High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

Science.gov (United States)

Sakai, Y; Goh, T K; Tani, Y

1993-06-01

We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.

Isolation of a conjugative F-like plasmid from a multidrug-resistant Escherichia coli strain CM6 using tandem shock wave-mediated transformation.

Science.gov (United States)

Soto-Alonso, G; Cruz-Medina, J A; Caballero-Pérez, J; Arvizu-Hernández, I; Ávalos-Esparza, L M; Cruz-Hernández, A; Romero-Gómez, S; Rodríguez, A L; Pastrana-Martínez, X; Fernández, F; Loske, A M; Campos-Guillén, J

2015-07-01

Genetic characterization of plasmids from bacterial strains provides insight about multidrug resistance. Ten wild type Escherichia coli (E. coli) strains isolated from cow fecal samples were characterized by their antibiotic resistance profile, plasmid patterns and three different identification methods. From one of the strains, a fertility factor-like plasmid was replicated using tandem shock wave-mediated transformation. Underwater shock waves with a positive pressure peak of up to approximately 40 MPa, followed by a pressure trough of approximately -19 MPa were generated using an experimental piezoelectric shock wave source. Three different shock wave energies and a fixed delay of 750 μs were used to study the relationship between energy and transformation efficiency (TE), as well as the influence of shock wave energy on the integrity of the plasmid. Our results showed that the mean shock wave-mediated TE and the integrity of the large plasmid (~70 kb) were reduced significantly at the energy levels tested. The sequencing analysis of the plasmid revealed a high identity to the pHK17a plasmid, including the replication system, which was similar to the plasmid incompatibility group FII. It also showed that it carried an extended spectrum beta-lactamase gene, ctx-m-14. Furthermore, diverse genes for the conjugative mechanism were identified. Our results may be helpful in improving methodologies for conjugative plasmid transfer and directly selecting the most interesting plasmids from environmental samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Deletion mutants of region E1 a of AD12 E1 plasmids: Effect on oncogenic transformation

NARCIS (Netherlands)

Bos, J.L.; Jochemsen, A.G.; Bernards, R.A.; Schrier, P.I.; Ormondt, H. van; Eb, A.J. van der

1983-01-01

Plasmids containing the El region of Ad12 DNA can transform certain rodent cells into oncogenic cells. To study the role of the Ela subregion in the process of oncogenic transformation, Ad12 region El mutants carrying deletions in the Ela region were constructed. Deletion mutants pR7 and pR8 affect

Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing.

Science.gov (United States)

Naito, Y; Naito, T; Kobayashi, I

1998-01-01

Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

Transformation frequency of γ irradiated plasmid DNA and the enzymatic double strand break formation by incubation in a protein extract of Escherichia coli

International Nuclear Information System (INIS)

Schulte-Frohlinde, D.; Mark, F.; Ventur, Y.

1994-01-01

It was found that incubation of γ-irradiated or DNaseI-treated plasmid DNA in a protein extract of Escherichia coli leads to enzyme-induced formation of double strand breaks (dsb) in competition with repair of precursors of these dsb. A survival curve of the plasmid DNA (as determined by transformation of E. coli) was calculated on the basis of enzyme-induced dsb as well as those produced by irradiation assuming that they are lethal. The calculated D O value was the same as that measured directly by transformation of irradiated plasmid DNA. Two models are presented that fit the experimental survival data as a function of dose. One is based on damage formation in the plasmid DNA including enzymatic conversion of single strand damage into dsb (U-model), the other is an enzymatic repair saturation model based on Michaelis-Menten kinetics. (Author)

Behavior of IncQ Plasmids in Agrobacterium tumefaciens

NARCIS (Netherlands)

Hille, Jacques; Schilperoort, Rob

1981-01-01

Inc-Q plasmids were introduced into Agrobacterium tumefuciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results

Identification of selected microorganisms from activated sludge capable of benzothiazole and benzotriazole transformation.

Science.gov (United States)

Kowalska, Katarzyna; Felis, Ewa

2015-01-01

Benzothiazole (BT) and benzotriazole (BTA) are present in the environment - especially in urban and industrial areas, usually as anthropogenic micropollutants. BT and BTA have been found in the municipal and industrial wastewater, rivers, soil, groundwater, sediments and sludge. The origins of those substances' presence in the environment are various industry branches (food, chemical, metallurgical, electrical), households and surface runoff from industrial areas. Increasingly strict regulations on water quality and the fact that the discussed compounds are poorly biodegradable, make them a serious problem in the environment. Considering this, it is important to look for environmentally friendly and socially acceptable ways to remove BT and BTA. The aim of this study was to identify microorganisms capable of BT and BTA transformation or/and degradation in aquatic environment. Selected microorganisms were isolated from activated sludge. The identification of microorganisms capable of BT and BTA removal was possible using molecular biology techniques (PCR, DNA sequencing). Among isolated microorganisms of activated sludge are bacteria potentially capable of BT and BTA biotransformation and/or removal. The most common bacteria capable of BT and BTA transformation were Rhodococcus sp., Enterobacter sp., Arthrobacter sp. They can grow in a medium with BT and BTA as the only carbon source. Microorganisms previously adapted to the presence of the studied substances at a concentration of 10 mg/l, showed a greater rate of growth of colonies on media than microorganisms unconditioned to the presence of such compounds. Results of the biodegradation test suggest that BT was degraded to a greater extent than BTA, 98-100% and 11-19%, respectively.

Liposome-enhanced transformation of Streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of Streptococcus lactis and Bacillus subtilis

NARCIS (Netherlands)

Vossen, Jos M.B.M. van der; Kok, Jan; Lelie, Daniel van der; Venema, Gerhardus

An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis. Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Emr)] with an efficiency of 3 × 10^5

Plasmid profiling of bacterial isolates from confined environments

Science.gov (United States)

van Houdt, Rob; Provoost, Ann; Coninx, Ilse; Leys, Natalie; Mergeay, Max

Plasmid profiling of bacterial isolates from confined environments R. Van Houdt, I. Coninx, A. Provoost, N. Leys, and M. Mergeay Expertise group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK•CEN), Boeretang 200, B-2400 Mol, Belgium. Human exploration of extreme and isolated hostile environments such as space requires special confined small volume habitats to protect and house the crew. However, human confinement in such small volume habitats has restrictions on waste disposal and personal hygiene and inevitably generates a particular community of microorganisms within the habitat. These microorganisms are mainly originating from the crew (skin, mucous membranes, upper respiratory tract, mouth, and gastrointestinal tract) but also include the residing environmental microorganisms. Earth-based confined habitats such as the Antarctic Research Station Concordia are used as test beds for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. The dynamics of the environmental microbial population in such a test bed could render additional insights in assessing the potential health risks in long-duration space missions. Not only total bacterial contamination levels are important, but it is essential to identify also the predominant microbial taxa and their mobile genetic elements (MGE). These MGEs could be exchanged between bacteria by horizontal gene transfer and may alter the pathogenic potential since they often carry antibiotic resistance or more in general adaptation-enhancing traits. In this study several bacterial strains isolated in the Concordia research station were examined for their plasmid content. An optimized protocol for extraction of large plasmids showed the present of at least one plasmid in 50% of the strains. For all strains the minimal inhibitory concentration of a range of antibiotics was determined indicating resistance to

CARTOGRAPHIE DU PLASMIDE pSU100, PLASMIDE CRYPTIQUE DE LACTOBACILLUS CASEI

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F BENSALAH

2003-06-01

Ce plasmide appelé pSU100 a été cloné dans le vecteur de transformation pUC18 au site EcoRI chez E. coli JM103. Les profils électrophorétiques de restriction obtenus par des digestions simples, doubles et triples sous l’action de 33 endonucléases, ont contribué à l’élaboration d’une carte de restriction de ce plasmide. Cinq sites uniques ont été identifiés, ainsi que d’autres sites doubles et multiples. Une étude préliminaire du rôle physiologique de ce plasmide a permis de déceler une résistance à la kanamycine.

Rapid detection of foodborne microorganisms on food surface using Fourier transform Raman spectroscopy

Science.gov (United States)

Yang, Hong; Irudayaraj, Joseph

2003-02-01

Fourier transform (FT) Raman spectroscopy was used for non-destructive characterization and differentiation of six different microorganisms including the pathogen Escherichia coli O157:H7 on whole apples. Mahalanobis distance metric was used to evaluate and quantify the statistical differences between the spectra of six different microorganisms. The same procedure was extended to discriminate six different strains of E. coli. The FT-Raman procedure was not only successful in discriminating the different E. coli strain but also accurately differentiated the pathogen from non-pathogens. Results demonstrate that FT-Raman spectroscopy can be an excellent tool for rapid examination of food surfaces for microorganism contamination and for the classification of microbial cultures.

Construction of Biologically Functional Bacterial Plasmids In Vitro

Science.gov (United States)

Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Helling, Robert B.

1973-01-01

The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins. Images PMID:4594039

Radioautographic test for genetic cotton transformation by pCaVItoxneo hybrid plasmid

International Nuclear Information System (INIS)

Imamkhodjaeva, A.S.

2006-01-01

Full text: Search for novel technologies in biology, application of up-to-date methods in gene engineering, manipulation with the recombinant DNA, in particular, open opportunities for experiments with plants. To identify some DNA fragments in an organism's genome, radioautographic methods, such as dot- and blot-hybridization are frequently used. As a rule, genomic DNA is first isolated from the plant's organ. Its purification and subsequent manipulation is followed by hybridization with a probe labeled with radioactive components. The purified DNA, cDNA of RNA reverse transcription or a DNA fragment cloned in E-coli could serve as the probe. Radioautography shows homologically hybridized fragments. We have performed express dot-hybridization analysis on hybrid plasmid transformation of G.Hirsutum L. (108F) and G. Barbadense L. (C-6037) cotton sorts. pCaVItoxneo plasmid obtained on the basis of independently replicated plasmid-like DNA of the G.Hirsutum L. (pGHm2) cotton mitochondria was used (Yusupov T., 1994). There are hybrid two-domain gene of insectotoxin and enzymatically active kanamycine - phosphotransferase in the plasmid. The whole content is controlled by the plant promoter of cauliflower mosaic virus (19 S SFMV). The plasmid in question was added to the pollen sprouting medium followed by the transfer of the suspension on the pistil stigmas of the pre-prepared cotton flowers. The seed budding as the result of the experiment were analyzed by means of dot-hybridization method. DNA probes used for radioactive hybridization were labeled by method of Fainberg and Vagelstein (1990). To perform that DNA was dissolved in Tris-EDTA (10:1), containing 10mM of Tris HCl and 1mM EDTA, denaturated at 100 d eg C for 2 minutes with subsequent addition of oligonucleotide primers and annealing. DNA synthesis in the presence of 32 P labeled dATP and dCTP (Tashkent) was performed in the reaction mixture of potassium-phosphate buffer containing 67mM of MgCl 2 , 1 mg/ml of

Conjugal plasmid transfer (pAM beta 1) in Lactobacillus plantarum.

OpenAIRE

Shrago, A W; Chassy, B M; Dobrogosz, W J

1986-01-01

The streptococcal plasmid pAM beta 1 (erythromycin resistance) was transferred via conjugation from Streptococcus faecalis to Lactobacillus plantarum and was transferred among L. plantarum strains. Streptococcus sanguis Challis was transformed with pAM beta 1 isolated from these transconjugants, and transformants harboring intact pAM beta 1 could conjugate the plasmid back to L. plantarum.

Plasmid-Mediated Resistance in Enterobacteriaceae Changing Landscape and Implications for Therapy

NARCIS (Netherlands)

Schultsz, Constance; Geerlings, Suzanne

2012-01-01

Antimicrobial resistance is increasing worldwide, and pathogenic microorganism's that are resistant to all available antimicrobial agents are increasingly reported. Emerging plasmid-encoded extended-spectrum beta-lactamases (ESBLs) and carbapenemases are increasingly reported worldwide.

INFLUENCE OF TECHNOGENIC LANDSCAPES RECULTIVATION ON FUNCTIONING OF SOIL MICROORGANISMS COMMUNITIES WHICH TAKE PART IN TRANSFORMATION OF NITROGEN COMPOUNDS

Directory of Open Access Journals (Sweden)

O. V. Syshchykova

2014-04-01

Full Text Available It is established that mining recultivation of tailings dams slimes promotes restoration of numerical structure of soil microorganisms community which take part in processes of nitrogen compounds transformation. The certificate of that is number restoration of the organotrophic bacteria of a nitrogen cycle to 0.3 million CFU/g of soil and increase by 2-3 times of streptomycetes quantity in blankets. The received results of quantitative structure of the microorganisms which are taking part in processes of nitrogen mineral compounds transformation in the chernozem usual allow to claim that in blankets the number of microorganisms makes 3.89 and 2.33 million CFU/g soil. It should be noted that the best conditions for microflora development are formed on slime with drawing 50 cm of loess-like loam and 30 cm of a fertile layer. The microorganism quantity on the specified monitoring area increases by 3-4 times in the soil of a fertile layer and by 1.3-1.6 times in loess-like loam in comparison with slime without recultivation. Increase of microbiological processes intensity, extremely important, considering strengthening of ecosystems self-regulation functions. It is established high level of microbiological transformation of organic substance, the indicator is made 7.3-11.1 in the edatopes of the recultivated slimes. Increasing indicators of microbiological transformation and mineralization of organic compounds in the technozems confirm restoration of a slimes biogenity at carrying out of recultivation that promotes an intensification of mineralization processes and assimilation by plants nitrogen compounds in the soil. Keywords: microorganisms, nitrogen compounds, technozems, mining recultivation.

Chlamydial plasmids and bacteriophages.

Science.gov (United States)

Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

2015-01-01

Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

Generation of a restriction minus enteropathogenic Escherichia coli E2348/69 strain that is efficiently transformed with large, low copy plasmids

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Ward Jordan D

2008-08-01

Full Text Available Abstract Background Many microbes possess restriction-modification systems that protect them from parasitic DNA molecules. Unfortunately, the presence of a restriction-modification system in a given microbe also hampers genetic analysis. Although plasmids can be successfully conjugated into the enteropathogenic Escherichia coli strain E2348/69 and optimized protocols for competent cell preparation have been developed, we found that a large, low copy (~15 bioluminescent reporter plasmid, pJW15, that we modified for use in EPEC, was exceedingly difficult to transform into E2348/69. We reasoned that a restriction-modification system could be responsible for the low transformation efficiency of E2348/69 and sought to identify and inactivate the responsible gene(s, with the goal of creating an easily transformable strain of EPEC that could complement existing protocols for genetic manipulation of this important pathogen. Results Using bioinformatics, we identified genes in the unfinished enteropathogenic Escherichia coli (EPEC strain E2348/69 genome whose predicted products bear homology to the HsdM methyltransferases, HsdS specificity subunits, and HsdR restriction endonucleases of type I restriction-modification systems. We constructed a strain carrying a deletion of the conserved enzymatic domain of the EPEC HsdR homologue, NH4, and showed that its transformation efficiency was up to four orders of magnitude higher than that of the parent strain. Further, the modification capacity of NH4 remained intact, since plasmids that were normally recalcitrant to transformation into E2348/69 could be transformed upon passage through NH4. NH4 was unaffected in virulence factor production, since bundle forming pilus (BFP subunits and type III secreted (T3S proteins were present at equivalent levels to those seen in E2348/69. Further, NH4 was indistinguishable from E2348/69 in tissue culture infection model assays of localized adherence and T3S. Conclusion We

INFLUENCE OF TECHNOGENIC LANDSCAPES RECULTIVATION ON FUNCTIONING OF SOIL MICROORGANISMS COMMUNITIES WHICH TAKE PART IN TRANSFORMATION OF NITROGEN COMPOUNDS

Directory of Open Access Journals (Sweden)

Syshchykova Oksana Vitalyevna

2014-04-01

Full Text Available It is established that mining recultivation of tailings dams slimes promotes restoration of numerical structure of soil microorganisms community which take part in processes of nitrogen compounds transformation. The certificate of that is number restoration of the organotrophic bacteria of a nitrogen cycle to 0.3 million CFU/g of soil and increase by 2-3 times of streptomycetes quantity in blankets. The received results of quantitative structure of the microorganisms which are taking part in processes of nitrogen mineral compounds transformation in the chernozem usual allow to claim that in blankets the number of microorganisms makes 3.89 and 2.33 million CFU/g soil. It should be noted that the best conditions for microflora development are formed on slime with drawing 50 cm of loess-like loam and 30 cm of a fertile layer. The microorganism quantity on the specified monitoring area increases by 3-4 times in the soil of a fertile layer and by 1.3-1.6 times in loess-like loam in comparison with slime without recultivation. Increase of microbiological processes intensity, extremely important, considering strengthening of ecosystems self-regulation functions. It is established high level of microbiological transformation of organic substance, the indicator is made 7.3-11.1 in the edatopes of the recultivated slimes. Increasing indicators of microbiological transformation and mineralization of organic compounds in the technozems confirm restoration of a slimes biogenity at carrying out of recultivation that promotes an intensification of mineralization processes and assimilation by plants nitrogen compounds in the soil.

Effect of phosphorus addition on the reductive transformation of pentachlorophenol (PCP) and iron reduction with microorganism involvement.

Science.gov (United States)

Wang, Yongkui; Liu, Xianli; Huang, Jiexun; Xiao, Wensheng; Zhang, Jiaquan; Yin, Chunqin

2017-10-01

The transformation of phosphorus added to the soil environment has been proven to be influenced by the Fe biochemical process, which thereby may affect the transformation of organic chlorinated contaminants. However, the amount of related literatures regarding this topic is limited. This study aimed to determine the effects of phosphorus addition on pentachlorophenol (PCP) anaerobic transformation, iron reduction, and paddy soil microbial community structure. Results showed that the transformation of phosphorus, iron, and PCP were closely related to the microorganisms. Moreover, phosphorus addition significantly influenced PCP transformation and iron reduction, which promoted and inhibited these processes at low and high concentrations, respectively. Both the maximum reaction rate of PCP transformation and the maximum Fe(II) amount produced were obtained at 1 mmol/L phosphorus concentration. Among the various phosphorus species, dissolved P and NaOH-P considerably changed, whereas only slight changes were observed for the remaining phosphorus species. Microbial community structure analysis demonstrated that adding low concentration of phosphorus promoted the growth of Clostridium bowmanii, Clostridium hungatei, and Clostridium intestinale and Pseudomonas veronii. By contrast, high-concentration phosphorus inhibited growth of these microorganisms, similar to the curves of PCP transformation and iron reduction. These observations indicated that Clostridium and P. veronii, especially Clostridium, played a vital role in the transformation of related substances in the system. All these findings may serve as a reference for the complicated reactions among the multiple components of soils.

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation

Science.gov (United States)

Rothschild, Lynn J.; Greenberg, Daniel T.; Takahashi, Jack R.; Thompson, Kirsten A.; Maheshwari, Akshay J.; Kent, Ryan E.; McCutcheon, Griffin; Shih, Joseph D.; Calvet, Charles; Devlin, Tyler D.;

Electron Resonance Decay into a Biological Function: Decrease in Viability of E. coli Transformed by Plasmid DNA Irradiated with 0.5-18 eV Electrons.

Science.gov (United States)

Kouass Sahbani, S; Cloutier, P; Bass, A D; Hunting, D J; Sanche, L

2015-10-01

Transient negative ions (TNIs) are ubiquitous in electron-molecule scattering at low electron impact energies (0-20 eV) and are particularly effective in damaging large biomolecules. Because ionizing radiation generates mostly 0-20 eV electrons, TNIs are expected to play important roles in cell mutagenesis and death during radiotherapeutic cancer treatment, although this hypothesis has never been directly verified. Here, we measure the efficiency of transforming E. coli bacteria by inserting into the cells, pGEM-3ZfL(-) plasmid DNA that confers resistance to the antibiotic ampicillin. Before transformation, plasmids are irradiated with electrons of specific energies between 0.5 and 18 eV. The loss of transformation efficiency plotted as a function of irradiation energy reveals TNIs at 5.5 and 9.5 eV, corresponding to similar states observed in the yields of DNA double strand breaks. We show that TNIs are detectable in the electron-energy dependence of a biological process and can decrease cell viability.

Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae.

Science.gov (United States)

Maglennon, Gareth A; Cook, Beth S; Matthews, Dominic; Deeney, Alannah S; Bossé, Janine T; Langford, Paul R; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N

2013-07-29

Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen.

Compatibility and entry exclusion of IncA and IncC plasmids revisited: IncA and IncC plasmids are compatible.

Science.gov (United States)

Ambrose, Stephanie J; Harmer, Christopher J; Hall, Ruth M

2018-02-24

In an early study, IncA and IncC plasmids that were reported to be compatible were grouped as the "A-C complex" based on similarities and on strong entry exclusion. However, recently, the term IncA/C has been used frequently to describe plasmids belonging to both of these two groups. Granted that the supporting data was not included in the original reports and that the consensus iteron sequences have since been shown to be essentially identical, we have addressed the question again. The original IncA plasmid, RA1, and the IncC plasmid pRMH760, were introduced into the same cell by transformation, and were found to be maintained stably for over 100 generations in the absence of selection for either plasmid, i.e. they were compatible. We conclude that use of the term IncA/C for this important plasmid group is indeed incorrect and it causes unnecessary confusion. Granted the importance of IncC plasmids in the spread of antibiotic resistance genes, we recommend that use of the misleading terms IncA/C, IncA/C 1 and IncA/C 2 should cease. In addition, RA1 and pRMH760 were shown to each completely prevent entry of the other via conjugative transfer into the cell they reside in. Copyright © 2018 Elsevier Inc. All rights reserved.

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

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Michelle D. Rodriguez

2017-12-01

Full Text Available Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus. These three reporter plasmids are available through BEI Resources.

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus.

Science.gov (United States)

Rodriguez, Michelle D; Paul, Zubin; Wood, Charles E; Rice, Kelly C; Triplett, Eric W

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus . These three reporter plasmids are available through BEI Resources.

Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

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Rika Etchuuya

Full Text Available Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain. In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5-10(-6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Ikai, K.; Tano, K.; Ohnishi, T.; Nozu, K.

1985-01-01

The repair of UV-irradiated DNA of plasmid YEp13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, rad1,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endonuclease increased repair. It was concluded that all the mutations of yeast were probably limited only to the nuclear DNA. (author)

Transfer of the pheromone-inducible plasmid pCF10 among Enterococcus faecalis microorganisms colonizing the intestine of mini-pigs

DEFF Research Database (Denmark)

Licht, Tine Rask; Laugesen, D.; Jensen, Lars Bogø

2002-01-01

A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG...

Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability

International Nuclear Information System (INIS)

Chopin, M.C.; Chopin, A.; Rouault, A.; Simon, D.

1986-01-01

Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains

Fast and efficient three-step target-specific curing of a virulence plasmid in Salmonella enterica.

Science.gov (United States)

de Moraes, Marcos H; Teplitski, Max

2015-12-01

Virulence plasmids borne by serovars of Salmonella enterica carry genes involved in its pathogenicity, as well as other functions. Characterization of phenotypes associated with virulence plasmids requires a system for efficiently curing strains of their virulence plasmids. Here, we developed a 3-step protocol for targeted curing of virulence plasmids. The protocol involves insertion of an I-SecI restriction site linked to an antibiotic resistance gene into the target plasmid using λ-Red mutagenesis, followed by the transformation with a temperature-sensitive auxiliary plasmid which carries I-SecI nuclease expressed from a tetracycline-inducible promoter. Finally, the auxiliary plasmid is removed by incubation at 42 °C and the plasmid-less strains are verified on antibiotic-containing media. This method is fast and very efficient: over 90 % of recovered colonies lacked their virulence plasmid.

Breaks in plasmid DNA strand induced by laser radiation at a wavelength of 193 nm

International Nuclear Information System (INIS)

Gurzadyan, G.G.; Shul'te Frolinde, D.

1996-01-01

DNA of plasmid pB322 irradiated with laser at a wavelength of 193 nm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type). The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks. The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks. A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks. The inactivation of the plasmid is mainly determined by the number of directly formed laser-induced single-strand breaks. 26 refs.; 2 figs

An improved ternary vector system for Agrobacterium-mediated rapid maize transformation.

