple reliable screening tool to determine the potential candidates for broad spectrum antimicrobial drugs. As a prototype, amp-1 and -2 were used, showing highest activity toward Gram-negative and -positive membranes respectively. In addition, in silico molecular docking studies with both peptides were carried out for the membranes. In silico results indicated that both peptides presented affinity for DPPG and DPPE phospholipids, constructed in order to emulate an in vivo membrane bilayer. As a result, amp-1 showed a higher complementary surface for Gram-negative while amp-2 showed higher affinity to Gram-positive membranes, corroborating MS analyses. In summary, results here obtained suggested that in vitro methodology using MALDI-TOF-MS in addition to theoretical studies may be able to improve AMP screening quality.

  8. Characterization of the COD removal, electricity generation, and bacterial communities in microbial fuel cells treating molasses wastewater.

    Science.gov (United States)

    Lee, Yun-Yeong; Kim, Tae G; Cho, Kyung-Suk

    2016-11-09

    The chemical oxygen demand (COD) removal, electricity generation, and microbial communities were compared in 3 types of microbial fuel cells (MFCs) treating molasses wastewater. Single-chamber MFCs without and with a proton exchange membrane (PEM), and double-chamber MFC were constructed. A total of 10,000 mg L(-1) COD of molasses wastewater was continuously fed. The COD removal, electricity generation, and microbial communities in the two types of single-chamber MFCs were similar, indicating that the PEM did not enhance the reactor performance. The COD removal in the single-chamber MFCs (89-90%) was higher than that in the double-chamber MFC (50%). However, electricity generation in the double-chamber MFC was higher than that in the single-chamber MFCs. The current density (80 mA m(-2)) and power density (17 mW m(-2)) in the double-chamber MFC were 1.4- and 2.2-times higher than those in the single-chamber MFCs, respectively. The bacterial community structures in single- and double-chamber MFCs were also distinguishable. The amount of Proteobacteria in the double-chamber MFC was 2-3 times higher than those in the single-chamber MFCs. For the archaeal community, Methanothrix (96.4%) was remarkably dominant in the single-chamber MFCs, but Methanobacterium (35.1%), Methanosarcina (28.3%), and Methanothrix (16.2%) were abundant in the double-chamber MFC.

  9. Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

    Indian Academy of Sciences (India)

    Arnab Roy; Umesh Varshney; Debnath Pal

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.

  10. HLH-30/TFEB-mediated autophagy functions in a cell-autonomous manner for epithelium intrinsic cellular defense against bacterial pore-forming toxin in C. elegans.

    Science.gov (United States)

    Chen, Huan-Da; Kao, Cheng-Yuan; Liu, Bang-Yu; Huang, Shin-Whei; Kuo, Cheng-Ju; Ruan, Jhen-Wei; Lin, Yen-Hung; Huang, Cheng-Rung; Chen, Yu-Hung; Wang, Horng-Dar; Aroian, Raffi V; Chen, Chang-Shi

    2017-02-01

    Autophagy is an evolutionarily conserved intracellular system that maintains cellular homeostasis by degrading and recycling damaged cellular components. The transcription factor HLH-30/TFEB-mediated autophagy has been reported to regulate tolerance to bacterial infection, but less is known about the bona fide bacterial effector that activates HLH-30 and autophagy. Here, we reveal that bacterial membrane pore-forming toxin (PFT) induces autophagy in an HLH-30-dependent manner in Caenorhabditis elegans. Moreover, autophagy controls the susceptibility of animals to PFT toxicity through xenophagic degradation of PFT and repair of membrane-pore cell-autonomously in the PFT-targeted intestinal cells in C. elegans. These results demonstrate that autophagic pathways and autophagy are induced partly at the transcriptional level through HLH-30 activation and are required to protect metazoan upon PFT intoxication. Together, our data show a new and powerful connection between HLH-30-mediated autophagy and epithelium intrinsic cellular defense against the single most common mode of bacterial attack in vivo.