Science.gov (United States)

Anand, Ajith; Bass, Steven H; Wu, Emily; Wang, Ning; McBride, Kevin E; Annaluru, Narayana; Miller, Michael; Hua, Mo; Jones, Todd J

2018-05-01

A simple and versatile ternary vector system that utilizes improved accessory plasmids for rapid maize transformation is described. This system facilitates high-throughput vector construction and plant transformation. The super binary plasmid pSB1 is a mainstay of maize transformation. However, the large size of the base vector makes it challenging to clone, the process of co-integration is cumbersome and inefficient, and some Agrobacterium strains are known to give rise to spontaneous mutants resistant to tetracycline. These limitations present substantial barriers to high throughput vector construction. Here we describe a smaller, simpler and versatile ternary vector system for maize transformation that utilizes improved accessory plasmids requiring no co-integration step. In addition, the newly described accessory plasmids have restored virulence genes found to be defective in pSB1, as well as added virulence genes. Testing of different configurations of the accessory plasmids in combination with T-DNA binary vector as ternary vectors nearly doubles both the raw transformation frequency and the number of transformation events of usable quality in difficult-to-transform maize inbreds. The newly described ternary vectors enabled the development of a rapid maize transformation method for elite inbreds. This vector system facilitated screening different origins of replication on the accessory plasmid and T-DNA vector, and four combinations were identified that have high (86-103%) raw transformation frequency in an elite maize inbred.

Plasmids in Gram negatives: molecular typing of resistance plasmids.

Science.gov (United States)

Carattoli, Alessandra

2011-12-01

A plasmid is defined as a double stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under non-stressed conditions but they have systems which guarantee their autonomous replication also controlling the copy number and ensuring stable inheritance during cell division. Most of the plasmids confer positively selectable phenotypes by the presence of antimicrobial resistance genes. Plasmids evolve as an integral part of the bacterial genome, providing resistance genes that can be easily exchanged among bacteria of different origin and source by conjugation. A multidisciplinary approach is currently applied to study the acquisition and spread of antimicrobial resistance in clinically relevant bacterial pathogens and the established surveillance can be implemented by replicon typing of plasmids. Particular plasmid families are more frequently detected among Enterobacteriaceae and play a major role in the diffusion of specific resistance genes. For instance, IncFII, IncA/C, IncL/M, IncN and IncI1 plasmids carrying extended-spectrum beta-lactamase genes and acquired AmpC genes are currently considered to be "epidemic resistance plasmids", being worldwide detected in Enterobacteriaceae of different origin and sources. The recognition of successful plasmids is an essential first step to design intervention strategies preventing their spread. Copyright © 2011 Elsevier GmbH. All rights reserved.

In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

DEFF Research Database (Denmark)

Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora

2014-01-01

In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

Genes from plasmid pKM101 in Haemophilus influenzae: separation of functions of mucA and mucB

International Nuclear Information System (INIS)

Balganesh, M.; Setlow, J.K.

1985-01-01

Haemophilus influenzae, normally not mutable by UV, became UV mutable with a recombinant plasmid insertion. A 7.8-kilobase-pair (kbp) fragment of the plasmid pKM101 containing the mucA and mucB genes was ligated to the shuttle vector pDM2, and a Rec- strain of H. influenzae was transformed with the ligated mixture. All of the transformants, unlike the parent Rec- strain, were resistant to UV, could carry out postreplication repair and Weigle reactivation, showed greatly increased spontaneous mutation, and contained a plasmid carrying an insert of only 1.2 rather than 7.8 kbp. This plasmid in a umuC mutant strain of Escherichia coli complemented a pKM101 derivative lacking mucA function but with an intact mucB gene, although there was no complementation with a mucA+ mucB- plasmid, suggesting that the newly constructed plasmid coded for the mucA protein; this is in accord with the restriction analysis and hybridization between the plasmid and a probe containing all of the mucA gene but only a small fraction of mucB. When one of the H. influenzae Rec- transformants lost the plasmid, the resistance to UV was retained but the high spontaneous mutation and UV mutability were not. The fact that there was hybridization between the chromosome of the cured strain and a probe containing both muc genes but none when almost no mucB was present suggested that at least part of the mucB gene had been integrated into the Rec- chromosome. Five different postreplication repair-proficient strains became UV mutable and had high spontaneous mutation rates caused by the putative mucA plasmid, indicating that these strains already possessed a chromosomal equivalent of the mucB gene

Induction and isolation of DNA transformation mutants in the yeast Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Hegerich, P.A.; Bruschi, C.V.

1987-01-01

The objective of this research was to induce and isolate mutants of the yeast Saccharomyces cerevisiae which have become transformable by purified plasmid DNA. Non-transformable yeast cells were mutagenized by ultraviolet light using a 65% lethal dose (480 ergs/mm 2 ). After a period of overnight liquid holding recovery, the irradiated cells were subjected to DNA transformation using our CaCl 2 protocol with the multi-marker shuttle plasmid pBB carrying the LEU 2 leucine gene. Following transformation the colonies that grew on selective leucineless medium were identified and subjected to further genetic analysis. From a total of 1 x 10 9 cells the authors have isolated 7 colonies deriving from putative mutants that have acquired the capability to uptake plasmid DNA. The transformants were cured from the plasmid by its mitotic loss on non-selective medium, then re-transformed to verify their genetic competence to give rise to a number of transformants comparable to transformable strains. We have identified and isolated one mutant, coded trs-1, which is able to reproduce a frequency of transformation comparable with the tranformable control. They, therefore, conclude that this mutant is specific for plasmid DNA transformation and that the mutation is mitotically stable

Expression of recombinant myostatin propeptide pPIC9K-Msp plasmid in Pichia pastoris.

Science.gov (United States)

Du, W; Xia, J; Zhang, Y; Liu, M J; Li, H B; Yan, X M; Zhang, J S; Li, N; Zhou, Z Y; Xie, W Z

2015-12-28

Myostatin propeptide can inhibit the biological activity of myostatin protein and promote muscle growth. To express myostatin propeptide in vitro with a higher biological activity, we performed codon optimization on the sheep myostatin propeptide gene sequence, and mutated aspartic acid-76 to alanine based on the codon usage bias of Pichia pastoris and the enhanced biological activity of myostatin propeptide mutant. Modified myostatin propeptide gene was cloned into the pPIC9K plasmid to form the recombinant plasmid pPIC9K-Msp. Recombinant plasmid pPIC9K-Msp was transformed into Pichia pastoris GS115 by electrotransformation. Transformed cells were screened, and methanol was used to induce expression. SDS-PAGE and western blotting were used to verify the successful expression of myostatin propeptide with biological activity in Pichia pastoris, providing the basis for characterization of this protein.

Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

Science.gov (United States)

Rhee, Mun Su; Kim, Jin-Woo; Qian, Yilei; Ingram, L O; Shanmugam, K T

2007-07-01

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

Effect of the atmospheric pressure nonequilibrium plasmas on the conformational changes of plasmid DNA

International Nuclear Information System (INIS)

Yan Xu; He Guangyuan; Shi Mengjun; Gao Xuan; Li Yin; Ma Fengyun; Yu Men; Wang Changdong; Wang Yuesheng; Yang Guangxiao; Zou Fei; Lu Xinpei; Xiong Qing; Xiong Zilan

2009-01-01

The cold atmospheric pressure plasma, which has been widely used for biomedical applications, may potentially affect the conformation of DNA. In this letter, an atmospheric pressure plasma plume is used to investigate its effects on the conformational changes of DNA of plasmid pAHC25. It is found that the plasma plume could cause plasmid DNA topology alteration, resulting in the percentage of the supercoiled plasmid DNA form decreased while that of the open circular and linearized form of plasmid DNA increased as detected by agrose gel electrophoresis. On the other hand, further investigation by using polymerase chain reaction method shows that the atmospheric pressure plasma jet treatments under proper conditions does not affect the genes of the plasmid DNA, which may have potential application in increasing the transformation frequency by genetic engineering.

A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae.

Science.gov (United States)

Ishag, H Z A; Liu, M J; Yang, R S; Xiong, Q Y; Feng, Z X; Shao, G Q

2016-04-28

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes porcine enzootic pneumonia (PEP) that significantly affects the pig industry worldwide. Despite the availability of the whole genome sequence, studies on the pathogenesis of this organism have been limited due to the lack of a genetic manipulation system. Therefore, the aim of the current study was to generate a general GFP reporter vector based on a replicating plasmid. Here, we describe the feasibility of GFP reporter expression in M. hyopneumoniae (strain 168L) controlled by the p97 gene promoter of this mycoplasma. An expression plasmid (pMD18-TOgfp) containing the p97 gene promoter, and origin of replication (oriC) of M. hyopneumoniae, tetracycline resistant marker (tetM), and GFP was constructed and used to transform competent M. hyopneumoniae cells. We observed green fluorescence in M. hyopneumoniae transformants under fluorescence microscopy, which indicates that there was expression of the GFP reporter that was driven by the p97 gene promoter. Additionally, an electroporation method for M. hyopneumoniae with an efficiency of approximately 1 x 10(-6) transformants/μg plasmid DNA was optimized and is described herein. In conclusion, our data demonstrate the susceptibility of M. hyopneumoniae to genetic manipulation whereby foreign genes are expressed. This work may encourage the development of genetic tools to manipulate the genome of M. hyopneumoniae for functional genomic analyses.

Agrobacterium-mediated transformation of lipomyces

Science.gov (United States)

Dai, Ziyu; Magnuson, Jon K.; Deng, Shuang; Bruno, Kenneth S.; Culley, David E.

2018-03-13

This disclosure provides Agrobacterium-mediated transformation methods for the oil-producing (oleaginous) yeast Lipomyces sp., as well as yeast produced by the method. Such methods utilize Agrobacterium sp. cells that have a T-DNA binary plasmid, wherein the T-DNA binary plasmid comprises a first nucleic acid molecule encoding a first protein and a second nucleic acid molecule encoding a selective marker that permits growth of transformed Lipomyces sp. cells in selective culture media comprising an antibiotic.

Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens, Mycoplasma bovis and Mycoplasma agalactiae

Science.gov (United States)

Sharma, Shukriti; Citti, Chistine; Sagné, Eveline; Marenda, Marc S.

2015-01-01

Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae. PMID:25746296

Development and host compatibility of plasmids for two important ruminant pathogens, Mycoplasma bovis and Mycoplasma agalactiae.

Directory of Open Access Journals (Sweden)

Shukriti Sharma

Full Text Available Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.

The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

International Nuclear Information System (INIS)

Roos, C; Santos, J N; Guimarães, O R; Geller, M; Fonseca, A S; Paoli, F

2013-01-01

Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm −2 ) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms. (paper)

Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA

Science.gov (United States)

Hershfield, Vickers; Boyer, Herbert W.; Yanofsky, Charles; Lovett, Michael A.; Helinski, Donald R.

1974-01-01

DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage ϕ80pt190 (trp+) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp- strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the ϕ80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes. Images PMID:4610576

Plasmid-Mediated Quinolone Resistance in Shigella flexneri Isolated From Macaques

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Anthony J. Mannion

2018-03-01

Full Text Available Non-human primates (NHPs for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants

Plasmid segregation mechanisms

DEFF Research Database (Denmark)

Ebersbach, G.; Gerdes, Kenn

2005-01-01

Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments...... that segregate plasmids paired at mid-cell to daughter cells. Like microtubules, ParM filaments exhibit dynamic instability (i.e., catastrophic decay) whose regulation is an important component of the DNA segregation process. The Walker box ParA ATPases are related to MinD and form highly dynamic, oscillating...... filaments that are required for the subcellular movement and positioning of plasmids. The role of the observed ATPase oscillation is not yet understood. However, we propose a simple model that couples plasmid segregation to ParA oscillation. The model is consistent with the observed movement...

An Enterobacter plasmid as a new genetic background for the transposon Tn1331

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Alavi MR

2011-11-01

Full Text Available Mohammad R Alavi1,2, Vlado Antonic2, Adrien Ravizee1, Peter J Weina3, Mina Izadjoo1,2, Alexander Stojadinovic21Division of Wound Biology and Translational Research, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC, 2Combat Wound Initiative Program, Walter Reed Army Medical Center, Washington DC, 3The Walter Reed Army Institute of Research, Silver Spring, MD, USABackground: Genus Enterobacter includes important opportunistic nosocomial pathogens that could infect complex wounds. The presence of antibiotic resistance genes in these microorganisms represents a challenging clinical problem in the treatment of these wounds. In the authors’ screening of antibiotic-resistant bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic-resistant plasmids conferring resistance to Escherichia coli. The aim of this study was to identify the resistance genes carried by one of these plasmids.Methods: The plasmids from the Enterobacter isolate were propagated in E. coli and one of the plasmids, designated as pR23, was sequenced by the Sanger method using fluorescent dye-terminator chemistry on a genetic analyzer. The assembled sequence was annotated by search of the GenBank database.Results: Plasmid pR23 is composed of the transposon Tn1331 and a backbone plasmid that is identical to the plasmid pPIGDM1 from Enterobacter agglomerans. The multidrug-resistance transposon Tn1331, which confers resistance to aminoglycoside and beta lactam antibiotics, has been previously isolated only from Klebsiella. The Enterobacter plasmid pPIGDM1, which carries a ColE1-like origin of replication and has no apparent selective marker, appears to provide a backbone for propagation of Tn1331 in Enterobacter. The recognition sequence of Tn1331 transposase for insertion into pPIGDM1 is the pentanucleotide TATTA, which occurs only once throughout the length of this plasmid.Conclusion: Transposition of Tn1331 into

Genetic Transformation of Bacteria.

Science.gov (United States)

Moss, Robert.

1991-01-01

An activity in which students transform an ampicillin-sensitive strain of E. coli with a plasmid containing a gene for ampicillin resistance is described. The procedure for the preparation of competent cells and the transformation of competent E. coli is provided. (KR)

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

Science.gov (United States)

Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

2017-01-01

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica

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Maria Hoffmann

2017-08-01

Full Text Available Determinants of multidrug resistance (MDR are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI, and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about

Test and research on the production of useful matter with carbon-dioxide-fixed microorganisms. Production of biodegradation plastics with photosynthesized microorganisms. Tansan gas kotei biseibutsu wo mochiita yuyo busshitsu seisan shiken kenkyu. Kogosei biseibutsu ni yoru seibunkaisei plastic seisan

Energy Technology Data Exchange (ETDEWEB)

Miyasaka, H. (The Kansai Electric Power Co. Inc., Osaka (Japan))

1994-07-10

The present report introduces the basic research on the production of PHB (one of the biodegradation plastics) by utilizing the photosynthesized microorganisms. The indigo algae are conveniently used for the genetic operation, because they are genetically inferior and have plasmid (extranuclear ringed DNA). Therefore, a certain type of oceanic indigo algae was used for the present research. As a vector to introduce the PHB-producing gene into the indigo alga cell, hybrid plasmid was prepared by combining the indigo alga plasmid pAQ1 with colon bacillus plasmid vectors pUC19. The thus prepared vectors had an introduction efficiency into about 1000 cells per one vector. A RuBisCO gene of indigo alga cell was selected as a promoter to heighten the activity of PHB-producing gene in that cell. The gene is being further studied in promotion efficiency by combining it with the above vector and PHB-producing gene. 4 figs.

Comparative symbiotic plasmid analysis indicates that symbiosis gene ancestor type affects plasmid genetic evolution.

Science.gov (United States)

Wang, X; Zhao, L; Zhang, L; Wu, Y; Chou, M; Wei, G

2018-07-01

Rhizobial symbiotic plasmids play vital roles in mutualistic symbiosis with legume plants by executing the functions of nodulation and nitrogen fixation. To explore the gene composition and genetic constitution of rhizobial symbiotic plasmids, comparison analyses of 24 rhizobial symbiotic plasmids derived from four rhizobial genera was carried out. Results illustrated that rhizobial symbiotic plasmids had higher proportion of functional genes participating in amino acid transport and metabolism, replication; recombination and repair; carbohydrate transport and metabolism; energy production and conversion and transcription. Mesorhizobium amorphae CCNWGS0123 symbiotic plasmid - pM0123d had similar gene composition with pR899b and pSNGR234a. All symbiotic plasmids shared 13 orthologous genes, including five nod and eight nif/fix genes which participate in the rhizobia-legume symbiosis process. These plasmids contained nod genes from four ancestors and fix genes from six ancestors. The ancestral type of pM0123d nod genes was similar with that of Rhizobium etli plasmids, while the ancestral type of pM0123d fix genes was same as that of pM7653Rb. The phylogenetic trees constructed based on nodCIJ and fixABC displayed different topological structures mainly due to nodCIJ and fixABC ancestral type discordance. The study presents valuable insights into mosaic structures and the evolution of rhizobial symbiotic plasmids. This study compared 24 rhizobial symbiotic plasmids that included four genera and 11 species, illuminating the functional gene composition and symbiosis gene ancestor types of symbiotic plasmids from higher taxonomy. It provides valuable insights into mosaic structures and the evolution of symbiotic plasmids. © 2018 The Society for Applied Microbiology.

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Science.gov (United States)

Pitard, Bruno; Aguerre, Olivier; Airiau, Marc; Lachagès, Anne-Marie; Boukhnikachvili, Tsiala; Byk, Gérardo; Dubertret, Catherine; Herviou, Christian; Scherman, Daniel; Mayaux, Jean-François; Crouzet, Joël

1997-01-01

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains. PMID:9405626

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

Science.gov (United States)

Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

2014-12-01

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

OpenAIRE

Serror, Pascale; Sasaki, Takashi; Ehrlich, S. Dusko; Maguin, Emmanuelle

2002-01-01

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 104 transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a goo...

[Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

Science.gov (United States)

Bolotin, A P; Sorokin, A V; Aleksandrov, N N; Danilenko, V N; Kozlov, Iu I

1985-11-01

The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

Development of a Candida glabrata dominant nutritional transformation marker utilizing the Aspergillus nidulans acetamidase gene (amdS).

Science.gov (United States)

Fu, Jianmin; Blaylock, Morganne; Wickes, Cameron F; Welte, William; Mehrtash, Adrian; Wiederhold, Nathan; Wickes, Brian L

2016-05-01

The gene encoding Aspergillus nidulans acetamidase (amdS) was placed under control of Candida albicans ACT1 promoter and terminator sequences and then cloned into a plasmid containing C. glabrata ARS10,CEN8 or ARS10+CEN8 sequences. All plasmids transformed C. glabrata wild-type cells to acetamide+, with the ARS-only containing plasmid transforming cells at the highest frequencies (>1.0 × 10(4) transformants μg(-1)). Plasmids were rapidly lost under non-selective conditions with the frequency dependent on chromosomal element, thus recycling the acetamide- phenotype. The amdS plasmid was used to transform a set of clinical isolates resistant to a variety of antifungal drugs. All strains were successfully transformed to the acetamide+ phenotype at high frequency, confirming that this plasmid construct could be used as a simple dominant marker on virtually any strain. Gap repair experiments demonstrated that just as in Saccharomyces cerevisiae, gap repair functions efficiently inC. glabrata, suggesting that C. glabrata has numerous similarities toS. cerevisiae with regard to ease of molecular manipulation. The amdS system is inexpensive and efficient, and combined with existing C. glabrata plasmid elements, confers a high transformation frequency for C. glabrata with a phenotype that can be easily recycled. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Fossil Microorganisms and Formation of Early Precambrian Weathering Profiles

Science.gov (United States)

Rozanov, A. Yu; Astafieva, M. M.; Vrevsky, A. B.; Alfimova, N. A.; Matrenichev, V. A.; Hoover, R. B.

2009-01-01

Weathering crusts are the only reliable evidences of the existence of continental conditions. Often they are the only source of information about exogenous processes and subsequently about conditions under which the development of the biosphere occurred. A complex of diverse fossil microorganisms was discovered as a result of Scanning Electron Microscope investigations. The chemical composition of the discovered fossils is identical to that of the host rocks and is represented by Si, Al, Fe, Ca and Mg. Probably, the microorganisms fixed in rocks played the role of catalyst. The decomposition of minerals comprising the rocks and their transformation into clayey (argillaceous) minerals, most likely occurred under the influence of microorganisms. And may be unique weathering crusts of Early Precambrian were formed due to interaction between specific composition of microorganism assemblage and conditions of hypergene transformations. So it is possible to speak about colonization of land by microbes already at that time and about existence of single raw from weathering crusts (Primitive soils) to real soils.

Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences

Science.gov (United States)

Garcillán-Barcia, M. Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M.; de la Cruz, Fernando

2014-01-01

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET, a new method for plasmid reconstruction from whole genome sequences.

Directory of Open Access Journals (Sweden)

Val F Lanza

2014-12-01

Full Text Available Bacterial whole genome sequence (WGS methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage, comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC, comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

Science.gov (United States)

Laine, M J; Nakhei, H; Dreier, J; Lehtilä, K; Meletzus, D; Eichenlaub, R; Metzler, M C

1996-05-01

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.

Dissemination of imipenem-resistant Acinetobacter baumannii with new plasmid-borne bla(OXA-72) in Taiwan.

Science.gov (United States)

Kuo, Shu-Chen; Yang, Su-Pen; Lee, Yi-Tzu; Chuang, Han-Chuan; Chen, Chien-Pei; Chang, Chi-Ling; Chen, Te-Li; Lu, Po-Liang; Hsueh, Po-Ren; Fung, Chang-Phone

2013-07-13

The systemic surveillance of imipenem-resistant Acinetobacter baumannii (IRAB) from multicenters in Taiwan revealed the emergence of isolates with bla(OXA-72). This study described their genetic makeup, mechanism of spread, and contribution to carbapenem resistance. Two hundred and ninety-one non-repetitive isolates of A. baumannii were collected from 10 teaching hospitals from different geographical regions in Taiwan from June 2007 to September 2007. Minimal inhibitory concentrations (MICs) were determined by agar dilution. Clonality was determined by pulsed-field gel electrophoresis. Plasmid was extracted and digested by restriction enzymes, and subsequently analyzed by electrophoresis and Southern blot for bla(OXA-72). The flanking regions of bla(OXA-72) were determined by inverse PCR. The contribution of bla(OXA-72) to imipenem MIC was determined by transforming plasmids carrying bla(OXA-72) into imipenem-susceptible A. baumannii. Among 142 IRAB in Taiwan, 27 harbored bla(OXA-72); 22 originated from Southern Taiwan, 5 from Central Taiwan, and none from Northern Taiwan. There were two major clones. The bla(OXA-72) was identified in the plasmids of all isolates. Two genetic structures flanking plasmid-borne bla(OXA-72) were identified and shared identical sequences in certain regions; the one described in previous literature was present in only one isolate, and the new one was present in the remaining isolates. Introduction of bla(OXA-72) resulted in an increase of imipenem MIC in the transformants. The overexpression of bla(OXA-72) mRNA in response to imipenem further supported the contribution of bla(OXA-72). In conclusion, isolates with new plasmid-borne blaOXA-72 were found to be disseminated successfully in Southern Taiwan. The spread of the resistance gene depended on clonal spread and dissemination of a new plasmid. Bla(OXA-72) in these isolates directly led to their imipenem-resistance.

Development of shuttle vectors for transformation of diverse Rickettsia species.

Directory of Open Access Journals (Sweden)

Nicole Y Burkhardt

Full Text Available Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies, and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.

Plasmids and packaging cell lines for use in phage display

Science.gov (United States)

Bradbury, Andrew M.

2012-07-24

The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene

NARCIS (Netherlands)

Heidekamp, F.; Dirkse, W.G.; Hille, J.; Ormondt, H. van

1983-01-01

The nucleotide sequence of the tmr gene, encoded by the octopine Ti plasmid from Agrobacterium tumefaciens (pTiAch5), was determined. The T-DNA, which encompasses this gene, is involved in tumor formation and maintenance, and probably mediates the cytokinin-independent growth of transformed plant

Transformation of group A streptococci by electroporation

NARCIS (Netherlands)

Suvorov, Alexander; Kok, Jan; Venema, Gerhardus

1988-01-01

The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 10^3 to 4 × 10^4 per µg of DNA. The method was also used to introduce an integrative plasmid and transformants were

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

Directory of Open Access Journals (Sweden)

Qureshi Nasib

2006-05-01

Full Text Available Abstract Background The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. Results We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved Cel

Origin and Evolution of Rickettsial Plasmids.

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Khalid El Karkouri

Full Text Available Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes.Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events.Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions.

Science.gov (United States)

Burbank, Lindsey P; Van Horn, Christopher R

2017-11-01

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa , but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb , putatively encoding a conjugative type IV secretion system, are found in some but not all X. fastidiosa isolates, often on native plasmids. X. fastidiosa strains that carry the conjugative transfer genes can belong to different subspecies and frequently differ in host ranges. Using X. fastidiosa strain M23 ( X. fastidiosa subsp. fastidiosa ) or Dixon ( X. fastidiosa subsp. multiplex ) as the donor strain and Temecula ( X. fastidiosa subsp. fastidiosa ) as the recipient strain, plasmid transfer was characterized using the mobilizable broad-host-range vector pBBR5pemIK. Transfer of plasmid pBBR5pemIK was observed under in vitro conditions with both donor strains and was dependent on both tra and trb operon functions. A conjugative mechanism likely contributes to gene transfer between diverse strains of X. fastidiosa , possibly facilitating adaptation to new environments or different hosts. IMPORTANCE Xylella fastidiosa is an important plant pathogen worldwide, infecting a wide range of different plant species. The emergence of new diseases caused by X. fastidiosa , or host switching of existing strains, is thought to be primarily due to the high frequency of HGT and recombination in this pathogen. Transfer of plasmids by a conjugative mechanism enables movement of larger amounts of genetic material at one time, compared with other routes of gene transfer such as natural transformation. Establishing the prevalence and functionality of this mechanism in X. fastidiosa contributes to a better understanding of HGT, adaptation, and disease emergence

Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human

DEFF Research Database (Denmark)

Shuyu, Wu; Dalsgaard, A.; Hammerum, A. M.