  11. Protein L. A bacterial Ig-binding protein that activates human basophils and mast cells.

    Science.gov (United States)

    Patella, V; Casolaro, V; Björck, L; Marone, G

    1990-11-01

    Peptostreptococcus magnus strain 312 (10(6) to 10(8)/ml), which synthesizes a protein capable of binding to kappa L chains of human Ig (protein L), stimulated the release of histamine from human basophils in vitro. P. magnus strain 644, which does not synthesize protein L, did not induce histamine secretion. Soluble protein L (3 x 10(-2) to 3 micrograms/ml) induced histamine release from human basophils. The characteristics of the release reaction were similar to those of rabbit IgG anti-Fc fragment of human IgE (anti-IgE): it was Ca2(+)- and temperature-dependent, optimal release occurring at 37 degrees C in the presence of 1.0 mM extracellular Ca2+. There was an excellent correlation (r = 0.82; p less than 0.001) between the maximal percent histamine release induced by protein L and that induced by anti-IgE, as well as between protein L and protein A from Staphylococcus aureus (r = 0.52; p less than 0.01). Preincubation of basophils with either protein L or anti-IgE resulted in complete cross-desensitization to a subsequent challenge with the heterologous stimulus. IgE purified from myeloma patients PS and PP (lambda-chains) blocked anti-IgE-induced histamine release but failed to block the histamine releasing activity of protein L. In contrast, IgE purified from myeloma patient ADZ (kappa-chains) blocked both anti-IgE- and protein L-induced releases, whereas human polyclonal IgG selectively blocked protein L-induced secretion. Protein L acted as a complete secretagogue, i.e., it activated basophils to release sulfidopeptide leukotriene C4 as well as histamine. Protein L (10(-1) to 3 micrograms/ml) also induced the release of preformed (histamine) and de novo synthesized mediators (leukotriene C4 and/or PGD2) from mast cells isolated from lung parenchyma and skin tissues. Intradermal injections of protein L (0.01 to 10 micrograms/ml) in nonallergic subjects caused a dose-dependent wheal-and-flare reaction. Protein L activates human basophils and mast cells in

  12. Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells1

    Science.gov (United States)

    Shpigel, Etai; Roiz, Levava; Goren, Raphael; Shoseyov, Oded

    1998-01-01

    Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control. PMID:9701575

  13. A novel approach to recycle bacterial culture waste for fermentation reuse via a microbial fuel cell-membrane bioreactor system.

    Science.gov (United States)

    Li, Jian; Zhu, Yuan; Zhuang, Liangpeng; Otsuka, Yuichiro; Nakamura, Masaya; Goodell, Barry; Sonoki, Tomonori; He, Zhen

    2015-09-01

    Biochemical production processes require water and nutrient resources for culture media preparation, but aqueous waste is generated after the target products are extracted. In this study, culture waste (including cells) produced from a lab-scale fermenter was fed into a microbial fuel cell-membrane bioreactor (MFC-MBR) system. Electrical energy was generated via the interaction between the microbial consortia and the solid electrode in the MFC. The treated wastewater was reclaimed in this process which was reused as a solvent and a nutrient source in subsequent fermentation. Polarization testing showed that the MFC produced a maximum current density of 37.53 A m(-3) with a maximum power density of 5.49 W m(-3). The MFC was able to generate 0.04 kWh of energy per cubic meter of culture waste treated. The lab-scale fermenters containing pure cultures of an engineered Pseudomonas spp. were used to generate 2-pyrone-4,6-dicarboxylic acid (PDC), a high value platform chemical. When the MFC-MBR-treated wastewater was used for the fermenter culture medium, a specific bacterial growth rate of 1.00 ± 0.05 h(-1) was obtained with a PDC production rate of 708.11 ± 64.70 mg PDC L(-1) h(-1). Comparable values for controls using pure water were 0.95 ± 0.06 h(-1) and 621.01 ± 22.09 mg PDC L(-1) h(-1) (P > 0.05), respectively. The results provide insight on a new approach for more sustainable bio-material production while at the same time generating energy, and suggest that the treated wastewater can be used as a solvent and a nutrient source for the fermentation production of high value platform chemicals.