2010-01-01

isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids...... and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids...

Application of genetically engineered microorganisms to bioremediation and wastewater treatment. Idenshi sosa biseibutsu no kankyo joka mizushori eno tekiyo

Energy Technology Data Exchange (ETDEWEB)

Fujita, M; Ike, M [Osaka University, Osaka (Japan). Faculty of Engineering

1993-11-10

This paper summarizes the following techniques: a gene engineering method for bioremediation and wastewater treatment, microorganism breeding using the former method, and a monitoring technique for genetical and ecological stability of genetically engineered microorganisms. Recombination bacteria reinforced with PH genes showed higher phenol removing rate than wild strains, but presented accumulation of catechol in such a large quantity as cannot be seen in wild strains, with the complete degradation rate rather decreased. Gene recombined bacteria structured by introducing the recombined plasmid, pBH500, had high genetic stability when P.putida BH-1 is used as a host. E.coli C600 having recombined plasmid and P.putida BH were added and cultivated in activated sludge. As a result, both recombined bacteria showed rapid logarithmic decrease just after the addition, then, maintained the relatively stable population groups, and remained in the activated sludge for an extended period of time. In monitoring techniques, the colony hybridization process detected clearly the gene recombined bacteria. 9 refs., 7 figs., 1 tab.

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

Science.gov (United States)

Serror, Pascale; Sasaki, Takashi; Ehrlich, S. Dusko; Maguin, Emmanuelle

2002-01-01

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 104 transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii. PMID:11772607

Fitness Advantage of mcr-1–Bearing IncI2 and IncX4 Plasmids in Vitro

Directory of Open Access Journals (Sweden)

Renjie Wu

2018-02-01

Full Text Available The objective of this study was to assess the impact of diverse plasmids bearing colistin resistance gene mcr-1 on host fitness. Forty-seven commensal E. coli isolates recovered from the pig farm where mcr-1 was first identified were screened for mcr-1. mcr-1-bearing plasmids were characterized by sequencing. The fitness impact of mcr-1-bearing plasmids was evaluated by in vitro competition assays. Twenty-seven (57.5% E. coli isolates were positive for mcr-1. The mcr-1 genes were mainly located on plasmids belonging to IncI2 (n = 5, IncX4 (n = 11, IncHI2/ST3 (n = 8, IncFII (n = 2, and IncY (n = 2. InHI2 plasmids also carried other resistance genes (floR, blaCTX−M, and fosA3 and were only detected in isolates from nursery pigs. Sequences of the representative mcr-1–bearing plasmids were almost identical to those of the corresponding plasmid types reported previously. An increase in the fitness of IncI2- and IncX4-carrying strains was observed, while the presence of IncHI2, IncFII and IncY plasmids showed a fitness cost although an insignificant fitness increase was initially observed in IncFII or IncY plasmids-containing strains. Acquisition of IncI2-type plasmid was more beneficial for host E. coli DH5α than either IncHI2 or IncX4 plasmid, while transformants with IncHI2-type plasmid presented a competitive disadvantage against IncI2 or IncX4 plasmid containing strains. In conclusion, IncI2, IncX4, and IncHI2 were the major plasmid types driving the dissemination of mcr-1 in this farm. Increased fitness or co-selection by other antimicrobials might contribute to the further dissemination of the three epidemic mcr-1–positive plasmids (IncI2, IncX4, and IncHI2 in this farm and worldwide.

Compost supplementation with nutrients and microorganisms in composting process.

Science.gov (United States)

Sánchez, Óscar J; Ospina, Diego A; Montoya, Sandra

2017-11-01

The composting is an aerobic, microorganism-mediated, solid-state fermentation process by which different organic materials are transformed into more stable compounds. The product obtained is the compost, which contributes to the improvement of physical, chemical and microbiological properties of the soil. However, the compost usage in agriculture is constrained because of its long-time action and reduced supply of nutrients to the crops. To enhance the content of nutrients assimilable by the plants in the compost, its supplementation with nutrients and inoculation with microorganisms have been proposed. The objective of this work was to review the state of the art on compost supplementation with nutrients and the role played by the microorganisms involved (or added) in their transformation during the composting process. The phases of composting are briefly compiled and different strategies for supplementation are analyzed. The utilization of nitrogenous materials and addition of microorganisms fixing nitrogen from the atmosphere or oxidizing ammonia into more assimilable for plants nitrogenous forms are analyzed. Several strategies for nitrogen conservation during composting are presented as well. The supplementation with phosphorus and utilization of microorganisms solubilizing phosphorus and potassium are also discussed. Main groups of microorganisms relevant during the composting process are described as well as most important strategies to identify them. In general, the development of this type of nutrient-enriched bio-inputs requires research and development not only in the supplementation of compost itself, but also in the isolation and identification of microorganisms and genes allowing the degradation and conversion of nitrogenous substances and materials containing potassium and phosphorus present in the feedstocks undergoing the composting process. In this sense, most important research trends and strategies to increase nutrient content in the compost

Selectable genes for transformation of the fungal plant pathogen Glomerella cingulata f. sp. phaseoli (Colletotrichum lindemuthianum).

Science.gov (United States)

Rodriguez, R J; Yoder, O C

1987-01-01

Glomerella cingulata f. sp. phaseoli (Gcp) was transformed using either of two selectable markers: the amdS + gene of Aspergillus nidulans, which encodes acetamidase and permits growth on acetamide as the sole nitrogen source and the hygBR gene of Escherichia coli which encodes hygromycin B (Hy) phosphotransferase and permits growth in the presence of the antibiotic Hy. The amdS+ gene functioned in Gcp under control of A. nidulans regulatory signals and hygBR was expressed after fusion to a promoter from Cochliobolus heterostrophus, another filamentous ascomycete. Protoplasts to be transformed were generated with the digestive enzyme complex Novozym 234 and then were exposed to plasmid DNA in the presence of 10 mM CaCl2 and polyethylene glycol. Transformation occurred by integration of single or multiple copies of either the amdS+ or hygBR plasmid into the fungal genome. There was no evidence of autonomous plasmid replication. Transformants were mitotically stable on selective and nonselective media. However, transforming DNA in hygBR transformants was observed to occasionally rearrange during nonselective growth, resulting in fewer copies of the plasmid per genome. These transformants were capable of infecting bean (Phaseolus vulgaris), the Gcp host plant, and after recovery from infected tissue were found to have retained both the transforming DNA unrearranged in their genomes and the Hy resistance phenotype. All single-conidial cultures derived from both amdS+ and hygBR transformants had the transplanted phenotype, suggesting that transformants were homokaryons.

Microbial genome-enabled insights into plant-microorganism interactions.

Science.gov (United States)

Guttman, David S; McHardy, Alice C; Schulze-Lefert, Paul

2014-12-01

Advances in genome-based studies on plant-associated microorganisms have transformed our understanding of many plant pathogens and are beginning to greatly widen our knowledge of plant interactions with mutualistic and commensal microorganisms. Pathogenomics has revealed how pathogenic microorganisms adapt to particular hosts, subvert innate immune responses and change host range, as well as how new pathogen species emerge. Similarly, culture-independent community profiling methods, coupled with metagenomic and metatranscriptomic studies, have provided the first insights into the emerging field of research on plant-associated microbial communities. Together, these approaches have the potential to bridge the gap between plant microbial ecology and plant pathology, which have traditionally been two distinct research fields.

Plasmid Mediated Antibiotic and Heavy Metal Resistance in Bacillus Strains Isolated From Soils in Rize, Turkey

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Elif SEVİM

2015-09-01

Full Text Available Fifteen Bacillus strains which were isolated from soil samples were examined for resistance to 17 different antibiotics (ampicillin, methicillin, erythromycin, norfloxacin, cephalotine, gentamycin, ciprofloxacin, streptomycin, tobramycin, chloramphenicol, trimethoprim-sulfamethoxazole, tetracycline, vancomycin, oxacilin, neomycin, kanamycin and, novabiocin and to 10 different heavy metals (copper, lead, cobalt, chrome, iron, mercury, zinc, nickel, manganese and, cadmium and for the presence of plasmid DNA. A total of eleven strains (67% were resistant to at least one antibiotic. The most common resistance was observed against methicillin and oxacillin. The most resistance strains were found as Bacillus sp. B3 and Bacillus sp. B11. High heavy metal resistance against copper, chromium, zinc, iron and nickel was detected, but mercury and cobalt resistance was not detected, except for 3 strains (B3, B11, and B12 which showed mercury resistance. It has been determined that seven Bacillus strains have plasmids. The isolated plasmids were transformed into the Bacillus subtilis W168 and it was shown that heavy metal and antibiotic resistance determinants were carried on these plasmids. These results showed that there was a correlation between plasmid content and resistance for both antibiotic and heavy metal resistance

Agrobacterium-mediated transformation of watermelon ( Citrullus ...

African Journals Online (AJOL)

Transformation of watermelon (Citrullus lanatus cv. Zaojia) using Agrobacterium tumefaciens strain EHA105 containing the plasmid pRD400 carrying Pti4 gene was studied in this work. Proximal cotyledons as explants were pre-cultivated for two day in the dark and it was found that the best condition for transformation of ...

Large-scale preparation of plasmid DNA.

Science.gov (United States)

Heilig, J S; Elbing, K L; Brent, R

2001-05-01

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

Rapid and inexpensive method for isolating plasmid DNA

International Nuclear Information System (INIS)

Aljanabi, S. M.; Al-Awadi, S. J.; Al-Kazaz, A. A.; Baghdad Univ.

1997-01-01

A small-scale and economical method for isolating plasmid DNA from bacteria is described. The method provides DNA of suitable quality for most DNA manipulation techniques. This DNA can be used for restriction endonuclease digestion, southern blot hybridization, nick translation and end labeling of DNA probes, Polymerase Chain Reaction (PCR) -based techniques, transformation, DNA cycle-sequencing, and Chain-termination method for DNA sequencing. The entire procedure is adapted to 1.5 ml microfuge tubes and takes approximately 30 mins. The DNA isolated by this method has the same purity produced by CTAB and cesium chloride precipitation and purification procedures respectively. The two previous methods require many hours to obtain the final product and require the use of very expensive equipment as ultracentrifuge. This method is well suited for the isolation of plasmid DNA from a large number of bacterial samples and in a very short time and low cost in laboratories where chemicals, expensive equipment and finance are limited factors in conducting molecular research. (authors). 11refs. 11refs

Plasmid and chromosome partitioning: surprises from phylogeny

DEFF Research Database (Denmark)

Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

2000-01-01

Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

Swimming of Microorganisms Viewed from String and Membrane Theories

OpenAIRE

Kawamura, Masako; Sugamoto, Akio; Nojiri, Shin'ichi

1993-01-01

Swimming of microorganisms is studied from a viewpoint of extended objects (strings and membranes) swimming in the incompressible f luid of low Reynolds number. The flagellated motion is analyzed in two dimensional fluid, by using the method developed in the ciliated motion with the Joukowski transformation. Discussion is given on the conserved charges and the algebra which are associated with the area (volume)- preserving diffeomorphisms giving the swimming motion of microorganisms. It is al...

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs

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Shuen Hon

2016-12-01

Full Text Available Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results. Keywords: Clostridium Thermocellum, Plasmid, adhE, Structural stability, Gene expression

Conjugal properties of the Sinorhizobium meliloti plasmid mobilome.

Science.gov (United States)

Pistorio, Mariano; Giusti, María A; Del Papa, María F; Draghi, Walter O; Lozano, Mauricio J; Tejerizo, Gonzalo Torres; Lagares, Antonio

2008-09-01

The biology and biochemistry of plasmid transfer in soil bacteria is currently under active investigation because of its central role in prokaryote adaptation and evolution. In this work, we examined the conjugal properties of the cryptic plasmids present in a collection of the N(2)-fixing legume-symbiont Sinorhizobium meliloti. The study was performed on 65 S. meliloti isolates recovered from 25 humic soils of Argentina, which were grouped into 22 plasmid-profile types [i.e. plasmid operational taxonomic units (OTUs)]. The cumulative Shannon index calculated for the observed plasmid profiles showed a clear saturation plateau, thus indicating an adequate representation of the S. meliloti plasmid-profile types in the isolates studied. The results show that isolates of nearly 14% of the plasmid OTUs hosted transmissible plasmids and that isolates of 29% of the plasmid OTUs were able to retransfer the previously characterized mobilizable-cryptic plasmid pSmeLPU88b to a third recipient strain. It is noteworthy that isolates belonging to 14% of the plasmid OTUs proved to be refractory to the entrance of the model plasmid pSmeLPU88b, suggesting either the presence of surface exclusion phenomena or the occurrence of restriction incompatibility with the incoming replicon. Incompatibility for replication between resident plasmids and plasmid pSmeLPU88b was observed in c. 20% of the OTUs. The results reported here reveal a widespread compatibility among the conjugal functions of the cryptic plasmids in S. meliloti, and this fact, together with the observed high proportion of existing donor genotypes, points to the extrachromosomal compartment of the species as being an extremely active plasmid mobilome.

Cloning of regions required for contact hemolysis and entry into LLC-MK2 cells from Shigella sonnei form I plasmid: virF is a positive regulator gene for these phenotypes.

OpenAIRE

Kato, J; Ito, K; Nakamura, A; Watanabe, H

1989-01-01

Two distinct regions required for both contact hemolysis and entry into LLC-MK2 cells were cloned into Escherichia coli from the Shigella sonnei form I plasmid, pSS120. The first region was cloned into an E. coli HB101 strain containing noninvasive Tn1 insertion mutants of the form I plasmid, and expression of ipa (invasion plasmid antigen) gene products was restored. The plasmid carrying the first region was then transformed into E. coli lacking the form I plasmid, and additional DNA fragmen...

Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

DEFF Research Database (Denmark)

Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela

2012-01-01

and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid...

Biolistics Transformation of Wheat

Science.gov (United States)

Sparks, Caroline A.; Jones, Huw D.

We present a complete, step-by-step guide to the production of transformed wheat plants using a particle bombardment device to deliver plasmid DNA into immature embryos and the regeneration of transgenic plants via somatic embryogenesis. Currently, this is the most commonly used method for transforming wheat and it offers some advantages. However, it will be interesting to see whether this position is challenged as facile methods are developed for delivering DNA by Agrobacterium tumefaciens or by the production of transformants via a germ-line process (see other chapters in this book).

Development of a novel rDNA based plasmid for enhanced cell surface display on Yarrowia lipolytica

CSIR Research Space (South Africa)

Bulani, S

2012-05-01

Full Text Available (YlCWP1). mCherry was used as a model protein to assess the efficiency of the constructed plasmid. Y. lipolytica transformants harbouring the expression cassettes showed a purple colour phenotype on selective YNB-casamino plates as compared to control...

Impact of co-carriage of IncA/C plasmids with additional plasmids on the transfer of antimicrobial resistance in Salmonella enterica isolates.

Science.gov (United States)

Han, Jing; Pendleton, Sean J; Deck, Joanna; Singh, Ruby; Gilbert, Jeffrey; Johnson, Timothy J; Sanad, Yasser M; Nayak, Rajesh; Foley, Steven L

2018-04-20

Antimicrobial resistance in Salmonella enterica is often plasmid encoded. A key resistance plasmid group is the incompatibility group (Inc) A/C plasmids that often carry multiple resistance determinants. Previous studies showed that IncA/C plasmids were often co-located with other plasmids. The current study was undertaken to evaluate the impact of plasmid co-carriage on antimicrobial resistance and plasmid transfer. A total of 1267 Salmonella isolates, representing multiple serotypes and sources were previously subjected to susceptibility testing and 251 isolates with resistance to at least 5 antimicrobial agents were identified for further study. Each isolate was subjected to PCR-based replicon typing, and those with IncA/C plasmids were selected for plasmid isolation, PCR-based mapping of IncA/C plasmid backbone genes, and conjugation assays to evaluate resistance plasmid transferability. Of the 87 identified IncA/C positive isolates, approximately 75% carried a plasmid with another identified replicon type, with the most common being I1 (39%), FIA, FIIA, FIB and HI2 (each 15%). PCR-based mapping indicated significant diversity in IncA/C backbone content, especially in regions encoding transfer-associated and hypothetical proteins. Conjugation experiments showed that nearly 68% of the isolates transferred resistance plasmids, with 90% containing additional identified plasmids or larger (>50 kb) non-typeable plasmids. The majority of IncA/C-positive strains were able to conjugally transfer antimicrobial resistance to the recipient, encoded by IncA/C and/or co-carried plasmids. These findings highlight the importance of co-located plasmids for resistance dissemination either by directly transferring resistance genes or by potentially providing the needed conjugation machinery for IncA/C plasmid transfer. Copyright © 2018. Published by Elsevier B.V.

Characterization of IncN plasmids carrying blaCTX-M-1 and qnr genes in Escherichia coli and Salmonella from animals, the environment and humans

DEFF Research Database (Denmark)

Dolejska, Monika; Villa, Laura; Hasman, Henrik

2013-01-01

were compared using restriction fragment length polymorphism (RFLP), plasmid multilocus sequence typing (pMLST) and hybridization with repN, qnrS1, qnrB19 or blaCTX-M-1 probes. Plasmids pKT58A and pHHA45 were sequenced using the 454-Genome Sequencer FLX platform on a library constructed from plasmid...... DNA purified from the respective E. coli transformants.Results Three types of IncN plasmids carrying blaCTX-M-1, qnrS1 and qnrB19 genes were identified in strains isolated from the Czech Republic, Poland, Slovakia, Denmark, Italy and the Netherlands, corresponding to pMLST sequence type (ST) 1, ST3...

Characterization of new plasmids from methylotrophic bacteria.

Science.gov (United States)

Brenner, V; Holubová, I; Benada, O; Hubácek, J

1991-07-01

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

Transfer and persistence of a multi-drug resistance plasmid in situ of the infant gut microbiota in the absence of antibiotic treatment

DEFF Research Database (Denmark)

Gumpert, Heidi; Kubicek-Sutherland, Jessica Z.; Porse, Andreas

2017-01-01

lineage was maintained for months, demonstrating that antibiotic resistance genes can disseminate and persist in the gut microbiome; even in absence of antibiotic selection. Furthermore, through in vivo competition assays, we suggest that the resistant transconjugant can persist through a fitness......The microbial ecosystem residing in the human gut is believed to play an important role in horizontal exchange of virulence and antibiotic resistance genes that threatens human health. While the diversity of gut-microorganisms and their genetic content has been studied extensively, high...... infections, as well as the loss and acquisition of plasmids in these lineages during their colonization of the human gut. In particular, we captured the exchange of multidrug resistance genes, and identified a clinically relevant conjugative plasmid mediating the transfer. This resistant transconjugant...

Explanatory chapter: how plasmid preparation kits work.

Science.gov (United States)

Koontz, Laura

2013-01-01

To isolate plasmid DNA from bacteria using a commercial plasmid miniprep kit (if interested, compare this protocol with Isolation of plasmid DNA from bacteria). Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

Science.gov (United States)

Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

2017-02-01

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including bla CMY and bla NDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a bla NDM-1 -positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of bla NDM -positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this bla NDM -containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

The detection and sequencing of a broad-host-range conjugative IncP-1β plasmid in an epidemic strain of Mycobacterium abscessus subsp. bolletii.

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Sylvia Cardoso Leão

Full Text Available BACKGROUND: An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004-2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE patterns differentiated by the presence of a ∼50 kb band. The nature of this band was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,267-bp [corrected] circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1β and is highly related to BRA100, pJP4, pAKD33 and pB10. The presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. The plasmid was visualized as a ∼50-kb band using PFGE and Southern blot hybridization in 12 isolates. The remaining 25 isolates did not exhibit any evidence of this plasmid. The plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc(2155 could not be demonstrated. CONCLUSIONS/SIGNIFICANCE: The occurrence of a broad-host-range IncP-1β plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease.

The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids.

Science.gov (United States)

Poulin-Laprade, Dominic; Carraro, Nicolas; Burrus, Vincent

2015-01-01

Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.

The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids.

Directory of Open Access Journals (Sweden)

Dominic ePoulin-Laprade

2015-08-01

Full Text Available Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs of the SXT/R391 family (SRIs and IncA/C conjugative plasmids (ACPs are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e. SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to Invasive Chlamydia trachomatis Biovars

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Michael John Patton

2018-01-01

Full Text Available Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars—lymphogranuloma venereum (LGV and trachoma—which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar, a L2 serovar (LGV biovar, and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.

Plasmid-mediated UV-protection in Streptococcus lactis

Energy Technology Data Exchange (ETDEWEB)

Chopin, M.C.; Rouault, A. (Institut National de la Recherche Agronomique, Rennes (France). Lab. de Recherches de Technologie Laitiere); Moillo-Batt, A. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital de Pontchaillon, 35 - Rennes (France))

1985-02-01

Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by co-transfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci.

Plasmid-mediated UV-protection in Streptococcus lactis

International Nuclear Information System (INIS)

Chopin, M.-C.; Rouault, A.

1985-01-01

Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by cotransfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci. (orig.)

Directly Transforming PCR-Amplified DNA Fragments into Plant Cells Is a Versatile System That Facilitates the Transient Expression Assay

Science.gov (United States)

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan

2013-01-01

A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.

Directory of Open Access Journals (Sweden)

Yuming Lu

Full Text Available A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments based transient expression system (PCR-TES for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells.

Persistence Mechanisms of Conjugative Plasmids

DEFF Research Database (Denmark)

Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

2009-01-01

Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting the suc...

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation

Energy Technology Data Exchange (ETDEWEB)

McIntyre, D.A.; Harlander, S.K. (Univ. of Minnesota, St. Paul (USA))

1989-03-01

The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit. Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 {mu}s to 1 s). Transformation efficiencies of 10{sup 3} transformants per {mu}g of DNA were obtained when dense suspensions (final concentration, 5 {times} 10{sup 10} CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30{degree}C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs).

Science.gov (United States)

Hon, Shuen; Lanahan, Anthony A; Tian, Liang; Giannone, Richard J; Hettich, Robert L; Olson, Daniel G; Lynd, Lee R

2016-12-01

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE . To explore the effects of overexpressing wild-type, mutant, and exogenous adhE s, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum . As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

OpenAIRE

Laine, M J; Nakhei, H; Dreier, J; Lehtilä, K; Meletzus, D; Eichenlaub, R; Metzler, M C

1996-01-01

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, a...

Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

NARCIS (Netherlands)

Romero, Diego; Perez-Garcia, Alejandro; Veening, Jan-Willem; de Vicente, Antonio; Kuipers, Oscar P.; de, Vicente A.

A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool

Plasmid-mediated mineralization of 4-chlorobiphenyl

International Nuclear Information System (INIS)

Shields, M.S.; Hooper, S.W.; Sayler, G.S.

1985-01-01

Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 x 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14 C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB

Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia.

Science.gov (United States)

Yeow, Tee Cian; Wong, Won Fen; Sabet, Negar Shafiei; Sulaiman, Sofiah; Shahhosseini, Fatemeh; Tan, Grace Min Yi; Movahed, Elaheh; Looi, Chung Yeng; Shankar, Esaki M; Gupta, Rishien; Arulanandam, Bernard P; Hassan, Jamiyah; Abu Bakar, Sazaly

2016-03-18

The 7.5 kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis-infected patients. A total of 180 female patients of child bearing age (mean 30.9 years old, IQR:27-35) with gynecological complications and subfertility issues, who visited Obstetrics and Gynecology clinics in Kuala Lumpur, Malaysia were recruited for the study. Prevalence of genital chlamydial infection among these patients was alarmingly high at 51.1% (92/180). Of the 92 chlamydia-infected patients, 93.5% (86/92) were infected with plasmid-bearing (+) C. trachomatis while the remaining 6.5% (6/92) were caused by the plasmid-free (-) variant. Our data showed that genital C. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract (mucopurulent cervicitis or endometriosis), irregular menstrual cycles and polycystic ovarian syndrome (PCOS). However, no statistical significance was detected among patients with plasmid (+) versus plasmid (-) C. trachomatis infection. Interestingly, plasmid (+) C. trachomatis was detected in all patients with PCOS, and the plasmid copy numbers were significantly higher among PCOS patients, relative to non-PCOS patients. Our findings show a high incidence of C. trachomatis infection among women with infertility or gynecological problems in Malaysia. However, due to the low number of plasmid (-) C. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort.

Genetic characterization of plasmid pRJ5 of Staphylococcus aureus compared to plasmid pE194

International Nuclear Information System (INIS)

Oliveira, S.S. de; Freire Bastos, M.C. de

1993-01-01

The pRJ5, a naturally occurring constitutive macrolide, lincosamide and streptogramin B (MLS) resistance plasmid of Staphylococcus aureus, was compared to pE194, a plasmid that confers the inducible phenotype. pRJ5 was stable in all strains of S. aureus tested, even under growth at 43 O C, which distinguished it from pE194 which was shown to be thermo-sensitive for replication. pRJ5, like pE194, was highly unstable in Bacillus subtilis when the cells were grown in nonselective conditions. Multimeric forms of pRJ5 DNA were detected in the few cells of B. subtilis that retained this plasmid. pE194 was transduced by phages φ 11 and φ 443 at frequencies 400 and 20-fold higher, respectively, than pRJ5. Both plasmids were co-transduced with the plasmid pRJ4. pRJ5 was shown to be compatible with pE194. Therefore they belong to distinct Inc groups. Hybridization studies revealed that pRJ5 shares a 1.35 kb region of homology to pE194, which is limited to the erm gene, conferring MLS resistance. (author)

Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

International Nuclear Information System (INIS)

Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

1984-01-01

Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. [ 3 H]Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes

Escherichia coli can be transformed by a liposome-mediated lipofection method.

Science.gov (United States)

Kawata, Yoshikazu; Yano, Shin-ichi; Kojima, Hiroyuki

2003-05-01

Transformation of Escherichia coli is a basic technique for genetic engineering. We used a liposome-mediated lipofection method to transform electrocompetent E. coli cells which has little natural competence of foreign DNA without electroporation treatment, and got transformants with simple and quick treatment by a plasmid or a transposon and transposase complex.

Plasmid fermentation process for DNA immunization applications.

Science.gov (United States)

Carnes, Aaron E; Williams, James A

2014-01-01

Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.

Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human.

Science.gov (United States)

Wu, Shuyu; Dalsgaard, Anders; Hammerum, Anette M; Porsbo, Lone J; Jensen, Lars B

2010-07-30

Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present. A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three sul genes. Sul genes were distributed widely in E. coli isolated

Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs, pig carcasses and human

Directory of Open Access Journals (Sweden)

Hammerum Anette M

2010-07-01

Full Text Available Abstract Background Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3 in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes. Methods A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present. Results A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%, while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively. Multireplicons were found associated with all three sul genes

Antimicrobial susceptibility pattern and plasmid-mediated ...

African Journals Online (AJOL)

negative Staphylococci (CoNS) were isolated from clinical samples and isolates subjected to antibiotic susceptibility testing, plasmid curing and plasmid DNA isolation. Result: The highest percentages isolates were recovered from urine samples and ...

Organotins and microorganisms. Yuki suzu kagobutsu to biseibutsu

Energy Technology Data Exchange (ETDEWEB)

Shinoda, S.; Onogi, H. (Okayama Univ., Okayama (Japan). Faculty of Pharmaceutical Sciences)

1992-08-10

Tributyltin (TBT) and triphenyltin have higher toxicity than other organotin compounds and are used as biocides, but in recent years the pollution of water and aquatic organisms has become a social problem. This paper describes the interaction between organotin compounds and microorganisms, centering on the decomposition of TBT. Part of microalgae whose activity is promoted by light or nutritive salts within an aquatic environment play an important role in TBT decomposition. Diatoms, and dinoflagellataes are mentioned as the examples. Moreover, an example in which microorganisms promote the transformation of inorganotin compounds to dimethyltin or trimethyltin is given. However, it is pointed out in this paper that the action of microorganisms relates greatly to the continuance of existence of organotins in environments, but the degradation efficiency is considered to be very low and prevention against pollution is of primary importance. 32 refs., 2 figs., 3 tabs.

Methylation by a unique α-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725.

Directory of Open Access Journals (Sweden)

Daehwan Chung

Full Text Available Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.

Agrobacterium tumefaciens-Mediated Transformation

DEFF Research Database (Denmark)

Frandsen, Rasmus John Normand

2015-01-01

The use of Agrobacterium tumefaciens-mediated transformation for achieving genetic transformation of fungi has steadily increased over the last decade, and has proven to be almost universally applicable technique once suitable selection markers have been developed. In recent years the major...... technical advances has been made within the initial steps of the process, more specifically the efficient construction of plasmids for performing targeted genome modifications. This chapter provides a generic protocol for performing genetic transformation of ascomycetes via A. tumefaciens......-mediated transformation (AMT) and guidelines for optimizing the AMT process with new fungal species. The chapter also includes a highly efficient vector construction system based on Uracil Specific Excisions Reagent (USER) cloning and specific PCR generated building blocks, which can be combined ad hoc to create complex...

Finding a facile way for the bacterial DNA transformation by biosynthesized gold nanoparticles.

Science.gov (United States)

Kumari, Madhuree; Pandey, Shipra; Mishra, Aradhana; Nautiyal, Chandra Shekhar

2017-07-03

The major problem encountered during genetic manipulation of bacteria is the inability to get transformed because of their natural non-competency. In this study, to overcome this problem, a cost-effective method was developed by combining the properties of gold nanoparticles (GNPs) and the Yoshida effect. Various parameters, including GNP:plasmid ratio, pH and time, were optimized for stability of the GNP-plasmid conjugate. With non-competent Gram-negative cells, the efficiency ranged between 0.1 and 0.45 × 104 transformants μg-1, while the range was (0.02-0.2) × 104 transformants μg-1 with Gram-positive bacteria. GNPs can serve efficiently as a vehicle for better transformation in bacteria. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

Science.gov (United States)

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

Directory of Open Access Journals (Sweden)

Mark Eppinger

Full Text Available Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

Tomato protoplast DNA transformation : physical linkage and recombination of exogenous DNA sequences

NARCIS (Netherlands)

Jongsma, Maarten; Koornneef, Maarten; Zabel, Pim; Hille, Jacques

1987-01-01

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There

Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

NARCIS (Netherlands)

Hille, Jacques; Goldbach, Rob

1986-01-01

A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

Microorganism immobilization

Science.gov (United States)

Compere, Alicia L.; Griffith, William L.

1981-01-01

Live metabolically active microorganisms are immobilized on a solid support by contacting particles of aggregate material with a water dispersible polyelectrolyte such as gelatin, crosslinking the polyelectrolyte by reacting it with a crosslinking agent such as glutaraldehyde to provide a crosslinked coating on the particles of aggregate material, contacting the coated particles with live microorganisms and incubating the microorganisms in contact with the crosslinked coating to provide a coating of metabolically active microorganisms. The immobilized microorganisms have continued growth and reproduction functions.

Transposition of a Ds element from a plasmid into the plant genome in Nicotiana plumbaginifolia protoplast-derived cells.

Science.gov (United States)

Houba-Hérin, N; Domin, M; Pédron, J

1994-07-01

Nicotiana plumbaginifolia haploid protoplasts were co-transformed with two plasmids, one with a NPT-II/Ds element and one with a gene encoding an amino-terminal truncated Ac transposase. It is shown that Ds can efficiently transpose from extrachromosomal DNA to N. plumbaginifolia chromosomes when the Ac transposase gene is present in trans. Ds has been shown to have transposed into the plant genome in a limited number of copies (1.9 copies per genome), for 21/32 transgenic lines tested. The flanking sequences present in the original plasmid are missing in these 21 plants. In only two of 21 plants was part of the transposase construct integrated. By segregation analysis of transgenic progeny, Ds was shown to be present in the heterozygous state in 10 lines even though haploid protoplasts had been originally transformed. This observation could indicate that integration occurred after or during DNA replication that leads to protoplast diploidization.

Multilocus sequence typing of IncN plasmids

DEFF Research Database (Denmark)

García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

2011-01-01

that spread and persistence of this particular IncN-carrying blaVIM-1 lineage in Greece. CONCLUSIONS: This study proposes the use of pMLST as a suitable and rapid method for identification of IncN epidemic plasmid lineages. The recent spread of blaCTX-M-1 among humans and animals seems to be associated......OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of blaVIM-1 in humans and blaCTX-M-1 in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid...... in different countries from both animals and humans belonged to ST1, suggesting dissemination of an epidemic plasmid through the food chain. Fifteen of 17 plasmids carrying blaVIM-1 from Klebsiella pneumoniae and Escherichia coli, isolated during a 5year period in Greece were assigned to ST10, suggesting...

Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

Science.gov (United States)

Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

2017-07-25

Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The β-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of

Cloning of the DNA repair gene, uvsF, by transformation of Aspergillus nidulans.

Science.gov (United States)

Oza, K; Käfer, E

1990-06-01

As a first step in the cloning of the DNA repair gene uvsF of Aspergillus nidulans, uvsF pyrG double mutant strains were transformed with a genomic library which carried the complementing Neurospora pyr-4 gene in the vector. Rare pyr+ uvs+ cotransformants were obtained on media lacking pyrimidines, overlayed with MMS (methyl-methane sulfonate) to which uvsF is hypersensitive. Among MMS-resistant transformants, Southerns revealed two types which showed single bands of different sizes when BglII-digested genomic DNA was probed with the vector. Both types produced uvsF- recombinants without vector sequences in homozygous crosses, but only those with the larger band also produced haploid uvs+ progeny. Using BglII-digested genomic DNA to transform Escherichia coli, plasmids of the corresponding two sizes could be rescued. Their inserts had a short internal region in common, giving evidence of rearrangement(s). In secondary transformation of uvsF mutants, only the plasmids with the larger insert showed complementation and these were used to screen Aspergillus libraries. Three types of genomic and two overlapping cDNA clones were identified. The cDNAs hybridized not only to each other, but also to the common region of the rescued plasmids. Therefore, cDNA subclones were used to map the putative uvsF sequences to a short segment in one genomic clone. In Northerns, the complementing large plasmid hybridized to three mRNAs, while the cDNA subclone identified one of these as the probable uvsF message.

Characterization of pLAC1, a cryptic plasmid isolated from Lactobacillus acidipiscis and comparative analysis with its related plasmids.

Science.gov (United States)

Asteri, Ioanna-Areti; Papadimitriou, Konstantinos; Boutou, Effrossyni; Anastasiou, Rania; Pot, Bruno; Vorgias, Constantinos E; Tsakalidou, Effie

2010-07-15

The pLAC1 plasmid of Lactobacillus acidipiscis ACA-DC 1533, a strain isolated from traditional Kopanisti cheese, was characterised. Nucleotide sequence analysis revealed a circular molecule of 3478bp with a G+C content of 37.2%. Ab initio annotation indicated four putative open reading frames (orfs). orf1 and orf4 were found to encode a replication initiation protein (Rep) and a mobilization protein (Mob), respectively. The deduced products of orf2 and orf3 revealed no significant homology to other known proteins. However, in silico examination of the plasmid sequence supported the existence of a novel operon that includes rep, orf2 and orf3 in pLAC1 and that this operon is highly conserved also in plasmids pLB925A02, pSMA23, pLC88 and pC7. RT-PCR experiments allowed us to verify that these three genes are co-transcribed as a single polycistronic mRNA species. Furthermore, phylogenetic analysis of pLAC1 Rep and Mob proteins demonstrated that they may have derived from different plasmid origins, suggesting that pLAC1 is a product of a modular evolution process. Comparative analysis of full length nucleotide sequences of pLAC1 and related Lactobacillus plasmids showed that pLAC1 shares a very similar replication backbone with pLB925A02, pSMA23 and pLC88. In contrast, mob of pLAC1 was almost identical with the respective gene of plasmids pLAB1000, pLB4 and pPB1. These findings lead to the conclusion that pLAC1 acquired mob probably via an ancestral recombination event. Our overall work highlights the importance of characterizing plasmids deriving from non-starter 'wild' isolates in order to better appreciate plasmid divergence and evolution of lactic acid bacteria. 2010 Elsevier B.V. All rights reserved.

Agrobacterium mediated transformation of Tunisian Cucumis melo ...

African Journals Online (AJOL)

Transgenic Cucumis melo cv. Maazoun containing the neomycin phosphotransferase II (NPT II) chimeric gene conferring resistance to kanamycin were obtained from cotyledons explants inoculated with Agrobacterium tumefaciens (GV3101) that contained the binary vector plasmid pADI. Transformed shoots were obtained ...

Incidence of plasmid-linked antibiotic-heavy metal resistant enterics in water-sediment from agricultural and harbor sites

Energy Technology Data Exchange (ETDEWEB)

Mietz, J.A.; Sjorgren, R.E.

1983-01-01

This study assesses the impact of antibiotics used in clinical and veterinary practices on the incidence of antibiotic-heavy metal resistant enterics in fresh water and sediment from agricultural and harbor sample sites. A total of 848 bacterial strains of the family Enterobacteriaceae was isolated from agricultural and lake harbor samples. These were examined for anitbiotic-heavy metal resistance. A select smaller number of these isolates were also examined for the presence of plasmids and ability to transfer antibiotic resistance via conjugation or transformation. More than 85% of the 848 isolates from all four sites were resistant to Pb, Zn, and Co while 5.6% to 16% were resistant to Te and 2.4% to 5.7% to Hg. Of the total isolates tested, 87% were resistant to six or more antibiotics and 74% were also simultaneously resistant to Co, Zn, and Pb. Testing the resistance of the water isolates to antibiotics used solely in animal husbandry-veterinary medicine indicated that 55.6% of the agricultural isolates possessed resistance to these antibiotics while only 31.9% of the isolates from harbor water showed resistance to the same antibiotics. Of 41 ampicillin resistant isolates examined, 16 (39%) were capable of transferring antibiotic-heavy resistance markers via conjugation. From this same group, plasmid DNA preparations were made. Of these latter preparations, 67% transformed recipient E. coli cells while 58% possessed discernible, often multiple plasmids when examined by gel electrophoresis.

Ecological and genetic determinants of plasmid distribution in Escherichia coli.

Science.gov (United States)

Medaney, Frances; Ellis, Richard J; Raymond, Ben

2016-11-01

Bacterial plasmids are important carriers of virulence and antibiotic resistance genes. Nevertheless, little is known of the determinants of plasmid distribution in bacterial populations. Here the factors affecting the diversity and distribution of the large plasmids of Escherichia coli were explored in cattle grazing on semi-natural grassland, a set of populations with low frequencies of antibiotic resistance genes. Critically, the population genetic structure of bacterial hosts was chararacterized. This revealed structured E. coli populations with high diversity between sites and individuals but low diversity within cattle hosts. Plasmid profiles, however, varied considerably within the same E. coli genotype. Both ecological and genetic factors affected plasmid distribution: plasmid profiles were affected by site, E. coli diversity, E. coli genotype and the presence of other large plasmids. Notably 3/26 E. coli serotypes accounted for half the observed plasmid-free isolates indicating that within species variation can substantially affect carriage of the major conjugative plasmids. The observed population structure suggest that most of the opportunities for within species plasmid transfer occur between different individuals of the same genotype and support recent experimental work indicating that plasmid-host coevolution, and epistatic interactions on fitness costs are likely to be important in determining occupancy. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis.

Science.gov (United States)

Walz, M; Kück, U

1995-12-01

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. macrospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora.

Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system.

Science.gov (United States)

Yamamoto, Shinji; Sakai, Ayako; Agustina, Vita; Moriguchi, Kazuki; Suzuki, Katsunori

2018-02-01

Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new "Ti/Ri eviction plasmids," each of which carries a repABC from one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacB and in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids into Agrobacterium cells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacB backbone. Furthermore, the highly stable cryptic plasmid pAtC58 in A. tumefaciens C58 was effectively evicted by the introduction of a pBBR1 vector containing the repABC of pAtC58. These results indicate that the set of pBBR1-repABC plasmids is a powerful tool for the removal of stable rhizobial plasmids.

The characteristics of micrococcus (deinococcus) radiodurans sark plasmids

International Nuclear Information System (INIS)

Sjarief, Sri Hariani; Kikuchi, Masahiro; Watanabe, Hiroshi.

1994-01-01

The characterization of micrococcus (deinococcus) radiodurans sark plasmids. This bacterium has been classified as a new genus deinococcus radiodurans which is resistant to gamma-rays. It can repair itself completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 KGy. To reveal the repair mechanism, several investigations had been done to develop a cloning vector available for the genetic analysis. For this purpose D. radiodurans Sark are to be prepared as a vector by studying the characteristics of its plasmid. Plasmids were isolated by electrophoresis using 0.6% low-melting-temperature agarose in TAE and run for 5.5 hours, followed by the identification. An antibiotic marker was also carried out in this experiment to identify its location in the genetic materials of the cell, beside making a restriction map of the plasmid. Results have shown that D. radiodurans Sark has 4 plasmids (P1, P2, P3, and P4) and the refampicin resistant genes were not found in the plasmid. (authors). 14 refs; 4 figs

The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family.

Science.gov (United States)

Li, Xiaobin; Top, Eva M; Wang, Yafei; Brown, Celeste J; Yao, Fei; Yang, Shan; Jiang, Yong; Li, Hui

2014-01-01

A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent "essential" plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.

The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family

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Xiaobin eLi

2015-01-01

Full Text Available A self-transmissible broad-host-range (BHR plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs, 28 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102 and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331, based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T and pTer331, suggesting these hypothetical orfs may represent ‘‘essential’’ plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.

[Evolution of pathogenic micro-organisms as a challenge for medicine].

Science.gov (United States)

Vaara, Martti

2009-01-01

Successful parasitic micro-organisms are able to adapt to the circumstances of the host's organ system, and it is usually not expedient for them to kill their host. Under selection pressure, the evolution of micro-organisms is vastly quicker that that of man. The selection pressure brought about by rapid ecological changes and alterations associated with human action provides for the development of new, dangerous pathogens and transformation of familiar pathogens to become more dangerous. Progress in molecular biology has thus far not yielded as many new tools for the treatment of infectious diseases as the hopes were in the early 2000's.

Transformation of Leuconostoc carnosum 4010 and evidence for natural competence of the organism

DEFF Research Database (Denmark)

Helmark, Søren; Hansen, Michael Edberg; Jelle, B.

2004-01-01

Plasmid transformation in Leuconostoc carnosum 4010 was analyzed. A successful transformation protocol for L. carnosum was established by modifying an existing protocol for Lactococcus lactis. Several parameters, including the number of generations that the cells had grown at the time of harvest...

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions

OpenAIRE

Burbank, Lindsey P.; Van Horn, Christopher R.

2017-01-01

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa, but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb, putatively encoding a conjugative type IV secretion system, are foun...

Plasmids foster diversification and adaptation of bacterial populations in soil.

Science.gov (United States)

Heuer, Holger; Smalla, Kornelia

2012-11-01

It is increasingly being recognized that the transfer of conjugative plasmids across species boundaries plays a vital role in the adaptability of bacterial populations in soil. There are specific driving forces and constraints of plasmid transfer within bacterial communities in soils. Plasmid-mediated genetic variation allows bacteria to respond rapidly with adaptive responses to challenges such as irregular antibiotic or metal concentrations, or opportunities such as the utilization of xenobiotic compounds. Cultivation-independent detection and capture of plasmids from soil bacteria, and complete sequencing have provided new insights into the role and ecology of plasmids. Broad host range plasmids such as those belonging to IncP-1 transfer a wealth of accessory functions which are carried by similar plasmid backbones. Plasmids with a narrower host range can be more specifically adapted to particular species and often transfer genes which complement chromosomally encoded functions. Plasmids seem to be an ancient and successful strategy to ensure survival of a soil population in spatial and temporal heterogeneous conditions with various environmental stresses or opportunities that occur irregularly or as a novel challenge in soil. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Plasmid profilling and similarities in identities of probable microbes isolated from crude oil contaminated agricultural soil

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Toochukwu Ekwutosi OGBULIE

2013-05-01

Full Text Available Plasmid analysis of bacteria isolated from agricultural soil experimentally contaminated with crude oil was carried out and the resultant bands’ depicting the different molecular sizes of the plasmid DNA molecules per isolate was obtained. There was no visible band observed for Klebsiella indicating that the organism lack plasmid DNA that confers degradative ability to it, possibly the gene could be borne on the chromosomal DNA which enabled its persistence in the polluted soil. Molecular characterization was undertaken to confirm the identities of the possible microorganisms that may be present in crude oil-contaminated soil. The result of the DNA extracted and amplified in a PCR using EcoRI and EcoRV restriction enzymes for cutting the DNA of the bacterial cells indicated no visible band for cuts made with EcoRV restriction enzyme showing that the enzyme is not specific for bacterial DNA of isolates in the samples, hence there was no amplification. By contrast though, visible bands of amplicons were observed using EcoRI restriction enzymes. The resultant visible bands of microbial profile obtained using the universal RAPD primer with nucleotide sequence of 5’—CTC AAA GCA TCT AGG TCC A---3’ showed that only Pseudomonas fluorescens and Bacillus mycoides had visible bands at identical position on the gel indicating that both species possibly had identical sequence or genes of negligible differences coding for degradation of hydrocarbons as shown by similar values in molecular weight and positions in the gel electrophoresis field.

Genetic transformation of olive somatic embryos through ...

African Journals Online (AJOL)

Administrator

2011-06-20

Jun 20, 2011 ... 2Department of Biochemistry, National Center of Genetic Engineering and Biotechnology, Tehran, Iran. Accepted 9 March, 2011. Transformed olive plants were regenerated from inoculated somatic embryos with Agrobacterium tumefacience strain GV3101, which carries the plasmid pBI-P5CS containing ...

Generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation

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Wang Genping

2016-09-01

Full Text Available Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154 and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154 were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of ‘clean’ GM wheat containing only the foreign genes of agronomic importance.

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Science.gov (United States)

Wang, Gen-Ping; Yu, Xiu-Dao; Sun, Yong-Wei; Jones, Huw D; Xia, Lan-Qin

2016-01-01

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium -mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

Plasmid DNA is released from nanosized acicular material surface by low molecular weight oligonucleotides: exogenous plasmid acquisition mechanism for penetration intermediates based on the Yoshida effect.

Science.gov (United States)

Yoshida, N; Ide, K

2008-10-01

When a colloidal solution consisting of nanosized acicular material and bacterial cells is stimulated with sliding friction at the interface between the hydrogel and interface-forming material where the frictional coefficient increases rapidly, the nanosized acicular material accompanying the bacterial cells forms a penetration intermediate. This effect is known as the Yoshida effect in honor of its discoverer. Through the Yoshida effect, a novel property in which penetration intermediates incorporate exogenous plasmid DNA has been identified. This report proposes a possible mechanism for exogenous plasmid acquisition by penetration intermediates in the Yoshida effect. Escherichia coli cells, pUC18, and chrysotile were used as recipient cells, plasmid DNA, and nanosized acicular material, respectively. Even when repeatedly washing the mixture consisting of pUC18 and chrysotile, transformation efficiency by pUC18 was stable. Accordingly, pUC18 adsorbed onto chrysotile was introduced into recipient E. coli cells. At saturation, the amount of pUC18 adsorbed onto chrysotile was 0.8-1.2 microg/mg. To investigate whether pUC18 adsorbed on chrysotile is replicated by polymerase, polymerase chain reaction (PCR) was carried out with the chrysotile. Amplification of the beta-lactamase gene coded in pUC18, which was adsorbed onto chrysotile, was strongly inhibited. This suggests that DNA adsorbed onto chrysotile is not replicated in vivo. When we searched for substances to release pUC18 adsorbed onto chrysotile, we found that a 300-bp single- or double-stranded segment of DNA releases pUC18 from chrysotile. Competitive adsorption onto chrysotile between double-stranded DNA and pUC18 was then examined through the Yoshida effect. The 310- and 603-bp double-stranded nucleotides caused 50% competitive inhibition at the same molar ratio with pUC18. Hence, the adsorbed region of pUC18 is about 300 bp in length. As the culture period for recipient cells increases, transformation

Strand breaks and lethal damage in plasmid DNA subjected to 60CO-γirradiation

International Nuclear Information System (INIS)

Klimczak, U.