  14. Bacterial surface adaptation

    Science.gov (United States)

    Utada, Andrew

    2014-03-01

    Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

  15. Link between Domoic Acid Production and Cell Physiology after Exchange of Bacterial Communities between Toxic Pseudo-nitzschia multiseries and Non-Toxic Pseudo-nitzschia delicatissima

    Directory of Open Access Journals (Sweden)

    Aurélie Lelong

    2014-06-01

    Full Text Available Bacteria are known to influence domoic acid (DA production by Pseudo-nitzschia spp., but the link between DA production and physiology of diatoms requires more investigation. We compared a toxic P. multiseries to a non-toxic P. delicatissima, investigating links between DA production, physiological parameters, and co-occurring bacteria. Bacterial communities in cultures of both species were reduced by antibiotic treatment, and each of the diatoms was inoculated with the bacterial community of the other species. The physiology of P. delicatissima was minimally affected by the absence of bacteria or the presence of alien bacteria, and no DA was detected. P. multiseries grew faster without bacteria, did not produce a significant amount of DA, and exhibited physiological characteristics of healthy cells. When grown with alien bacteria, P. multiseries did not grow and produced more DA; the physiology of these cells was affected, with decreases in chlorophyll content and photosynthetic efficiency, an increase in esterase activity, and almost 50% mortality of the cells. The alien bacterial community had morphological and cellular characteristics very different from the original bacteria, and the number of free-living bacteria per algal cell was much higher, suggesting the involvement of bacteria in DA production.

  16. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.; BEWLEY,M.C.

    2001-12-03

    /or misfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins.

  17. PROTEIN QUALITY CONTROL IN BACTERIAL CELLS: INTEGRATED NETWORKS OF CHAPERONES AND ATP-DEPENDENT PROTEASES.

    Energy Technology Data Exchange (ETDEWEB)

    FLANAGAN,J.M.BEWLEY,M.C.

    2002-10-01

    aggregation and/or mislfolding. Thus it is not surprising that, in cells, the protein folding process is error prone and organisms have evolved ''editing'' or quality control (QC) systems to assist in the folding, maintenance and, when necessary, selective removal of damaged proteins. In fact, there is growing evidence that failure of these QC-systems contributes to a number of disease states (5-8). This chapter describes our current understanding of the nature and mechanisms of the protein quality control systems in the cytosol of bacteria. Parallel systems are exploited in the cytosol and mitochondria of eukaryotes to prevent the accumulation of misfolded proteins.

  18. Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes

    DEFF Research Database (Denmark)

    Li, Yiping; Ingmer, Hanne; Madsen, Mogens;

    2008-01-01

    Background Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression....... jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria......-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs. Conclusion We show that under...

  19. Microbubble-mediated ultrasound promotes accumulation of bone marrow mesenchymal stem cell to the prostate for treating chronic bacterial prostatitis in rats

    OpenAIRE

    2016-01-01

    Chronic bacterial prostatitis (CBP) is an intractable disease. Although bone marrow mesenchymal stem cells (BMMSCs) are able to regulate inflammation in CBP, the effect of microbubble-mediated ultrasound- induced accumulation of BMMSCs on CBP remains unclear. To address this gap, a model of CBP was established in SD rats, which were then treated with BMMSCs alone (BMMSC group), BMMSCs with ultrasound (ultrasound group), BMMSCs with microbubble-mediated ultrasound (MMUS group) and compared wit...

  20. Antimicrobials for bacterial bioterrorism agents.

    Science.gov (United States)

    Sarkar-Tyson, Mitali; Atkins, Helen S

    2011-06-01

    The limitations of current antimicrobials for highly virulent pathogens considered as potential bioterrorism agents drives the requirement for new antimicrobials that are suitable for use in populations in the event of a deliberate release. Strategies targeting bacterial virulence offer the potential for new countermeasures to combat bacterial bioterrorism agents, including those active against a broad spectrum of pathogens. Although early in the development of antivirulence approaches, inhibitors of bacterial type III secretion systems and cell division mechanisms show promise for the future.