1992-01-01

Experiments with calf thymus DNA subjected to extracellular irradiation yield information on the role of direct and indirect effects in single-strand breakage, if this is evaluated with reference to the scavenger activity in respect of OH radicals. The role of the two processes in the occurrence of double-stand breaks and further damage leading to cell decay has so far remained largely obscure. It was the aim of the study described here to contribute to research in this field by performing in vitro experiments on biologically active DNA. For this purpose, DNA from pBR322 plasmids was irradiated in the presence of OH-radical scavengers. The number of single-strand and double-strand breaks was determined on the basis of the system's ability to eliminate OH radicals. In order to asses the influence of irradiation processes on the biological activity of DNA, investigations were carried out in E. coli for transformations caused by irradiated plasmid DNA. The results were interpreted in the light of theories about inhomogenous reaction kinetics put forward by Mark et al. (1989). It was finally discussed, which of the gamma-irradiation injuries occurring in DNA was to be held responsible for the inactivation of plasmid DNA and which enzymatic processes were additionally at work here. (orig./MG) [de

Humus application produced by organic waste transformation in growing lands

International Nuclear Information System (INIS)

Gutierrez Martinez, M. G.; Morales Osorio, A.; Gutierrez Martinez, R.; Marquez Monsalvo, J. J.; Reyes Reyes, G.

2009-01-01

Every year in Mexico are produced more tons of garbage (19 874 259 tons) than tons of corn (18 309 000 tons). Fifty percent of domestic garbage is constituted by organic remainders. In december 2005 began de project Biotransformation of Organic Remainders in Humus for its Application in growing Lands with the purpose to prove the methods of: 1) Natural outdoors transformation, 2) Accelerated fermentation with thermophilic and mesophilic microorganisms, and 3) Transformation with microorganisms such as californian red worm, Eisenia andrei, to transform efficiently fruits and vegetables remainders. (Author)

Mutation in ESBL Plasmid from Escherichia coli O104:H4 Leads Autoagglutination and Enhanced Plasmid Dissemination

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Mickaël Poidevin

2018-02-01

Full Text Available Conjugative plasmids are one of the main driving force of wide-spreading of multidrug resistance (MDR bacteria. They are self-transmittable via conjugation as carrying the required set of genes and cis-acting DNA locus for direct cell-to-cell transfer. IncI incompatibility plasmids are nowadays often associated with extended-spectrum beta-lactamases producing Enterobacteria in clinic and environment. pESBL-EA11 was isolated from Escherichia coli O104:H4 outbreak strain in Germany in 2011. During the previous study identifying transfer genes of pESBL-EA11, it was shown that transposon insertion at certain DNA region of the plasmid, referred to as Hft, resulted in great enhancement of transfer ability. This suggested that genetic modifications can enhance dissemination of MDR plasmids. Such ‘superspreader’ mutations have attracted little attention so far despite their high potential to worsen MDR spreading. Present study aimed to gain our understanding on regulatory elements that involved pESBL transfer. While previous studies of IncI plasmids indicated that immediate downstream gene of Hft, traA, is not essential for conjugative transfer, here we showed that overexpression of TraA in host cell elevated transfer rate of pESBL-EA11. Transposon insertion or certain nucleotide substitutions in Hft led strong TraA overexpression which resulted in activation of essential regulator TraB and likely overexpression of conjugative pili. Atmospheric Scanning Electron Microscopy observation suggested that IncI pili are distinct from other types of conjugative pili (such as long filamentous F-type pili and rather expressed throughout the cell surface. High transfer efficiency in the mutant pESBL-EA11 was involved with hyperpiliation which facilitates cell-to-cell adhesion, including autoagglutination. The capability of plasmids to evolve to highly transmissible mutant is alarming, particularly it might also have adverse effect on host pathogenicity.

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

International Nuclear Information System (INIS)

Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I.

1990-01-01

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores

Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA

International Nuclear Information System (INIS)

Paramio, J.M.; Bauluz, C.; Vidania, R. de

1986-01-01

Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs

Plasmid-Mediated Antimicrobial Resistance in Staphylococci and Other Firmicutes.

Science.gov (United States)

Schwarz, Stefan; Shen, Jianzhong; Wendlandt, Sarah; Fessler, Andrea T; Wang, Yang; Kadlec, Kristina; Wu, Cong-Ming

2014-12-01

In staphylococci and other Firmicutes, resistance to numerous classes of antimicrobial agents, which are commonly used in human and veterinary medicine, is mediated by genes that are associated with mobile genetic elements. The gene products of some of these antimicrobial resistance genes confer resistance to only specific members of a certain class of antimicrobial agents, whereas others confer resistance to the entire class or even to members of different classes of antimicrobial agents. The resistance mechanisms specified by the resistance genes fall into any of three major categories: active efflux, enzymatic inactivation, and modification/replacement/protection of the target sites of the antimicrobial agents. Among the mobile genetic elements that carry such resistance genes, plasmids play an important role as carriers of primarily plasmid-borne resistance genes, but also as vectors for nonconjugative and conjugative transposons that harbor resistance genes. Plasmids can be exchanged by horizontal gene transfer between members of the same species but also between bacteria belonging to different species and genera. Plasmids are highly flexible elements, and various mechanisms exist by which plasmids can recombine, form cointegrates, or become integrated in part or in toto into the chromosomal DNA or into other plasmids. As such, plasmids play a key role in the dissemination of antimicrobial resistance genes within the gene pool to which staphylococci and other Firmicutes have access. This chapter is intended to provide an overview of the current knowledge of plasmid-mediated antimicrobial resistance in staphylococci and other Firmicutes.

Genetic transformation of midgut bacteria from the red imported fire ant (Solenopsis invicta).

Science.gov (United States)

Medina, Freder; Li, Haiwen; Vinson, S Bradleigh; Coates, Craig J

2009-05-01

In our previous study we isolated 10 bacterial species from fourth-instar larval midguts of the red imported fire ant, Solenopsis invicta. Here we report the genetic transformation and reintroduction of three species (Kluyvera cryocrescens, Serratia marcescens, and isolate 38) into the fire ant host. All three species were transformed with the plasmid vector, pZeoDsRed. High expression levels of DsRed were observed and the plasmid is maintained in these bacteria at 37 degrees C in the absence of antibiotic selection for at least 9 days of subculturing. The transformed bacteria were successfully reintroduced into fire ant larvae and survived in the fire ant gut for at least 7 days. Upon pupal emergence, 7 days after reintroduction, transformed bacteria can still be isolated, however, most were passed out in the meconium. We further demonstrated that the engineered bacteria could be spread within the colony by feeding this meconium to naive larvae with the aid of worker fire ants.

Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

Science.gov (United States)

Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

2015-01-01

Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

Comparative genomics of the IncA/C multidrug resistance plasmid family.

Science.gov (United States)

Fricke, W Florian; Welch, Timothy J; McDermott, Patrick F; Mammel, Mark K; LeClerc, J Eugene; White, David G; Cebula, Thomas A; Ravel, Jacques

2009-08-01

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

A new approach to produce amino-carbon nanotubes as plasmid transfection vector by [2 + 1] cycloaddition of nitrenes

International Nuclear Information System (INIS)

Jiang Yongjian; Jin Chen; Yang Feng; Yu Xianjun; Wang Guojian; Cheng Si; Di, Yang; Li, Ji; Fu, Deliang; N, Quanxing

2011-01-01

Amino-carbon nanotubes (amino-CNTs) can conjugate with the DNA by electrostatic interactions and shuttle the DNA to the cell cytoplasm or even the nucleus. Here we report a new approach to produce amino-CNTs by cycloaddition of nitrenes. Fourier transform infrared spectroscopy was used to verify the success of the functionalization, and the functionalization degree was calculated by thermal gravity analysis. Transmission electron microscope (TEM) was used to observe the solubility of the CNTs and the interactions of the amino-CNTs with the plasmids. Cell ultrathin sections were made and observed under the TEM to confirm the amino-CNTs enter the cells. Transfection experiments ultimately verify the amino-CNTs produced through cycloadditions of nitrenes can serve as plasmid vector.

Frequency and diversity of small cryptic plasmids in the genus Rahnella

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Summers David K

2010-02-01

Full Text Available Abstract Background Rahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids. Results In total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified. Conclusions For the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to diffent groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the

Plasmid stability in dried cells of the desert cyanobacterium Chroococcidiopsis and its potential for GFP imaging of survivors on Earth and in space.

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Billi, Daniela

2012-06-01

Two GFP-based plasmids, namely pTTQ18-GFP-pDU1(mini) and pDUCA7-GFP, of about 7 kbp and 15 kbp respectively, able to replicate in Chroococcidiopsis sp. CCMEE 029 and CCMEE 123, were developed. Both plasmids were maintained in Chroococcidiopsis cells after 18 months of dry storage as demonstrated by colony PCR, plasmid restriction analysis, GFP imaging and colony-forming ability under selection of dried transformants; thus suggesting that strategies employed by this cyanobacterium to stabilize dried chromosomal DNA, must have protected plasmid DNA. The suitability of pDU1(mini)-plasmid for GFP tagging in Chroococcidiopsis was investigated by using the RecA homolog of Synechocystis sp. PCC 6803. After 2 months of dry storage, the presence of dried cells with a GFP-RecA(Syn) distribution resembling that of hydrated cells, supported its capability of preventing desiccation-induced genome damage, whereas the rewetted cells with filamentous GFP-RecA(Syn) structures revealed sub-lethal DNA damage. The long-term stability of plasmid DNA in dried Chroococcidiopsis has implication for space research, for example when investigating the recovery of dried cells after Martian and space simulations or when developing life support systems based on phototrophs with genetically enhanced stress tolerance and stored in the dry state for prolonged periods.

Plasmid mediated enhancement of uv resistance in Streptococcus faecalis

International Nuclear Information System (INIS)

Miehl, R.; Miller, M.; Yasbin, R.E.

1980-01-01

A 38.5-Mdal plasmid of Streptococcus faecalis subdp. zymogenes has been shown to enhance survival following uv irradiation. In addition, the presence of this plasmid increases the mutation frequencies following uv irradiation and enhanced W-reactivation. The data presented indicate that S. faecalis has an inducible error-prone repair system and that the plasmid enhances these repair functions

Multiple drug resistant carbapenemases producing Acinetobacter baumannii isolates harbours multiple R-plasmids

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Rajagopalan Saranathan

2014-01-01

Full Text Available Background & objectives: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR, consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common β-lactamases encoding genes on these plasmids. Methods: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23 , blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA and metallo-β-lactamase (MBL type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing. Results: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥ 25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36% carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance. Interpretation & conclusions: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in

Transformation of lettuce (Lactuca sativa) mediated by Agrobacterium tumefaciens.

Science.gov (United States)

Michelmore, R; Marsh, E; Seely, S; Landry, B

1987-12-01

Lactuca sativa can be routinely transformed using Ti plasmids of Agrobacterium tumefaciens containing a chimeric kanamycin resistance gene (NOS.NPTII.NOS). Critical experimental variables were plant genotype, bacterial concentration, presence of a nurse culture and timing of transfers between tissue culture media. Transformation was confirmed by the ability to callus and root in the presence of kanamycin, nopaline production, and by hybridization in Southern blots. Transformation has been achieved with several Ti vectors. Several hundred transformed plants have been regenerated. Kanamycin resistance was inherited monogenically. Homozygotes can be selected by growing R2 seedlings on media containing G418.

Movement and equipositioning of plasmids by ParA filament disassembly

DEFF Research Database (Denmark)

Ringgaard, Simon; van Zon, Jeroen; Howard, Martin

2009-01-01

, plasmids consistently migrate behind disassembling ParA cytoskeletal structures, suggesting that ParA filaments pull plasmids by depolymerization. The perpetual cycles of ParA assembly and disassembly result in continuous relocation of plasmids, which, on time averaging, results in equidistribution...

The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains

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Ilana Teruszkin Balassiano

2007-11-01

Full Text Available This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5% of the samples were positive for tetA, and also 37.5% were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.

Construction of rat cell lines that contain potential morphologically transforming regions of the herpes simplex virus type 2 genome

NARCIS (Netherlands)

van den Berg, F. M.; van Amstel, P. J.; Walboomers, J. M.

1985-01-01

Hybrid recombinant plasmids were constructed; they were composed of the herpes simplex virus type 2 (HSV2) thymidine kinase (tk) gene and DNA sequences of HSV2 that have been reported to induce morphological and/or oncogenic transformation of rodent cells in culture. Several plasmids were made in

Selection of mesophilic microorganisms with biodesulfuration capacity

International Nuclear Information System (INIS)

Madero, A; Mogollon, L. I; Mora, A.L; Osorio, L.F

1998-01-01

The development of bio desulfurization (BDS) processes for hydrocarbons requires fast and reliable methods for the screening of microorganisms. This work shows the results of the screening process for indigenous Colombian strains with a BDS potential capacity. The main criteria for the screening were the qualitative and quantitative determination of 2-hydroxybiphenyl (2-HBP) as the typical metabolite of the 4S specific pathway. Microorganisms were cultured by two methodologies, A and B, using DBT as the model compound. The quantitative determination of metabolites was made by HPLC. Thirteen strains were evaluated, including the strain Rhodococcus rhodocrous IGTS8, by methods A and B. In method A, the inoculum was exposed to DBT since the beginning of the culture. Method B, employed two stages: (i) Growth period under limiting sulfur conditions, (ii) Transforming period, in which the pre-grown inoculum was exposed to the organic sulfur substrate. The culture of mesophilic microorganisms isolated by method B, served to find a mechanism for the organic sulfur metabolism, and the evaluation of the sulfur removal capability of five indigenous strains. In the cultures of these strains, 2- hydroxybiphenyl (2-HBP) was detected as a byproduct of DBT metabolism, both qualitatively and quantitatively

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

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Tang Xian-Lai

2010-11-01

Full Text Available Abstract Background Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. Thermostable and high-activity fumarases from organisms that inhabit extreme environments may have great potential in industry, biotechnology, and basic research. The marine environment is highly complex and considered one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms are inaccessible in nature and are not easily cultivated in the laboratory. Metagenomic approaches provide a powerful tool to isolate and identify enzymes with novel biocatalytic activities for various biotechnological applications. Results A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF was isolated. Amino acid sequence analysis revealed that the FumF protein shared the greatest homology with Class II fumarate hydratases from Bacteroides sp. 2_1_33B and Parabacteroides distasonis ATCC 8503 (26% identical and 43% similar. The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The maximum activity for FumF protein occurred at pH 8.5 and 55°C in 5 mM Mg2+. The enzyme showed higher affinity and catalytic efficiency under optimal reaction conditions: Km= 0.48 mM, Vmax = 827 μM/min/mg, and kcat/Km = 1900 mM/s. Conclusions We isolated a novel fumarase gene, fumF, from a sequence-based screen of a plasmid metagenomic library from uncultivated

Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

Science.gov (United States)

Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

1999-01-01

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world. PMID:10488224

Plasmid DNA Delivery: Nanotopography Matters.

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Song, Hao; Yu, Meihua; Lu, Yao; Gu, Zhengying; Yang, Yannan; Zhang, Min; Fu, Jianye; Yu, Chengzhong

2017-12-20

Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.

DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

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Jing Han

Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

Simple method for identification of plasmid-coded proteins

International Nuclear Information System (INIS)

Sancar, A.; Hack, A.M.; Rupp, W.D.

1979-01-01

Proteins encoded by plasmid DNA are specifically labeled in uv-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA

Presence and analysis of plasmids in human and animal associated arcobacter species.

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Laid Douidah

Full Text Available In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.

THE ENDOGENOUS BACILLUS-SUBTILIS (NATTO) PLASMIDS PTA1015 AND PTA1040 CONTAIN SIGNAL PEPTIDASE-ENCODING GENES - IDENTIFICATION OF A NEW STRUCTURAL MODULE ON CRYPTIC PLASMIDS

NARCIS (Netherlands)

MEIJER, WJJ; DEJONG, A; BEA, G; WISMAN, A; TJALSMA, H; VENEMA, G; BRON, S; MAARTEN, J; VANDIJL, JM

Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis

Agrobacterium-mediated transformation of modified antifreeze protein gene in strawberry

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Srisulak Dheeranupattana

2005-07-01

Full Text Available The optimum condition for shoot regeneration from leaf explants of strawberry cultivar Tiogar was investigated. It was found that the best regeneration condition was MS medium containing N6-Benzyladenine (BA and 2,4-Dichlorophenoxy acetic acid (2,4-D at concentrations of 1 mg.l-1 and 0.2 mg.l-1, respectively. Antibiotics sensitivity test found that shoot regeneration from leaf explant was inhibited more than 90% at the concentration of kanamycin (Km as low as 5 mg.l-1. The modified gene encoding antifreeze protein isoform HPLC 6 was successfully constructed using codons which were optimally expressed in the strawberry plant. The antifreeze protein genes, naturally in plasmid pSW1 and modified in plasmid BB, were transformed to strawberry leaf explants by Agrobacterium tumefaciens LBA 4404. The strawberry plants, transformed with both AFP genes, were able to root in MS media containing 50 mg.l-1 Km, while no roots grew from nontransformed plant in this condition. Polymerase chain reaction indicated that the transgenes were integrated in the genome of transformants.

A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host.

Science.gov (United States)

Purser, Joye E; Lawrenz, Matthew B; Caimano, Melissa J; Howell, Jerrilyn K; Radolf, Justin D; Norris, Steven J

2003-05-01

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25-B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.

Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects.

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Monika Stimac

Full Text Available Vascular targeted therapies, targeting specific endothelial cell markers, are promising approaches for the treatment of cancer. One of the targets is endoglin, transforming growth factor-β (TGF-β co-receptor, which mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. However, its specific, safe and long-lasting targeting remains the challenge. Therefore, in our study we evaluated the transfection efficacy, vascular targeted effects and therapeutic potential of the plasmid silencing endoglin with the tissue specific promoter, specific for endothelial cells marker endothelin-1 (ET (TS plasmid, in comparison to the plasmid with constitutive promoter (CON plasmid, in vitro and in vivo. Tissue specificity of TS plasmid was demonstrated in vitro on several cell lines, and its antiangiogenic efficacy was demonstrated by reducing tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of gene electrotransfer (GET of either of both plasmids in treatment of smaller tumors still in avascular phase of growth, as well as on bigger tumors, already well vascularized. In support to the observations on predominantly vascular targeted effects of endoglin, histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. A significant antitumor effect was observed in tumors in avascular and vascular phase of growth, possibly due to both, the antiangiogenic and the vascular disrupting effect. Furthermore, the study indicates on the potential use of TS plasmid in cancer gene therapy since the same efficacy as of CON plasmid was determined.

Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects.

Science.gov (United States)

Stimac, Monika; Dolinsek, Tanja; Lampreht, Ursa; Cemazar, Maja; Sersa, Gregor

2015-01-01

Vascular targeted therapies, targeting specific endothelial cell markers, are promising approaches for the treatment of cancer. One of the targets is endoglin, transforming growth factor-β (TGF-β) co-receptor, which mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. However, its specific, safe and long-lasting targeting remains the challenge. Therefore, in our study we evaluated the transfection efficacy, vascular targeted effects and therapeutic potential of the plasmid silencing endoglin with the tissue specific promoter, specific for endothelial cells marker endothelin-1 (ET) (TS plasmid), in comparison to the plasmid with constitutive promoter (CON plasmid), in vitro and in vivo. Tissue specificity of TS plasmid was demonstrated in vitro on several cell lines, and its antiangiogenic efficacy was demonstrated by reducing tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of gene electrotransfer (GET) of either of both plasmids in treatment of smaller tumors still in avascular phase of growth, as well as on bigger tumors, already well vascularized. In support to the observations on predominantly vascular targeted effects of endoglin, histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. A significant antitumor effect was observed in tumors in avascular and vascular phase of growth, possibly due to both, the antiangiogenic and the vascular disrupting effect. Furthermore, the study indicates on the potential use of TS plasmid in cancer gene therapy since the same efficacy as of CON plasmid was determined.

Investigation of diversity of plasmids carrying the blaTEM-52 gene

DEFF Research Database (Denmark)

Bielak, Eliza Maria; Bergenholtz, Rikke D.; Jørgensen, Mikael Skaanning

2011-01-01

OBJECTIVES: To investigate the diversity of plasmids that carry blaTEM-52 genes among Escherichia coli and Salmonella enterica originating from animals, meat products and humans. METHODS: A collection of 22 blaTEM-52-encoding plasmids was characterized by restriction fragment length polymorphism...... of self-transfer to a plasmid-free E. coli recipient. CONCLUSIONS: The blaTEM-52 gene found in humans could have been transmitted on transferable plasmids originating from animal sources. Some of the blaTEM-52 plasmids carry replicons that differ from the classical ones. Two novel replicons were detected...

Development of a Novel Escherichia coli–Kocuria Shuttle Vector Using the Cryptic pKPAL3 Plasmid from K. palustris IPUFS-1 and Its Utilization in Producing Enantiopure (S-Styrene Oxide

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Hiroshi Toda

2017-11-01

Full Text Available The novel cryptic pKPAL3 plasmid was isolated from the Gram-positive microorganism Kocuria palustris IPUFS-1 and characterized in detail. pKPAL3 is a circular plasmid that is 4,443 bp in length. Open reading frame (ORF and homology search analyses indicated that pKPAL3 possesses four ORFs; however, there were no replication protein coding genes predicted in the plasmid. Instead, there were two nucleotide sequence regions that showed significant identities with untranslated regions of K. rhizophila DC2201 (NBRC 103217 genomic sequences, and these sequences were essential for autonomous replication of pKPAL3 in Kocuria cells. Based on these findings, we constructed the novel Escherichia coli–Kocuria shuttle vectors pKITE301 (kanamycin resistant and pKITE303 (thiostrepton resistant from pKPAL3. The copy numbers of the constructed shuttle vectors were estimated to be 20 per cell, and they exhibited low segregation stability in Kocuria transformant cells in the absence of antibiotics. Moreover, constructed vectors showed compatibility with the other K. rhizophila shuttle vector pKITE103. We successfully expressed multiple heterologous genes, including the styrene monooxygenase gene from Rhodococcus sp. ST-10 (rhsmo and alcohol dehydrogenase gene from Leifsonia sp. S749 (lsadh, in K. rhizophila DC2201 using the pKITE301P and pKITE103P vectors under the control of the glyceraldehyde 3-phosphate dehydrogenase (gapdh promotor. The RhSMO–LSADH co-expressing K. rhizophila was used as a biocatalyst in an organic solvent–water biphasic reaction system to efficiently convert styrene into (S-styrene oxide with 99% ee in the presence of 2-propanol as a hydrogen donor. The product concentration of the reaction in the organic solvent reached 235 mM after 30 h under optimum conditions. Thus, we demonstrated that this novel shuttle vector is useful for developing biocatalysts based on organic solvent-tolerant Kocuria cells.

Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727

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Mahillon Jacques

2005-07-01

Full Text Available Abstract Background Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the genetically close-knit Bacillus cereus sensu lato group, a family of rod-shaped Gram-positive bacteria. pAW63 is the first conjugative plasmid from the B. cereus group to be completely sequenced. Results The 71,777 bp nucleotide sequence of pAW63 reveals a modular structure, including a 42 kb tra region encoding homologs of the Type IV secretion systems components VirB11, VirB4 and VirD4, as well as homologs of Gram-positive conjugation genes from Enterococcus, Lactococcus, Listeria, Streptococcus and Staphylococcus species. It also firmly establishes the existence of a common backbone between pAW63, pXO2 from Bacillus anthracis and pBT9727 from the pathogenic Bacillus thuringiensis serovar konkukian strain 97-27. The alignment of these three plasmids highlights the presence of well conserved segments, in contrast to distinct regions of high sequence plasticity. The study of their specific differences has provided a three-point reference framework that can be exploited to formulate solid hypotheses concerning the functionalities and the molecular evolution of these three closely related plasmids. This has provided insight into the chronology of their divergence, and led to the discovery of two Type II introns on pAW63, matching copies of the mobile element IS231L in different loci of pXO2 and pBT9727, and the identification on pXO2 of a 37 kb pathogenicity island (PAI containing the anthrax capsule genes. Conclusion The complete sequence determination of pAW63 has led to a functional map of the plasmid yielding insights into its conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large plasmids of Gram-positive bacteria. Of particular interest is the extensive homology shared between pAW63 and pXO2, the second virulence plasmid of B. anthracis, as well as pBT9727 from the pathogenic strain B. thuringiensis

Permissiveness of soil microbial communities towards broad host range plasmids

DEFF Research Database (Denmark)

Klümper, Uli

. Plasmids are implicated in the rapid spread of antibiotic resistance and the emergence of multi-resistant pathogenic bacteria, making it crucial to be able to quantify, understand, and, ideally, control plasmid transfer in mixed microbial communities. The fate of plasmids in microbial communities...... of microbial communities may be directly interconnected through transfer of BHR plasmids at a so far unrecognized level. The developed method furthermore enabled me to explore how agronomic practices may affect gene transfer in soil microbial communities. I compared bacterial communities extracted from plots...

Microbial monoterpene transformations – A review

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Robert eMarmulla

2014-07-01

Full Text Available Isoprene and monoterpenes constitute a significant fraction of new plant biomass. Emission rates into the atmosphere alone are estimated to be over 500 Tg per year. These natural hydrocarbons are mineralized annually in similar quantities. In the atmosphere, abiotic photochemical processes cause lifetimes of minutes to hours. Microorganisms encounter isoprene, monoterpenes and other volatiles of plant origin while living in and on plants, in the soil and in aquatic habitats. Below toxic concentrations, the compounds can serve as carbon and energy source for aerobic and anaerobic microorganisms. Besides these catabolic reactions, transformations may occur as part of detoxification processes. Initial transformations of monoterpenes involve the introduction of functional groups, oxidation reactions and molecular rearrangements catalyzed by various enzymes. Pseudomonas and Rhodococcus strains and members of the genera Castellaniella and Thauera have become model organisms for the elucidation of biochemical pathways. We review here the enzymes and their genes together with microorganisms known for a monoterpene metabolism, with a strong focus on microorganisms that are taxonomically validly described and currently available from culture collections. Metagenomes of microbiomes with a monoterpene-rich diet confirmed the ecological relevance of monoterpene metabolism and raised concerns on the quality of our insights based on the limited biochemical knowledge.

Drug resistance plasmids in Lactobacillus acidophilus and Lactobacillus reuteri.

OpenAIRE

Vescovo, M; Morelli, L; Bottazzi, V

1982-01-01

Sixteen strains of Lactobacillus reuteri and 20 strains of Lactobacillus acidophilus were tested for resistance to 22 antibiotics by using commercially available sensitivity disks. Evidence suggesting linkage of these resistances to plasmids was obtained by "curing" experiments with acridine dyes and high growth temperatures. Examination of plasmid patterns of agarose gel electrophoresis provided further evidence of loss in plasmid DNA under curing conditions in some of the strains examined.

Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe.

Directory of Open Access Journals (Sweden)

Ayako Chino

Full Text Available BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC, is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp. No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different

Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids.

Science.gov (United States)

Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi; Neuman, Keir C; Liu, Jian

2017-04-11

Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian ratchet mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation

Plasmid P1 replication: negative control by repeated DNA sequences.

OpenAIRE

Chattoraj, D; Cordes, K; Abeles, A

1984-01-01

The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

Micro-Organ Device

Science.gov (United States)

Gonda, Steve R. (Inventor); Chang, Robert C. (Inventor); Starly, Binil (Inventor); Culbertson, Christopher (Inventor); Holtorf, Heidi L. (Inventor); Sun, Wei (Inventor); Leslie, Julia (Inventor)

2013-01-01

A method for fabricating a micro-organ device comprises providing a microscale support having one or more microfluidic channels and one or more micro-chambers for housing a micro-organ and printing a micro-organ on the microscale support using a cell suspension in a syringe controlled by a computer-aided tissue engineering system, wherein the cell suspension comprises cells suspended in a solution containing a material that functions as a three-dimensional scaffold. The printing is performed with the computer-aided tissue engineering system according to a particular pattern. The micro-organ device comprises at least one micro-chamber each housing a micro-organ; and at least one microfluidic channel connected to the micro-chamber, wherein the micro-organ comprises cells arranged in a configuration that includes microscale spacing between portions of the cells to facilitate diffusion exchange between the cells and a medium supplied from the at least one microfluidic channel.

Genetic transformation of garlic ( Allium sativum L.) with tobacco ...

African Journals Online (AJOL)

Garlic yield and quality have decreased due to white rot disease caused by Sclerotium cepivorum Berk. A transformation protocol to introduce tobacco chitinase and glucanase genes into garlic embryogenic calli using Agrobacterium tumefaciens has been established. LBA4404 strain having pC2301CHGLU plasmid with ...

Antibiotic resistance plasmids of Staphylococcus aureus and their clinical importance

International Nuclear Information System (INIS)

Lacey, R.W.

1975-01-01

A variety of plasmids were isolated physically, and most antibiotic resistance is thought to be plasmid mediated. A number of characters (e.g., resistance to erythromycin or methicillin, and production of pigment) are determined by genes that do not give clear indications of either plasmid or chromosomal location. Although the formation of a particular plasmid is probably, even in bacterial terms, a very rare event, once formed such an element can spread rapidly among the bacterial population. The spectacular increase in the incidence of penicillinase-producing hospital strains in the late 1940's could have been due in part to this process. Evidence is stronger, however, for the intercell transfer of recently isolated plasmids coding for resistance to fusidic acid (and penicillinase production), or for neomycin, or for tetracycline resistance. Study of bacterial plasmids can resolve fundamental biochemical problems, and give some insight into the life of the cell at the molecular level. But the immediate application of the study of staphylococcal plasmids may be directed towards improving the effectiveness of antibiotic therapy. The most important aspect of future anti-staphylococcal chemotherapy should thus be the limitation of the use of antibiotics, particularly for application to the skin and nose. (U.S.)

Characterization of a Large Antibiotic Resistance Plasmid Found in Enteropathogenic Escherichia coli Strain B171 and Its Relatedness to Plasmids of Diverse E. coli and Shigella Strains.

Science.gov (United States)

Hazen, Tracy H; Michalski, Jane; Nagaraj, Sushma; Okeke, Iruka N; Rasko, David A

2017-09-01

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline ( tetA ), sulfonamides ( sulI ), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor ( csi ). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 ( csi and traI ) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli . Copyright © 2017 American Society for Microbiology.

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration, and Acquisition of the Plasmid Results in a Variable Cost of Fitness.

Science.gov (United States)

Johnson, Timothy J; Singer, Randall S; Isaacson, Richard E; Danzeisen, Jessica L; Lang, Kevin; Kobluk, Kristi; Rivet, Bernadette; Borewicz, Klaudyna; Frye, Jonathan G; Englen, Mark; Anderson, Janet; Davies, Peter R

2015-05-15

IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Homologous gene targeting of a carotenoids biosynthetic gene in Rhodosporidium toruloides by Agrobacterium-mediated transformation.

Science.gov (United States)

Sun, Wenyi; Yang, Xiaobing; Wang, Xueying; Lin, Xinping; Wang, Yanan; Zhang, Sufang; Luan, Yushi; Zhao, Zongbao K

2017-07-01

To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method. The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11. Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

Complete sequencing of IncI1 sequence type 2 plasmid pJIE512b indicates mobilization of blaCMY-2 from an IncA/C plasmid.

Science.gov (United States)

Tagg, Kaitlin A; Iredell, Jonathan R; Partridge, Sally R

2014-08-01

Sequencing of pJIE512b, a 92.3-kb IncI1 sequence type 2 (ST2) plasmid carrying bla(CMY-2), revealed a bla(CMY-2) context that appeared to have been mobilized from an IncA/C plasmid by the insertion sequence IS1294. A comparison with published plasmids suggests that bla(CMY-2) has been mobilized from IncA/C to IncI1 plasmids more than once by IS1294-like elements. Alignment of pJIE512b with the only other available IncI1 ST2 plasmid revealed differences across the backbones, indicating variability within this sequence type. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Carriage of extended-spectrum beta-lactamase-plasmids does not reduce fitness but enhances virulence in some strains of pandemic E. coli lineages

Directory of Open Access Journals (Sweden)

Katharina eSchaufler

2016-03-01

Full Text Available Pathogenic ESBL-producing E. coli lineages occur frequently worldwide, not only in a human health context but in animals and the environment, also in settings with low antimicrobial pressures. This study investigated the fitness costs of ESBL-plasmids and their influence on chromosomally encoded features associated with virulence, such as those involved in the planktonic and sessile behaviors of ST131 and ST648 E. coli. ESBL-plasmid-carrying wild-type E. coli strains, their corresponding ESBL-plasmid-cured variants (PCV, and complementary ESBL-carrying transformants were comparatively analyzed using growth curves, Omnilog® phenotype microarray (PM assays, macrocolony and biofilm formation, swimming motility, and RNA sequence analysis. Growth curves and PM results pointed towards similar growth and metabolic behaviors among the strains. Phenotypic differences in some strains were detected, including enhanced curli fimbriae and/or cellulose production as well as a reduced swimming capacity of some ESBL-carrying strains, as compared to their respective PCVs. RNA sequencing mostly confirmed the phenotypic results, suggesting that the chromosomally encoded csgD pathway is a key factor involved. These results contradict the hypothesis that ESBL-plasmid-carriage leads to a fitness loss in ESBL-carrying strains. Instead, the results indicate an influence of some ESBL-plasmids on chromosomally encoded features associated with virulence in some E. coli strains. In conclusion, apart from antibiotic resistance selective advantages, ESBL-plasmid-carriage may also lead to enhanced virulence or adaption to specific habitats in some strains of pandemic ESBL-producing E. coli lineages.

Novel assay to measure the plasmid mobilizing potential of mixed microbial communities

DEFF Research Database (Denmark)

Klümper, Uli; Droumpali, Ariadni; Dechesne, Arnaud

2014-01-01

Mobilizable plasmids lack necessary genes for complete conjugation and are therefore non-self-transmissible. Instead, they rely on the conjugation system of conjugal plasmids to be horizontally transferred to new recipients. While community permissiveness, the fraction of a mixed microbial...... community that can receive self-transmissible conjugal plasmids, has been studied, the intrinsic ability of a community to mobilize plasmids that lack conjugation systems is unexplored. Here, we present a novel framework and experimental method to estimate the mobilization potential of mixed communities. We...... of the donors receiving the conjugal plasmid in the first step. Further work is needed to establish how plasmid mobilization potential varies within and across microbial communities....

Characterization of a novel plasmid type and various genetic contexts of bla OXA-58 in Acinetobacter spp. from multiple cities in China.

Directory of Open Access Journals (Sweden)

Yiqi Fu

Full Text Available BACKGROUND/OBJECTIVE: Several studies have described the epidemiological distribution of blaOXA-58-harboring Acinetobacter baumannii in China. However, there is limited data concerning the replicon types of blaOXA-58-carrying plasmids and the genetic context surrounding blaOXA-58 in Acinetobacter spp. in China. METHODOLOGY/PRINCIPAL FINDINGS: Twelve non-duplicated blaOXA-58-harboring Acinetobacter spp. isolates were collected from six hospitals in five different cities between 2005 and 2010. The molecular epidemiology of the isolates was carried out using PFGE and multilocus sequence typing. Carbapenemase-encoding genes and plasmid replicase genes were identified by PCR. The genetic location of blaOXA-58 was analyzed using S1-nuclease method. Plasmid conjugation and electrotransformation were performed to evaluate the transferability of blaOXA-58-harboring plasmids. The genetic structure surrounding blaOXA-58 was determined by cloning experiments. The twelve isolates included two Acinetobacter pittii isolates (belong to one pulsotype, three Acinetobacter nosocomialis isolates (belong to two pulsotypes and seven Acinetobacter baumannii isolates (belong to two pulsotypes/sequence types. A. baumannii ST91 was found to be a potential multidrug resistant risk clone carrying both blaOXA-58 and blaOXA-23. blaOXA-58 located on plasmids varied from ca. 52 kb to ca. 143 kb. All plasmids can be electrotransformed to A. baumannii recipient, but were untypeable by the current replicon typing scheme. A novel plasmid replicase named repAci10 was identified in blaOXA-58-harboring plasmids of two A. pittii isolates, three A. nosocomialis isolates and two A. baumannii isolates. Four kinds of genetic contexts of blaOXA-58 were identified. The transformants of plasmids with structure of IS6 family insertion sequence (ISOur1, IS1008 or IS15-ΔISAba3-like element-blaOXA-58 displayed carbapenem nonsusceptible, while others with structure of intact ISAba3-like element

Production and pharmaceutical formulation of plasmid DNA vaccines

NARCIS (Netherlands)

van der Heijden, I.

2013-01-01

Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human

Photoinduced silver nanoparticles/nanorings on plasmid DNA scaffolds.

Science.gov (United States)

Liu, Jianhua; Zhang, Xiaoliang; Yu, Mei; Li, Songmei; Zhang, Jindan

2012-01-23

Biological scaffolds are being actively explored for the synthesis of nanomaterials with novel structures and unexpected properties. Toroidal plasmid DNA separated from the Bacillus host is applied as a sacrificial mold for the synthesis of silver nanoparticles and nanorings. The photoirradiation method is applied to reduce Ag(I) on the plasmid. The nanoparticles are obtained by varying the concentration of the Ag(I) ion solution and the exposure time of the plasmid-Ag(I) complex under UV light at 254 nm and room temperature. It is found that the plasmid serves not only as a template but also as a reductant to drive the silver nucleation and deposition. The resulting nanoparticles have a face-centered cubic (fcc) crystal structure and 20-30 nm average diameter. The detailed mechanism is discussed, and other metals or alloys could also be synthesized with this method. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design and evaluation of protein expression in a recombinant plasmid encoding epitope gp 350/220 of the Epstein-Barr virus (EBV)

Science.gov (United States)

Himmah, Karimatul; Dluha, Nurul; Anyndita, Nadya V. M.; Rifa'i, Muhaimin; Widodo

2017-05-01

The Epstein - Barr virus (EBV) causes severe infections that may lead to cancers such as nasopharyngeal carcinoma. Development of effective EBV vaccines is necessary to prevent the virus spreading throughout the community. TheEBV has a surface protein gp 350/220, which serves as an antigen to help interact with host cells. Epitopes of the protein can potentially serve as bases for a vaccine. In a previous study, we have found a conserved epitope of gp 350/220 from all strains EBV through an in silico approach. The aim of this study is to design and overproduce a recombinant peptide of epitope gp 350/220 in E. coli. DNA encoding the conserved epitope was synthesized and cloned into plasmid pET-22b(+); the recombinant plasmid was transformed into E. coli strains DH5α and BL21. The transformed plasmid DNA was isolated and confirmed by restriction using XbaI and PstI enzymes followed by DNA sequencing. Protein expression was induced by isopropyl-D-thiogalactopyranoside (IPTG) with final concentrations of 0.1, 0.2, 1, and 2 mM in consecutive times. An osmotic shock method was used to isolate protein from periplasmic fraction of E. coli DH5α and BL21. The SDS-PAGE analysis was carried out to detect peptide target (3.4 kDa). Based on this result, the induction process did not work properly, and thus needs further investigation.

Genetic transformation of carnation (Dianthus caryophylus L.).

Science.gov (United States)

Nontaswatsri, Chalermsri; Fukai, Seiichi

2010-01-01

This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

Plasmids and rickettsial evolution: insight from Rickettsia felis.

Directory of Open Access Journals (Sweden)

Joseph J Gillespie

2007-03-01

Full Text Available The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG or spotted fever group (SFG rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria.Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives also occur in AG (but not SFG rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFdelta, is an artifact of the original genome assembly.Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the

A fresh method of DNA transformation to the seeds irradiated by Co ...

African Journals Online (AJOL)

Jane

2011-06-29

Jun 29, 2011 ... To find out a simpler method that can directly transfer the aim gene into plant ... Key words: DNA transformation, irradiated seeds, purple medic, salt screening. ..... characterization of a maize mitochondrial plasmid-like DNA.

Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

Energy Technology Data Exchange (ETDEWEB)

Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.; Eakes, Thomas C.; Eto, Karina Yui; Kwong, Stephen M.; Ramsay, Joshua P.; Firth, Neville; Redinbo, Matthew R. (Curtin U.); (Sydney); (UNC)

2016-01-04

Antimicrobial resistance inStaphylococcus aureuspresents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at theoriTregion of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES proteinin vitro. Second, pSK156 and pCA347 are nonconjugativeStaphylococcus aureusplasmids that contain sequences similar to theoriTregion of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate theoriTsequences of these nonconjugative plasmidsin vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognateoriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variantoriTmimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-likeoriT. These data indicate that the conjugative relaxase intransmechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids.

IMPORTANCEUnderstanding the

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

Science.gov (United States)

A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

Nature of transforming deoxyribonucleic acid in calcium-treated Escherichia coli

International Nuclear Information System (INIS)

Strike, P.; Humphreys, G.O.; Roberts, R.J.

1979-01-01

A study of the reactivation of ultraviolet-irradiated plasmid and phage deoxyribonucleic acid molecules after transformation into Escherichia coli strains indicated that, when double-stranded deoxyribonucleic acid was used as the donor species, it was taken up without conversion to the single-stranded form

A replicative plasmid vector allows efficient complementation of pathogenic Leptospira strains.

Science.gov (United States)

Pappas, Christopher J; Benaroudj, Nadia; Picardeau, Mathieu

2015-05-01

Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids.

Science.gov (United States)

He, Susu; Chandler, Michael; Varani, Alessandro M; Hickman, Alison B; Dekker, John P; Dyda, Fred

2016-12-06

The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content) of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE) from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance. The spread of antibiotic resistance among Gram-negative bacteria is a serious public health threat, as it can critically limit the types of drugs that can be used to treat infected patients. In particular, carbapenem-resistant members of the Enterobacteriaceae family are responsible for a significant and growing burden of morbidity and mortality. Here, we report on the mechanisms underlying the evolution of several plasmids carried by previously sequenced clinical Enterobacteriaceae isolates from the National Institutes of Health Clinical Center (NIH CC). Our ability to track genetic rearrangements that occurred within resistance plasmids was dependent on accurate annotation of the mobile genetic elements within the plasmids, which was greatly aided by access to long-read DNA sequencing data and knowledge of their mechanisms. Mobile genetic elements such as

Type 3 Fimbriae Encoded on Plasmids Are Expressed from a Unique Promoter without Affecting Host Motility, Facilitating an Exceptional Phenotype That Enhances Conjugal Plasmid Transfer

DEFF Research Database (Denmark)

Madsen, Jonas Stenlokke; Riber, Leise; Kot, Witold

2016-01-01

Horizontal gene transfer (HGT), the transmission of genetic material to a recipient that is not the progeny of the donor, is fundamental in bacterial evolution. HGT is often mediated by mobile genetic elements such as conjugative plasmids, which may be in conflict with the chromosomal elements...... of the genome because they are independent replicons that may petition their own evolutionary strategy. Here we study differences between type 3 fimbriae encoded on wild type plasmids and in chromosomes. Using known and newly characterized plasmids we show that the expression of type 3 fimbriae encoded...... on plasmids is systematically different, as MrkH, a c-di-GMP dependent transcriptional activator is not needed for strong expression of the fimbriae. MrkH is required for expression of type 3 fimbriae of the Klebsiella pneumoniae chromosome, wherefrom the fimbriae operon (mrkABCDF) of plasmids is believed...

Earthworm-microorganism interactions: a strategy to stabilize domestic wastewater sludge.

Science.gov (United States)

Zhao, Limin; Wang, Yayi; Yang, Jian; Xing, Meiyan; Li, Xiaowei; Yi, Danghao; Deng, Dehan

2010-04-01

The performance of a conventional biofilter (BF) and a vermifilter containing the earthworm, Eisenia foetida, (VF) for the treatment of domestic wastewater sludge were compared with the earthworm-microorganism interaction mechanisms involved in sludge stabilization. The results revealed that the presence of earthworms in the VF led to significant stabilization of the sludge by enhancing the reduction in volatile suspended solids (VSS) by 25.1%. Digestion by earthworms and the earthworm-microorganism interactions were responsible for 54% and 46% of this increase, respectively. Specifically, earthworms in the VF were capable of transforming insoluble organic materials to a soluble form and then selectively digesting the sludge particles of 10-200 microm to finer particles of 0-2 microm, which led to the further degradation of organic materials by the microorganisms in the reactor. Additionally, denaturing gradient gel electrophoresis (DGGE) profiles showed that there was an intensified bacterial diversity in the vermifilter due to the presence of earthworms, especially in response to the nutrients in their casts. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Resistant plasmid profile analysis of multidrug resistant Escherichia ...

African Journals Online (AJOL)

Multiple drug resistance isolates causing UTI has seri- ous implications for the empiric therapy against patho- genic isolates and for the possible co-selection of antimicrobial resistant mediated by multi drug resistant plasmids21,22. E. coli from clinical isolates are known to harbour plasmids of different molecular sizes23.

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

Science.gov (United States)

Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

1990-01-01

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

IncA/C plasmids: An emerging threat to human and animal health?

Science.gov (United States)

Johnson, Timothy J; Lang, Kevin S

2012-01-01

Incompatibility group IncA/C plasmids are large, low copy, theta-replicating plasmids that have been described in the literature for over 40 years. However, they have only recently been intensively studied on the genomic level because of their associations with the emergence of multidrug resistance in enteric pathogens of humans and animals. These plasmids are unique among other enterobacterial plasmids in many aspects, including their modular structure and gene content. While the IncA/C plasmid genome structure has now been well defined, many questions remain pertaining to their basic biological mechanisms of dissemination and regulation. Here, we discuss the history of IncA/C plasmids in light of our recent understanding of their population distribution, genomics, and effects on host bacteria.

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Science.gov (United States)

Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

2017-06-01

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

Functional analysis of three plasmids from Lactobacillus plantarum

NARCIS (Netherlands)

Kranenburg, R. van; Golic, N.; Bongers, R.; Leer, R.J.; Vos, W.M. de; Siezen, R.J.; Kleerebezem, M.

2005-01-01

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism.

The technology of large-scale pharmaceutical plasmid purification ...

African Journals Online (AJOL)

Further test demonstrated that the pcDNAlacZ purified with CTAB and authoritative endotoxin-free plasmid Kit had the similar transfection efficiency in vivo and in vitro. CTAB can be used for plasmid purification; the main advantages of the DNAs purified with CTAB include the avoidance of animal-derived enzymes, toxic ...

Stable transformation of Hi-II maize using the particle inflow gun

CSIR Research Space (South Africa)

O'Kennedy, MM

1998-04-01

Full Text Available Focuses on the transformation of the embryogenic type II callus, initiated from cultured immature zygotic embryos of the maize line Hi-II, by microprojectile bombardment using a particle inflow gun. Use of the plasmid pAHC25; Tissue culture...

Role of plasmids in Lactobacillus brevis BSO 464 hop tolerance and beer spoilage.

Science.gov (United States)

Bergsveinson, Jordyn; Baecker, Nina; Pittet, Vanessa; Ziola, Barry

2015-02-01

Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate.

Evaluation of terrestrial microcosms for assessing the fate and effects of genetically engineered microorganisms on ecological processes

Energy Technology Data Exchange (ETDEWEB)

Fredrickson, J.K.; Bentjen, S.A.; Bolton, H. Jr.; Li, S.W.; Ligotke, M.W.; McFadden, K.M.; Van Voris, P.

1989-04-01

This project evaluates and modifies the existing US Environmental Protection Agency's Office of Pesticides and Toxic Substances (EPA/OPTS) terrestrial microcosm test system and test protocols such that they can be used to determine the environmental fate and ecological hazards of genetically engineered microorganisms (GEMs). The intact soil-core microcosm represents terrestrial ecosystems, and when coupled with appropriate test protocols, such microcosms may be appropriate to define and limit risks associated with the intentional release of GEMs. The terrestrial microcosm test system was used to investigate the survival and transport of two model GEMs (Azospirillum lipoferum and Pseudomonas sp. Tn5 mutants) to various trophic levels and niches and through intact soil cores. Subsequent effects on nutrient cycling and displacement of indigenous microorganisms were evaluated. The model organisms were a diazotrophic root-colonizing bacterium (A. lipoferum) and a wheat root growth-inhibiting rhizobacterium (Pseudomonas sp.). The transposable element Tn5 was used as a genetic marker for both microorganisms in two separate experiments. The organisms were subjected to transposon mutagenesis using a broad host-range-mobilizable suicide plasmid. The transposon Tn5 conferred levels of kanamycin resistance up to 500 ..mu..g/ml (Pseudomonas sp.), which allowed for selection of the bacteria from environmental samples. The presence of Tn5 DNA in the genome of the model GEMs also allowed the use of Tn5 gene probes to confirm and enumerate the microorganisms in different samples from the microcosms. Two types of root growth-inhibiting Pseudomonas sp. Tn5 mutants were obtained and used in microcosm studies: those that lacked the ability to inhibit either wheat root growth or the growth of other microorganisms in vitro (tox/sup /minus//) and those which retained these properties (tox/sup +/). 53 refs., 7 figs., 6 tabs.