  1. tmRNA-mediated trans-translation as the major ribosome rescue system in a bacterial cell

    Directory of Open Access Journals (Sweden)

    Hyouta eHimeno

    2014-04-01

    Full Text Available tmRNA (transfer messenger RNA; also known as 10Sa RNA or SsrA RNA is a small RNA molecule that is conserved among bacteria. It has structural and functional similarities to tRNA: it has an upper half of the tRNA-like structure, its 5’ end is processed by RNase P, it has typical tRNA-specific base modifications, it is aminoacylated with alanine, it binds to EF-Tu after aminoacylation and it enters the ribosome with EF-Tu and GTP. However, tmRNA lacks an anticodon, and instead it has a coding sequence for a short peptide called tag-peptide. An elaborate interplay of actions of tmRNA as both tRNA and mRNA with the help of a tmRNA-binding protein, SmpB, facilitates trans-translation, which produces a single polypeptide from two mRNA molecules. Initially alanyl-tmRNA in complex with EF-Tu and SmpB enters the vacant A-site of the stalled ribosome like aminoacyl-tRNA but without a codon-anticodon interaction, and subsequently truncated mRNA is replaced with the tag-encoding region of tmRNA. During these processes, not only tmRNA but also SmpB structurally and functionally mimics both tRNA and mRNA. Thus trans-translation rescues the stalled ribosome, thereby allowing recycling of the ribosome. Since the tag-peptide serves as a target of AAA+ proteases, the trans-translation products are preferentially degraded so that they do not accumulate in the cell. Although alternative rescue systems have recently been revealed, trans-translation is the only system that universally exists in bacteria. Furthermore, it is unique in that it employs a small RNA and that it prevents accumulation of nonfunctional proteins from truncated mRNA in the cell. It might play the major role in rescuing the stalled translation in the bacterial cell.

  2. Effects of structure on the interactions between five natural antimicrobial compounds and phospholipids of bacterial cell membrane on model monolayers

    Science.gov (United States)

    Monolayers composed of bacterial phospholipids were used as model membranes to study interactions of naturally occurring phenolic compounds 2,5-dihydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde and the plant essential oil compounds carvacrol, cinnamaldehyde, and geraniol, previously found to be...

  3. Bacterial gastroenteritis

    Science.gov (United States)

    Bacterial gastroenteritis is present when bacteria cause an infection of the stomach and intestines ... has not been treated Many different types of bacteria can cause ... Campylobacter jejuni E coli Salmonella Shigella Staphylococcus ...

  4. Bacterial wall products induce downregulation of vascular endothelial growth factor receptors on endothelial cells via a CD14-dependent mechanism: implications for surgical wound healing.

    LENUS (Irish Health Repository)

    Power, C

    2012-02-03

    INTRODUCTION: Vascular endothelial growth factor (VEGF) is a potent mitogenic cytokine which has been identified as the principal polypeptide growth factor influencing endothelial cell (EC) migration and proliferation. Ordered progression of these two processes is an absolute prerequisite for initiating and maintaining the proliferative phase of wound healing. The response of ECs to circulating VEGF is determined by, and directly proportional to, the functional expression of VEGF receptors (KDR\\/Flt-1) on the EC surface membrane. Systemic sepsis and wound contamination due to bacterial infection are associated with significant retardation of the proliferative phase of wound repair. The effects of the Gram-negative bacterial wall components lipopolysaccharide (LPS) and bacterial lipoprotein (BLP) on VEGF receptor function and expression are unknown and may represent an important biological mechanism predisposing to delayed wound healing in the presence of localized or systemic sepsis. MATERIALS AND METHODS: We designed a series of in vitro experiments investigating this phenomenon and its potential implications for infective wound repair. VEGF receptor density on ECs in the presence of LPS and BLP was assessed using flow cytometry. These parameters