Transfer and Persistence of a Multi-Drug Resistance Plasmid in situ of the Infant Gut Microbiota in the Absence of Antibiotic Treatment

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Heidi Gumpert

2017-09-01

Full Text Available The microbial ecosystem residing in the human gut is believed to play an important role in horizontal exchange of virulence and antibiotic resistance genes that threatens human health. While the diversity of gut-microorganisms and their genetic content has been studied extensively, high-resolution insight into the plasticity, and selective forces shaping individual genomes is scarce. In a longitudinal study, we followed the dynamics of co-existing Escherichia coli lineages in an infant not receiving antibiotics. Using whole genome sequencing, we observed large genomic deletions, bacteriophage infections, as well as the loss and acquisition of plasmids in these lineages during their colonization of the human gut. In particular, we captured the exchange of multidrug resistance genes, and identified a clinically relevant conjugative plasmid mediating the transfer. This resistant transconjugant lineage was maintained for months, demonstrating that antibiotic resistance genes can disseminate and persist in the gut microbiome; even in absence of antibiotic selection. Furthermore, through in vivo competition assays, we suggest that the resistant transconjugant can persist through a fitness advantage in the mouse gut in spite of a fitness cost in vitro. Our findings highlight the dynamic nature of the human gut microbiota and provide the first genomic description of antibiotic resistance gene transfer between bacteria in the unperturbed human gut. These results exemplify that conjugative plasmids, harboring resistance determinants, can transfer and persists in the gut in the absence of antibiotic treatment.

Long- term manure exposure increases soil bacterial community potential for plasmid uptake

DEFF Research Database (Denmark)

Musovic, Sanin; Klümper, Uli; Dechesne, Arnaud

2014-01-01

Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive and main......Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive...... and maintain the plasmids. The community permissiveness increased up to 100% in communities derived from manured soil. While the plasmid transfer frequency was significantly influenced by both the type of plasmid and the agronomic treatment, the diversity of the transconjugal pools was purely plasmid dependent...

Monitoring of phytopathogenic Ralstonia solanacearum cells using green fluorescent protein-expressing plasmid derived from bacteriophage phiRSS1.

Science.gov (United States)

Kawasaki, Takeru; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

2007-12-01

A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.

Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia

OpenAIRE

Yeow, Tee Cian; Wong, Won Fen; Sabet, Negar Shafiei; Sulaiman, Sofiah; Shahhosseini, Fatemeh; Tan, Grace Min Yi; Movahed, Elaheh; Looi, Chung Yeng; Shankar, Esaki M.; Gupta, Rishien; Arulanandam, Bernard P.; Hassan, Jamiyah; Abu Bakar, Sazaly

2016-01-01

Background The 7.5?kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis?infected patie...

Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids

Energy Technology Data Exchange (ETDEWEB)

Casjens S. R.; Dunn J.; Mongodin, E. F.; Qiu, W.-G.; Luft, B. J.; Schutzer, S. E.; Gilcrease, E. B.; Huang, W. M.; Vujadinovic, M.; Aron, J. K.; Vargas, L. C.; Freeman, S.; Radune, D.; Weidman, J. F.; Dimitrov, G. I.; Khouri, H. M.; Sosa, J. E.; Halpin, R. A.; Fraser, C. M.

2012-03-14

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi {approx}900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short {le}20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.

Plasmid Conjugation in E. coli and Drug Resistance | Igwe ...

African Journals Online (AJOL)

This study aimed at determining the antibiotics susceptibility pattern of E. coli isolates claimed to be multidrug resistance using disc diffusion method. It also determined the presence of transferable resistance plasmids through conjugation and evaluated the medical significance of plasmid encoding E. coli and drug ...

Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

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Susu He

2016-12-01

Full Text Available The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance.

Plasmids of Staphylococcus cohnii isolated from the intensive-care unit.

Science.gov (United States)

Szewczyk, E M; Rózalska, M; Cieślikowski, T; Nowak, T

2004-01-01

Numerous isolates of both subspecies of Staphylococcus cohnii were found in the environment of the intensive-care unit of a pediatric hospital. These isolates carried in their cells many plasmids, up to fourteen, of a wide range of sizes ( 56 kb). Striking was the occurrence of large plasmids not very common in staphylococci. These were present in > 80% of S. cohnii isolates. Fifty-two different plasmid profiles were found in 79 investigated isolates belonging to S. cohnii ssp. cohnii and S. cohnii ssp. urealyticus. Isolates similar in plasmid profiles were grouped in antibiotic-resistance clusters established for 9 antibiotics (gentamicin, ciprofloxacin, clindamycin, erythromycin, tetracycline, chloramphenicol, mupirocin, trimethoprim-sulfamethoxazole, vancomycin) using the method of unweighted pair group mathematical averages (UPGMA). Many isolates were multiresistant to antibiotics and produced bacteriocins.

Genetic transformation of Bacillus strains close to bacillus subtilis and isolated from the soil

International Nuclear Information System (INIS)

Van, C.K.; Kuzin, Yu.Yu.; Kozlovskii, Yu.E.; Prozorov, A.A.

1986-01-01

Chromosomal and plasmid transformation was found in five out of 118 Bacillus strains, close or identical to Bacillus subtilis, and isolated from soil in Moscow or in the Moscow district. The efficiency of transformation in these strains was lower than that in derivatives of Bac. subtilis strain 168. In these strains the ability to undergo transformation was dependent on the rate of sporulation and the presence of restrictases. As in the case of Bac. subtilis 168 the strains isolated may be used as models in genetic transformation studies on Bac. subtilis

Quantification bias caused by plasmid DNA conformation in quantitative real-time PCR assay.

Science.gov (United States)

Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification.

Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

DEFF Research Database (Denmark)

Klümper, Uli; Riber, Leise; Dechesne, Arnaud

2014-01-01

and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse...

The Asd+-DadB+ Dual-Plasmid System Offers a Novel Means To Deliver Multiple Protective Antigens by a Recombinant Attenuated Salmonella Vaccine

Science.gov (United States)

Xin, Wei; Wanda, Soo-Young; Zhang, Xiangmin; Santander, Javier; Scarpellini, Giorgio; Ellis, Karen; Alamuri, Praveen

2012-01-01

We developed means to deliver multiple heterologous antigens on dual plasmids with non-antibiotic-resistance markers in a single recombinant attenuated vaccine strain of Salmonella enterica serotype Typhimurium. The first component of this delivery system is a strain of S. Typhimurium carrying genomic deletions in alr, dadB, and asd, resulting in obligate requirements for diaminopimelic acid (DAP) and d-alanine for growth. The second component is the Asd+-DadB+ plasmid pair carrying wild-type copies of asdA and dadB, respectively, to complement the mutations. To evaluate the protection efficacy of the dual-plasmid vaccine, S. Typhimurium strain χ9760 (a strain with multiple attenuating mutations: Δasd Δalr ΔdadB ΔrecF) was transformed with Asd+ and DadB+ plasmids specifying pneumococcal antigens PspA and PspC, respectively. Both plasmids were stable in χ9760 for 50 generations when grown in nonselective medium. This was significantly (P < 0.05) greater than the stability seen in its recF+ counterpart χ9590 and could be attributed to reduced interplasmid recombination in χ9760. Oral immunization of BALB/c mice with 1 × 109 CFU of χ9760 (carrying Asd+-PspA and DadB+-PspC plasmids) elicited a dominant Th1-type serum IgG response against both antigens and protected mice against intraperitoneal challenge with 200 50% lethal doses (LD50s) of virulent Streptococcus pneumoniae strain WU2 or intravenous challenge with 100 LD50s of virulent S. pneumoniae strain L81905 or intranasal challenge with a lethal dose of S. pneumoniae A66.1 in a pneumonia model. Protection offered by χ9760 was superior to that offered by the mixture of two strains, χ9828 (Asd+-PspA) and χ11026 (DadB+-PspC). This novel dual-plasmid system marks a remarkable improvement in the development of live bacterial vaccines. PMID:22868499

The Asd(+)-DadB(+) dual-plasmid system offers a novel means to deliver multiple protective antigens by a recombinant attenuated Salmonella vaccine.

Science.gov (United States)

Xin, Wei; Wanda, Soo-Young; Zhang, Xiangmin; Santander, Javier; Scarpellini, Giorgio; Ellis, Karen; Alamuri, Praveen; Curtiss, Roy

2012-10-01

We developed means to deliver multiple heterologous antigens on dual plasmids with non-antibiotic-resistance markers in a single recombinant attenuated vaccine strain of Salmonella enterica serotype Typhimurium. The first component of this delivery system is a strain of S. Typhimurium carrying genomic deletions in alr, dadB, and asd, resulting in obligate requirements for diaminopimelic acid (DAP) and d-alanine for growth. The second component is the Asd(+)-DadB(+) plasmid pair carrying wild-type copies of asdA and dadB, respectively, to complement the mutations. To evaluate the protection efficacy of the dual-plasmid vaccine, S. Typhimurium strain χ9760 (a strain with multiple attenuating mutations: Δasd Δalr ΔdadB ΔrecF) was transformed with Asd(+) and DadB(+) plasmids specifying pneumococcal antigens PspA and PspC, respectively. Both plasmids were stable in χ9760 for 50 generations when grown in nonselective medium. This was significantly (P < 0.05) greater than the stability seen in its recF(+) counterpart χ9590 and could be attributed to reduced interplasmid recombination in χ9760. Oral immunization of BALB/c mice with 1 × 10(9) CFU of χ9760 (carrying Asd(+)-PspA and DadB(+)-PspC plasmids) elicited a dominant Th1-type serum IgG response against both antigens and protected mice against intraperitoneal challenge with 200 50% lethal doses (LD(50)s) of virulent Streptococcus pneumoniae strain WU2 or intravenous challenge with 100 LD(50)s of virulent S. pneumoniae strain L81905 or intranasal challenge with a lethal dose of S. pneumoniae A66.1 in a pneumonia model. Protection offered by χ9760 was superior to that offered by the mixture of two strains, χ9828 (Asd(+)-PspA) and χ11026 (DadB(+)-PspC). This novel dual-plasmid system marks a remarkable improvement in the development of live bacterial vaccines.

Plasmids in Vibrio parahemolyticus strains isolated in Japan and Bangladesh with special reference to different distributions.

Science.gov (United States)

Arai, T; Ando, T; Kusakabe, A; Ullah, M A

1983-01-01

We surveyed plasmids in naturally occurring Vibrio parahemolyticus strains isolated in Japan and Bangladesh. Among the strains isolated in Japan, about half of the strains isolated from stools of patients of domestic diarrhea outbreaks as well as of travelers returning from East Asia were found to have plasmids, but no strains from foods had plasmids. In contrast, among the strains isolated in Bangladesh, none of the four strains isolated from patients had plasmids, but two out of eight strains isolated from water had plasmids, suggesting that plasmids are common in strains from the water in Bangladesh. All plasmids so far reported in V. parahemolyticus were detected in strains isolated from stools of patients. Incidences of plasmids in this organism were not so high in either area. In Japan, all plasmids were detected in strains from human intestines at 37 C, but in Bangladesh, where the temperature is around 30-40 C, the plasmids were detected in strains from the natural environment. These results suggested the possibility that these plasmids can come from different bacteria under rather high temperatures and that incidences of plasmids are influenced by the incidences of plasmids in bacteria present in the vicinity of V. parahemolyticus strains. None of these plasmids were found to have any relation to the biological characters tested.

Generation of doubled haploid transgenic wheat lines by microspore transformation.

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Rhoda A T Brew-Appiah

Full Text Available Microspores can be induced to develop homozygous doubled haploid plants in a single generation. In the present experiments androgenic microspores of wheat have been genetically transformed and developed into mature homozygous transgenic plants. Two different transformation techniques were investigated, one employing electroporation and the other co-cultivation with Agrobacterium tumefaciens. Different tissue culture and transfection conditions were tested on nine different wheat cultivars using four different constructs. A total of 19 fertile transformants in five genotypes from four market classes of common wheat were recovered by the two procedures. PCR followed by DNA sequencing of the products, Southern blot analyses and bio/histo-chemical and histological assays of the recombinant enzymes confirmed the presence of the transgenes in the T0 transformants and their stable inheritance in homozygous T1∶2 doubled haploid progenies. Several decisive factors determining the transformation and regeneration efficiency with the two procedures were determined: (i pretreatment of immature spikes with CuSO4 solution (500 mg/L at 4°C for 10 days; (ii electroporation of plasmid DNA in enlarged microspores by a single pulse of ∼375 V; (iii induction of microspores after transfection at 28°C in NPB-99 medium and regeneration at 26°C in MMS5 medium; (iv co-cultivation with Agrobacterium AGL-1 cells for transfer of plasmid T-DNA into microspores at day 0 for <24 hours; and (v elimination of AGL-1 cells after co-cultivation with timentin (200-400 mg/L.

Characterization of Plasmid pPO1 from the Hyperacidophile Picrophilus oshimae

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Angel Angelov

2011-01-01

Full Text Available Picrophilus oshimae and Picrophilus torridus are free-living, moderately thermophilic and acidophilic organisms from the lineage of Euryarchaeota. With a pH optimum of growth at pH 0.7 and the ability to even withstand molar concentrations of sulphuric acid, these organisms represent the most extreme acidophiles known. So far, nothing is known about plasmid biology in these hyperacidophiles. Also, there are no genetic tools available for this genus. We have mobilized the 7.6 Kbp plasmid from P. oshimae in E. coli by introducing origin-containing transposons and described the plasmid in terms of its nucleotide sequence, copy number in the native host, mode of replication, and transcriptional start sites of the encoded ORFs. Plasmid pPO1 may encode a restriction/modification system in addition to its replication functions. The information gained from the pPO1 plasmid may prove useful in developing a cloning system for this group of extreme acidophiles.

Sequential acquisition of R-plasmids in vivo by Salmonella typhimurium.

Science.gov (United States)

Platt, D J; Sommerville, J S; Gribben, J

1984-01-01

Salmonella typhimurium, resistant only to trimethoprim and sulphamethoxazole, was isolated from the faeces and blood of a chronic alcoholic patient in acute renal failure. The isolates harboured an 18 Md non-conjugative plasmid. He was dialysed peritoneally and treated with ampicillin; four days later there was no clinical improvement and his peritoneal dialysis fluid (PDF) had become infected. Salm. typhimurium was isolated from faeces and PDF. Both isolates were additionally resistant to ampicillin and contained two plasmids (55 Md and 18 Md). Therapy was changed to chloramphenicol and gentamicin was added to the PDF. Two weeks later Salm. typhimurium was again isolated from PDF and faeces. The PDF isolate was unchanged but 4% of the colonies isolated from this faecal specimen were resistant to chloramphenicol and had acquired an additional 62 Md plasmid. From all PDF and faecal specimens two different strains of Escherichia coli and one strain of Klebsiella pneumoniae were isolated which contained plasmids indistinguishable, on the basis of molecular weight and transferable resistance markers, from those acquired by Salm. typhimurium. The transferability of these plasmids in vitro to E. coli K12 and to the patient's initial Salm. typhimurium was studied and the results discussed.

Characterization of plasmids in a human clinical strain of Lactococcus garvieae.

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Mónica Aguado-Urda

Full Text Available The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25 encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

Broad-Host-Range IncP-1 plasmids and their resistance potential

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Magdalena ePopowska

2013-03-01

Full Text Available The plasmids of the incompatibility group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad host range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

Non-additive costs and interactions alter the competitive dynamics of co-occurring ecologically distinct plasmids.

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Morton, Elise R; Platt, Thomas G; Fuqua, Clay; Bever, James D

2014-03-22

Plasmids play an important role in shaping bacterial evolution and adaptation to heterogeneous environments. As modular genetic elements that are often conjugative, the selective pressures that act on plasmid-borne genes are distinct from those that act on the chromosome. Many bacteria are co-infected by multiple plasmids that impart niche-specific phenotypes. Thus, in addition to host-plasmid dynamics, interactions between co-infecting plasmids are likely to be important drivers of plasmid population dynamics, evolution and ecology. Agrobacterium tumefaciens is a facultative plant pathogen that commonly harbours two distinct megaplasmids. Virulence depends on the presence of the tumour-inducing (Ti) plasmid, with benefits that are primarily restricted to the disease environment. Here, we demonstrate that a second megaplasmid, the At plasmid, confers a competitive advantage in the rhizosphere. To assess the individual and interactive costs of these plasmids, we generated four isogenic derivatives: plasmidless, pAt only, pTi only and pAtpTi, and performed pairwise competitions under carbon-limiting conditions. These studies reveal a low cost to the virulence plasmid when outside of the disease environment, and a strikingly high cost to the At plasmid. In addition, the costs of pAt and pTi in the same host were significantly lower than predicted based on single plasmid costs, signifying the first demonstration of non-additivity between naturally occurring co-resident plasmids. Based on these empirically demonstrated costs and benefits, we developed a resource-consumer model to generate predictions about the frequencies of these genotypes in relevant environments, showing that non-additivity between co-residing plasmids allows for their stable coexistence across environments.

Reconstructing the complex evolutionary history of mobile plasmids in red algal genomes

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Lee, JunMo; Kim, Kyeong Mi; Yang, Eun Chan; Miller, Kathy Ann; Boo, Sung Min; Bhattacharya, Debashish; Yoon, Hwan Su

2016-01-01

The integration of foreign DNA into algal and plant plastid genomes is a rare event, with only a few known examples of horizontal gene transfer (HGT). Plasmids, which are well-studied drivers of HGT in prokaryotes, have been reported previously in red algae (Rhodophyta). However, the distribution of these mobile DNA elements and their sites of integration into the plastid (ptDNA), mitochondrial (mtDNA), and nuclear genomes of Rhodophyta remain unknown. Here we reconstructed the complex evolutionary history of plasmid-derived DNAs in red algae. Comparative analysis of 21 rhodophyte ptDNAs, including new genome data for 5 species, turned up 22 plasmid-derived open reading frames (ORFs) that showed syntenic and copy number variation among species, but were conserved within different individuals in three lineages. Several plasmid-derived homologs were found not only in ptDNA but also in mtDNA and in the nuclear genome of green plants, stramenopiles, and rhizarians. Phylogenetic and plasmid-derived ORF analyses showed that the majority of plasmid DNAs originated within red algae, whereas others were derived from cyanobacteria, other bacteria, and viruses. Our results elucidate the evolution of plasmid DNAs in red algae and suggest that they spread as parasitic genetic elements. This hypothesis is consistent with their sporadic distribution within Rhodophyta. PMID:27030297

Bacteria isolated from pristine high altitude environments in the Argentinean Andean wetlands: plasmid profile and multiple antibiotic resistance

International Nuclear Information System (INIS)

Dib, J.R.; Martinez, M.A.; Sineriz, F.; Farias, M.E.

2005-01-01

Full text: Andean wetlands, placed in the North-Western Argentine at 4,600 m altitude, are attractive for both, environmental and biotechnology studies. Most of these wetlands are completely remote and inaccessible, having a high salinity and metal contents, a wide range of daily temperature changes, and an important intensity of solar UV-B radiation. Bacteria isolated from these environments were identified by 16SrDNA sequence and resulted in Gram-positive colored bacteria. Interesting features, to our knowledge never reported so far from bacteria isolates from these pristine high altitude lake-environments, such as similar plasmids profiles and multiple antibiotic resistances are the focus of this work. At least two plasmids were found in all isolates studied by using modifications of the alkaline Iysis method. Their preliminary characterization in this work includes size, incompatibility group through PCR, genetic transference to suitable hosts by transformation and conjugation, and studies of possible relationships of them with antibiotic resistances. (author)

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

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Chen, Yuan-Jyue; Rao, Sundipta D; Seelig, Georg

2015-11-25

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Strategies and approaches in plasmidome studies—uncovering plasmid diversity disregarding of linear elements?

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Dib, Julián R.; Wagenknecht, Martin; Farías, María E.; Meinhardt, Friedhelm

2015-01-01

The term plasmid was originally coined for circular, extrachromosomal genetic elements. Today, plasmids are widely recognized not only as important factors facilitating genome restructuring but also as vehicles for the dissemination of beneficial characters within bacterial communities. Plasmid diversity has been uncovered by means of culture-dependent or -independent approaches, such as endogenous or exogenous plasmid isolation as well as PCR-based detection or transposon-aided capture, respectively. High-throughput-sequencing made possible to cover total plasmid populations in a given environment, i.e., the plasmidome, and allowed to address the quality and significance of self-replicating genetic elements. Since such efforts were and still are rather restricted to circular molecules, here we put equal emphasis on the linear plasmids which—despite their frequent occurrence in a large number of bacteria—are largely neglected in prevalent plasmidome conceptions. PMID:26074886

Recent trends in ionic liquid (IL) tolerant enzymes and microorganisms for biomass conversion.

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Portillo, Maria Del Carmen; Saadeddin, Anas

2015-01-01

Second generation biofuel production depends on lignocellulosic (LC) biomass transformation into simple sugars and their subsequent fermentation into alcohols. However, the main obstacle in this process is the efficient breakdown of the recalcitrant cellulose to sugar monomers. Hence, efficient feedstock pretreatment and hydrolysis are necessary to produce a cost effective biofuel. Recently, ionic liquids (ILs) have been recognized as a promising solvent able to dissolve different biomass feedstocks, providing higher sugar yields. However, most of the hydrolytic enzymes and microorganisms are inactivated, completely or partially, in the presence of even low concentrations of IL, making necessary the discovery of novel hydrolytic enzymes and fermentative microorganisms that are tolerant to ILs. In this review, the current state and the challenges of using ILs as a pretreatment of LC biomass was evaluated, underlining the advances in the discovery and identification of new IL-tolerant enzymes and microorganisms that could improve the bioprocessing of biomass to fuels and chemicals.

Repair promoted by plasmid pKM101 is different from SOS repair

International Nuclear Information System (INIS)

Goze, A.; Devoret, R.

1979-01-01

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid PKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although Wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions. (Auth.)

The complete sequence and comparative analysis of a multidrug- resistance and virulence multireplicon IncFII plasmid pEC302/04 from an extraintestinal pathogenic Escherichia coli EC302/04 indicate extensive diversity of IncFII plasmids

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Wing Sze eHo

2016-01-01

Full Text Available Extraintestinal pathogenic Escherichia coli (ExPEC that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA and FIB with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok and a plasmid partitioning system, ParAB and PsiAB, which are important for plasmid maintenance were also found.Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of

The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids.

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Ho, Wing Sze; Yap, Kien-Pong; Yeo, Chew Chieng; Rajasekaram, Ganeswrie; Thong, Kwai Lin

2015-01-01

Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as bla TEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical

Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1

DEFF Research Database (Denmark)

Basta, Tamara; Smyth, John; Forterre, Patrick

2009-01-01

. Although nucleotide sequence comparisons revealed extensive intergenomic exchange during the evolution of archaeal conjugative plasmids, pAH1 was shown to be stably maintained suggesting that the host system is suitable for studying plasmid-virus interactions. AFV1 infection and propagation leads to a loss...... of the circular form of pAH1 and this effect correlates positively with the increase in the intracellular quantity of AFV1 DNA. We infer that the virus inhibits plasmid replication since no pAH1 degradation was observed. This mechanism of archaeal viral inhibition of plasmid propagation is not observed...... in bacteria where relevant bacteriophages either are dependent on a conjugative plasmid for successful infection or are excluded by a resident plasmid....

Effects of 32 P incorporated in plasmid DNA: strand breaks and mutagenesis

International Nuclear Information System (INIS)

Fonseca, Adenilson de S. da; Felzenszwalb, Israel

1996-01-01

In order to study the 32 P decay effects in DNA, bacterial plasmid were labeled with different activities of the radioisotope in vivo: 1,2 and 6 x 10 5 Bk/ml of bacterial culture, leading to 1,2 and 6 x 10 3 Bk/μg of nucleic acid or in vitro: 0.7, 1.5 and 3.5 x 10 3 Bk/μg of nucleic acid, stored at -20 deg C and its electroforetic profiles, transformation capacity of wild type and DNA repair. E. coli mutants cells and mutagenesis, were followed during three months. The results achieved in this work suggest that: the decay of the incorporated 32 P in vivo is able to change the pBR322 electroforetic profile, we detected a decrease on the form III (super coiled) and increase on the form II (circular), indicating single strands breaks; the decay incorporated 32 in vitro does not modify the electrophoretic profile of pBR322, suggesting that in some way the effects of the radioactive decay of incorporated 32 P is dependent of the DNA topology, the damages induced by 32 P decay increase mutation frequency in pAC189 plasmids. MRF is increased by a factor of three after 6 t 1/2 of storage, indicating direct or indirect action through mismatch DNA repair pathway. (author)

Agrobacterium-medicated transformation of mature Prunus serotina (black cherry) and regeneration of trangenic shoots

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Xiaomei Liu; Paula Pijut

2010-01-01

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced...

An Efficient Agrobacterium-Mediated Transformation of Strawberry cv. Camarosa by a Dual Plasmid System

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Fatemeh Haddadi

2015-02-01

Full Text Available An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100% of shoot formation and the highest mean number of shoots (24 produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86% in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

Novel methods for genetic transformation of natural Bacillus subtilis isolates used to study the regulation of the mycosubtilin and surfactin synthetases

NARCIS (Netherlands)

Duitman, Erwin H.; Wyczawski, Dobek; Boven, Ludolf G.; Venema, Gerard; Kuipers, Oscar P.; Hamoen, Leendert W.

Natural isolates of Bacillus subtilis are often difficult to transform due to their low genetic competence levels. Here we describe two methods that stimulate natural transformation. The first method uses plasmid pGSP12, which expresses the competence transcription factor ComK and stimulates

Comparative metagenomic analysis of plasmid encoded functions in the human gut microbiome

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Marchesi Julian R

2010-01-01

Full Text Available Abstract Background Little is known regarding the pool of mobile genetic elements associated with the human gut microbiome. In this study we employed the culture independent TRACA system to isolate novel plasmids from the human gut microbiota, and a comparative metagenomic analysis to investigate the distribution and relative abundance of functions encoded by these plasmids in the human gut microbiome. Results Novel plasmids were acquired from the human gut microbiome, and homologous nucleotide sequences with high identity (>90% to two plasmids (pTRACA10 and pTRACA22 were identified in the multiple human gut microbiomes analysed here. However, no homologous nucleotide sequences to these plasmids were identified in the murine gut or environmental metagenomes. Functions encoded by the plasmids pTRACA10 and pTRACA22 were found to be more prevalent in the human gut microbiome when compared to microbial communities from other environments. Among the most prevalent functions identified was a putative RelBE toxin-antitoxin (TA addiction module, and subsequent analysis revealed that this was most closely related to putative TA modules from gut associated bacteria belonging to the Firmicutes. A broad phylogenetic distribution of RelE toxin genes was observed in gut associated bacterial species (Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, but no RelE homologues were identified in gut associated archaeal species. We also provide indirect evidence for the horizontal transfer of these genes between bacterial species belonging to disparate phylogenetic divisions, namely Gram negative Proteobacteria and Gram positive species from the Firmicutes division. Conclusions The application of a culture independent system to capture novel plasmids from the human gut mobile metagenome, coupled with subsequent comparative metagenomic analysis, highlighted the unexpected prevalence of plasmid encoded functions in the gut microbial ecosystem. In

Magnetohydrodynamics Nanofluid Flow Containing Gyrotactic Microorganisms Propagating Over a Stretching Surface by Successive Taylor Series Linearization Method

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Shahid, A.; Zhou, Z.; Bhatti, M. M.; Tripathi, D.

2018-03-01

Nanofluid dynamics with magnetohydrodynamics has tremendously contributed in industrial applications recently since presence of nanoparticle in base fluids enhances the specific chemical and physical properties. Owing to the relevance of nanofluid dynamics, we analyze the nanofluid flow in the presence of gyrotactic microorganism and magnetohydrodynamics through a stretching/shrinking plate. The impacts of chemical reaction and thermal radiation on flow characteristics are also studied. To simplify the governing equations of microorganisms, velocity, concentration and temperature, the similarity transformations are employed. The couple governing equations are numerically solved using Successive Taylor Series Linearization Method (STSLM). The velocity profile, motile microorganism density profile, concentration profile, temperature profile as well as Nusselt number, skin friction coefficient, Sherwood number and density number of motile microorganisms are discussed using tables and graphs against all the sundry parameters. A numerical comparison is also given for Nusselt number, Sherwood number, skin friction, and density number of motile microorganisms with previously published results to validate the present model. The results show that Nusselt number, Sherwood number and density number diminish with increasing the magnetic field effects.

Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid

NARCIS (Netherlands)

Okker, Robert J.H.; Spaink, Herman; Hille, Jacques; Brussel, Ton A.N. van; Lugtenberg, Ben; Schilperoort, Rob A.

1984-01-01

Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for

High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

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Yanping Wen

Full Text Available Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4% were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2 and 32 isolates (17.0% were positive for aac(6'-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6'-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05. In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05. All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6'-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.

Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar.

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Yo Sugawara

Full Text Available The bacterial enzyme New Delhi metallo-β-lactamase hydrolyzes almost all β-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-β-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1, IncX3 plasmids harboring blaNDM-4 (n = 2 or blaNDM-7 (n = 1, IncFII plasmids harboring blaNDM-4 (n = 1 or blaNDM-5 (n = 3, and a multireplicon F plasmid harboring blaNDM-5 (n = 1. Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.

Factors affecting the efficient transformation of Colletotrichum species

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Redman, Regina S.; Rodriguez, Rusty J.

1994-01-01

Factors affecting the efficient transformation of Colletotrichum species. Experimental Mycology, 18, 230-246. Twelve isolates representing four species of Colletotrichum were transformed either by enhanced protoplast, restriction enzyme-mediated integration (REMI), or electroporation-mediated protocols. The enhanced protoplast transformation protocol resulted in 100- and 50-fold increases in the transformation efficiencies of Colletotrichum lindemuthianum and C. magna , respectively. REMI transformation involved the use of Hin dIII and vector DNA linearized with HindIII to increase the number of integration events and potential gene disruptions in the fungal genome. Combining the enhanced protoplast and the REMI protocols resulted in a 22-fold increase in the number of hygromycin/nystatin-resistant mutants in C. lindemuthianum . Electroporation-mediated transformation was performed on mycelial fragments and spores of four Colletotrichum species, resulting in efficiencies of up to 1000 transformants/μg DNA. The pHA1.3 vector which confers hygromycin resistance contains telomeric sequences from Fusarium oxysporum , transforms by autonomous replication and genomic integration, and was essential for elevated transformation efficiencies of 100 to 10,000 transformants/μg DNA. Modifications of pHA1.3 occurred during bacterial amplification and post fungal transformation resulting in plasmids capable of significantly elevated transformation efficiencies in C. lindemuthianum.

Loss of cellular transformation efficiency induced by DNA irradiation with low-energy (10 eV) electrons.

Science.gov (United States)

Kouass Sahbani, Saloua; Sanche, Leon; Cloutier, Pierre; Bass, Andrew D; Hunting, Darel J

2014-11-20

Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in biological matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (-)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap(2+)). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to transforming cluster damage into DSBs by digestion with repair enzymes, also occurred relatively infrequently. The exact nature of the lethal damage remains unknown, but it is probably a form of compact cluster damage in which the lesions are too close to be revealed by purified repair enzymes. In addition, this damage is either not repaired or is misrepaired by E. coli, since it results in plasmid inactivation, when they contain an average of three lesions. Comparison with previous results from a similar experiment performed with γ-irradiated plasmids indicates that the type of clustered DNA lesions, created directly on cellular DNA by LEEs, may be more difficult to repair than those produced by other species from radiolysis.

Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

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Mart Krupovic

Full Text Available Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1, with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

Two novel conjugative plasmids from a single strain of Sulfolobus

NARCIS (Netherlands)

Erauso, G.; Stedman, K.M.; Werken, van de H.J.G.; Zillig, W.; Oost, van der J.

2006-01-01

Two conjugative plasmids (CPs) were isolated and characterized from the same 'Sulfolobus islandicus' strain, SOG2/4, The plasmids were separated from each other and transferred into Sulfolobus soltataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Science.gov (United States)

Ahn, Young-Beom; Rhee, Sung-Keun; Fennell, Donna E.; Kerkhof, Lee J.; Hentschel, Ute; Häggblom, Max M.

2003-01-01

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration. PMID:12839794

Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

NARCIS (Netherlands)

Diepeningen, van A.D.; Debets, A.J.M.; Slakhorst-Wandel, S.M.; Hoekstra, R.F.

2008-01-01

Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with

Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence

NARCIS (Netherlands)

van Diepeningen, Anne D; Debets, Alfons J M; Slakhorst, S Marijke; Hoekstra, Rolf F

Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and homologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with

Plasmid profiles and antibiotic susceptibility patterns of Staphylococcus aureus isolates from Nigeria.

Science.gov (United States)

Olukoya, D K; Asielue, J O; Olasupo, N A; Ikea, J K

1995-06-01

In an investigation into the problems of infections due to Staphylococcus aureus in Nigeria, 100 strains were isolated from various hospitals in Lagos. The strains were screened for the presence of plasmids and for susceptibility to antimicrobial agents. Plasmids were extracted by modification of the method of Takahashi and Nagono[1]. The plasmids were diverse in nature. The strains were found to be highly resistant to commonly prescribed antibiotics.

Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles

Directory of Open Access Journals (Sweden)

Olson Daniel G

2012-05-01

Full Text Available Abstract Background Temperature-sensitive (Ts plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996 for predicting Ts mutants based on the amino-acid sequence of the protein. Results A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.

Giant linear plasmids in Streptomyces: a treasure trove of antibiotic biosynthetic clusters.

Science.gov (United States)

Kinashi, Haruyasu

2011-01-01

Many giant linear plasmids have been isolated from Streptomyces by using pulsed-field gel electrophoresis and some of them were found to carry an antibiotic biosynthetic cluster(s); SCP1 carries biosynthetic genes for methylenomycin, pSLA2-L for lankacidin and lankamycin, and pKSL for lasalocid and echinomycin. Accumulated data suggest that giant linear plasmids have played critical roles in genome evolution and horizontal transfer of secondary metabolism. In this review, I summarize typical examples of giant linear plasmids whose involvement in antibiotic production has been studied in some detail, emphasizing their finding processes and interaction with the host chromosomes. A hypothesis on horizontal transfer of secondary metabolism involving giant linear plasmids is proposed at the end.

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.

Science.gov (United States)

Cao, Qing-Hua; Shao, Huan-Huan; Qiu, Hui; Li, Tao; Zhang, Yi-Zheng; Tan, Xue-Mei

2017-03-01

The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.

Transfer of conjugative plasmids among bacteria under environmentally relevant conditions

DEFF Research Database (Denmark)

Musovic, Sanin

Mobile genetiske elementer (f.eks. plasmider), der ofte bærer ekstra funktioner såsom antibiotikaresistens, eller kataboliske- og xenobiotiske nedbrydnings gener, antages at have en meget vigtigt evolutionær rolle for bakterier. I denne PhD afhandling undersøgte jeg størrelsen af plasmid overførs...

Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate.

Science.gov (United States)

Bastaki, Nasmah K; Cullis, Christopher A

2014-12-19

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation. In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

OpenAIRE

Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

2008-01-01

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK h...

Effects of Atrazine on Soil Microorganisms

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Ljiljana Radivojević

2006-01-01

Full Text Available Effects of the herbicide atrazine on soil microorganisms was investigated. Trials were set up in laboratory, on a clay loam soil. Atrazine was applied at 8.0, 40.0 and 80.0 mg/kg soil rates. The abundance of total microorganisms, fungi, actinomycetes, cellulolytic microorganisms and amino-heterotrophs was recorded. Soil samples were collected 1, 7, 14, 21, 30 and 60 days after atrazine treatment for microbiological analyses.The results showed that the intensity of atrazine effect on soil microorganisms depended on treatment rate, exposure time and group of microorganisms. Atrazine had an inhibiting effect on cellulolytic microorganisms and amino-heterotrophs. Initially, it inhibited fungiand actinomycetes but its effect turned into a stimulating one once a population recovered. Atrazine had a stimulating effect on total abundance of microorganisms.

Quantification of plasmid DNA reference materials for Shiga toxin-producing Escherichia coli based on UV, HR-ICP-MS and digital PCR.

Science.gov (United States)

Liang, Wen; Xu, Li; Sui, Zhiwei; Li, Yan; Li, Lanying; Wen, Yanli; Li, Chunhua; Ren, Shuzhen; Liu, Gang

2016-01-01

The accuracy and metrology traceability of DNA quantification is becoming a critical theme in many fields, including diagnosis, forensic analysis, microorganism detection etc. Thus the research of DNA reference materials (RMs) and consistency of DNA quantification methods has attracted considerable research interest. In this work, we developed 3 plasmid candidate RMs, containing 3 target genes of Escherichia coli O157:H7 (E. coli O157:H7) and other Shiga toxin-producing Escherichia coli (STEC): stx1, stx2, and fliC (h7) respectively. Comprehensive investigation of the plasmid RMs was performed for their sequence, purity, homogeneity and stability, and then the concentration was quantified by three different methods: ultraviolet spectrophotometer (UV), high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) and digital PCR. As a routinely applied method for DNA analysis, UV was utilized for the quantification (OD260) and purity analysis for the plasmids. HR-ICP-MS quantified the plasmid DNA through analysing the phosphorus in DNA molecules. Digital PCR distributed the DNA samples onto a microarray chip containing thousands of reaction chambers, and quantified the DNA copy numbers by analysing the number of positive signals without any calibration curves needed. Based on the high purification of the DNA reference materials and the optimization of dPCR analysis, we successfully achieved good consistency between UV, HR-ICP-MS and dPCR, with relative deviations lower than 10 %. We then performed the co-quantification of 3 DNA RMs with three different methods together, and the uncertainties of their concentration were evaluated. Finally, the certified values and expanded uncertainties for 3 DNA RMs (pFliC, pStx1 and pStx2) were (1.60 ± 0.10) × 10(10) copies/μL, (1.53 ± 0.10) × 10(10) copies/μL and (1.70 ± 0.11) × 10(10) copies/μL respectively.Graphical abstractWe developed 3 plasmid candidate RMs, containing 3 target genes of

Monitoring growth and movement of Ralstonia solanacearum cells harboring plasmid pRSS12 derived from bacteriophage phiRSS1.

Science.gov (United States)

Fujie, Makoto; Takamoto, Hirofumi; Kawasaki, Takeru; Fujiwara, Akiko; Yamada, Takashi

2010-02-01

We monitored growth and movement of Ralstonia solanacearum harboring the plasmid pRSS12 in tomato seedlings. The plasmid contains a gene for green fluorescent protein (GFP) and is stably maintained in R. solanacearum cells without selection pressure. Bacteria harboring the plasmid can be tracked in planta by visualizing GFP fluorescence. Stems of seedlings were infected with R. solanacearum cells transformed with pRSS12, and bacterial growth and movement, particularly around the vascular bundles, were monitored for more than 7 days. Our results showed that vascular bundles are independent of each other within the stem, and that it takes a long time for R. solanacearum cells to migrate from one vascular bundle to another. For real-time monitoring of bacteria in planta, tomato seedlings were grown on agar medium and bacterial suspension was applied to the root apex. The bacterial invasion process was monitored by fluorescent microscopy. Bacteria invaded taproots within 6 h, and movement of the bacteria was observed until 144 h after inoculation. In susceptible tomato cultivars, strong GFP fluorescence was observed in hypocotyls and lateral roots as well as the taproot. In resistant cultivars, however, GFP fluorescence was rarely observed on lateral roots. Our results show that this monitoring system can be used to assess bacterial pathogenicity efficiently. Copyright (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Isolation and properties of plasmids from Deinococcus radiodurans Sark

International Nuclear Information System (INIS)

Sjarief, S.H.; Kikuchi, Masahiro; Kurita, Hiromi; Kitayama, Shigeru; Watanabe, Hiroshi.

1990-05-01

Radioresistant bacterium, Deinococcus radiodurans, can repair completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 kGy. In order to reveal the repair mechanism, it is necessary to develop a cloning vector available for the genetic analysis. We tried to isolate plasmids from D.radiodurans Sark strain. In the present paper the isolation and properties of plasmids were described. (author)

Molecular processes as basis for plasmid-mediated bacterial UV-light resistance and mutagenesis

International Nuclear Information System (INIS)

Aleshkin, G.I.; Brukhanskij, G.V.; Skavronskaya, A.G.

1985-01-01

The increase of UV-resistance and UV-induced mutagenesis by lambda 1 pint intmid as well as molecular-genetic mechanisms of plasmid participation in reparation and DNA replication and its degradation after UV-irradiation in plasmid cells on pKM101 plasmid model have been investigated. Data testifying to the necessity of intmid integration in chromosome as obligatory stage of intmid participation in increasing UV-resistance of bacterial cells are obtained. It has been found that intmid raises UV-resistance of cells and increases respectively the UV-induced reverants efficiency. On the basis of the experiment data the conclusion is drawn that the intmid capacity to raise UV-resistance and, possibly, mutagenesis is bound not only with its integration into chromosome but also with pol A + chromosome replication by dependendent imtmid replication complex. It is shown that pKM101 plasmid ensures functioning in E coli cells of inducible, chloroamphenicol-resistant DNA replication, highly resistant to UV-light harmful effect and that the volume of excision reparation in E. coli cells carrying pKM101 plasmid is increased as compared with the volume of reparation in plasmid legs cells. The combination of the data obtained gives grounds to the authors to assume that inducible replication, inducible reparation of DNA and inducible decrease of DNA degradation determined by pKM101 plasmid may serve as recA + lexA + basis dependent increase of UV-resistance and mutagenesis and that these processes provide the possibility of functioning of integrative replication mechanism of plasmid participation in ensuring UV-resistance and mutagenesis of plants

Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains.

Science.gov (United States)

Jones, M K; Iwanejko, L A; Longden, M S

1989-09-01

Plasmid analysis of over 120 strains of Clostridium perfringens, isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6.2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.

Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

DEFF Research Database (Denmark)

Migura, Lourdes Garcia; Valenzuela, Antonio Jesus Sanchez; Jensen, Lars Bogø

2011-01-01

developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids....

Infectious alphavirus production from a simple plasmid transfection+

Directory of Open Access Journals (Sweden)

Olson Ken E

2011-07-01

Full Text Available Abstract We have developed a new method for producing infectious double subgenomic alphaviruses from plasmids transfected into mammalian cells. A double subgenomic Sindbis virus (TE3'2J was transcribed from a cytomegalovirus PolII promoter, which results in the production of infectious virus. Transfection of as little as 125 ng of plasmid is able to produce 1 × 108 plaque forming units/ml (PFU/ml of infectious virus 48 hours post-transfection. This system represents a more efficient method for producing recombinant Sindbis viruses.

Optimization of agrobacterium tumefaciens mediated transformation in eucalyptus camaldulensis

International Nuclear Information System (INIS)

Ahad, A.; Maqbool, A.; Malik, K.A.

2014-01-01

This study was conducted to optimize Agrobacterium tumefaciens mediated transformation for Eucalyptus camaldulensis. Transformation was done by using LBA4404 containing binary plasmid pGA482 with uidA (Gus) gene under CamV35S promoter and nptII gene under nos promoter. For optimization, different explants (Cotyledonary leaves, plantlet leaves and hypocotyls of young In vitro plants and calli) with and without preculture were infected with a range of optical densities (O.D.600nm=0.3-0.6). Effect of different concentrations of Acetosyringone, infection time and co-cultivation time on transformation efficiency was evaluated. Confirmation of transformation was done through GUS histochemical staining and through PCR. Callogenesis and regeneration was found fast on MS medium containing 0.5 mg/L NAA and 1.5 mg/L BAP. Highest transformation efficiency was obtained with bacterial suspension of O.D.600nm = 0.5 for non-precultured explants and O.D.600nm=0.3 for precultured explants. (author)

Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

DEFF Research Database (Denmark)

Porse, Andreas; Schønning, Kristian; Munck, Christian

2016-01-01

sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions...... consistently followed by all evolved E. coli lineages exposes a trade-off between horizontal and vertical transmission that may ultimately limit the dissemination potential of clinical multidrug resistance plasmids in these hosts....

Plasmid DNA Analysis of Pasteurella multocida Serotype B isolated from Haemorrhagic Septicaemia outbreaks in Malaysia

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Jamal, H.

2005-01-01

Full Text Available A total of 150 purified isolates of Pasteurella multocida serotype B were used (Salmah, 2004 for plasmid DNA curing experiment to determine hyaluronidase activity, antibiotic resistance pattern (ARP and mice lethality test (LD50 for their role of pathogenicity. A plasmid curing experiment was carried out by using the intercalating agent; ethidium bromide and rifampicin, where it was found all the plasmids had been cured (plasmidless from Pasteurella multocida. All of these plasmidless isolates maintained their phenotypic characteristics. They showed the same antibiotic resistancepattern as before curing, produced hyaluronidase and possessed lethality activity in mice when injected intraperitoneally(i.p. Based on this observation, the antibiotic resistance, hyaluronidase activity and mice virulence could probably be chromosomal-mediated. Plasmids were detected 100% in all P. multocida isolates with identical profile of 2 plasmids size 3.0 and 5.5 kb. No large plasmids could be detected in all isolates. Since all the isolates appeared to have identicalplasmid profiles, they were subjected to restriction enzyme(RE analysis. From RE analysis results obtained, it can be concluded that the plasmid DNA in serotype B isolates are identical. Only 4 of 32 REs were found to cleave these plasmids with identical restriction fingerprints; BglII, HaeIII, RsaI and SspI. From RE analysis results, it can be concluded that the plasmid DNA isolates are identical. This plasmid might not played any role in pathogenicity of Pasteurella multocida serotype B, however this information is important for the construction of shuttle vectors in genetic studies of the pathogenicity of haemorrhagic septicaemia(HS.

Microorganisms in food technology

Energy Technology Data Exchange (ETDEWEB)

Rose, A H

1981-11-01

Man has been using microorganisms for thousands of years to make bread, cheese, beer, wine, etc. Today, microorganisms can be specially grown or genetically manipulated so as to synthesize high-quality proteins even from low-grade basic materials.

Centromere pairing by a plasmid-encoded type I ParB protein

DEFF Research Database (Denmark)

Ringgaard, Simon; Löwe, Jan; Gerdes, Kenn

2007-01-01

The par2 locus of Escherichia coli plasmid pB171 encodes two trans-acting proteins, ParA and ParB, and two cis-acting sites, parC1 and parC2, to which ParB binds cooperatively. ParA is related to MinD and oscillates in helical structures and thereby positions ParB/parC-carrying plasmids regularly......, hence identifying the N terminus of ParB as a requirement for ParB-mediated centromere pairing. These observations suggest that centromere pairing is an important intermediate step in plasmid partitioning mediated by the common type I loci....

Fossil Microorganisms in Archaean

Science.gov (United States)

Astafleva, Marina; Hoover, Richard; Rozanov, Alexei; Vrevskiy, A.

2006-01-01

Ancient Archean and Proterozoic rocks are the model objects for investigation of rocks comprising astromaterials. The first of Archean fossil microorganisms from Baltic shield have been reported at the last SPIE Conference in 2005. Since this confeence biomorphic structures have been revealed in Archean rocks of Karelia. It was determined that there are 3 types of such bion structures: 1. structures found in situ, in other words microorganisms even-aged with rock matrix, that is real Archean fossils biomorphic structures, that is to say forms inhabited early formed rocks, and 3. younger than Archean-Protherozoic minerali microorganisms, that is later contamination. We made attempt to differentiate these 3 types of findings and tried to understand of burial of microorganisms. The structures belongs (from our point of view) to the first type, or real Archean, forms were under examination. Practical investigation of ancient microorganisms from Green-Stone-Belt of Northern Karelia turns to be very perspective. It shows that even in such ancient time as Archean ancient diverse world existed. Moreover probably such relatively highly organized cyanobacteria and perhaps eukaryotic formes existed in Archean world.

Analysis of Redox Responses During TNT Transformation by Clostridium acetobutylicum ATCC 824 and Mutants Exhibiting Altered Metabolism

Science.gov (United States)

2012-01-01

vivo methylation in Escherichia coli by the Bacillus subtilis phage ϕ3T methyltrans- ferase to protect plasmids from restriction upon transformation of...buffered CGM medium with appropriate antibiotics and incubated overnight as seed cul- tures. An appropriate amount from seed culture was used for the...protocol (Mermelstein and Papoutsakis 1993); positive transformants were isolated on agar-solidified CGM supple- mented with the appropriate antibiotic

Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

Science.gov (United States)

Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

2001-01-01

This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

Biosurfactants from marine microorganisms

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Suppasil Maneerat

2005-11-01

Full Text Available Biosurfactants are the surface-active molecules synthesized by microorganisms. With the advantage of environmental compatibility, the demand for biosurfactants has been steadily increasing and may eventually replace their chemically synthesized counterparts. Marine biosurfactants produced by some marine microorganisms have been paid more attention, particularly for the bioremediation of the sea polluted by crude oil. This review describes screening of biosurfactant-producing microorganisms, the determination of biosurfactant activity as well as the recovery of marine surfactant. The uses of marine biosurfactants for bioremediation are also discussed.

Quorum-Dependent Mannopine-Inducible Conjugative Transfer of an Agrobacterium Opine-Catabolic Plasmid

Science.gov (United States)

Wetzel, Margaret E.; Kim, Kun-Soo; Miller, Marilyn; Olsen, Gary J.

2014-01-01

The Ti plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related plasmids. Sequence analysis of one such plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the plasmid and produced little to no quormone even when grown with MOP, indicating that TraRpAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved. PMID:24363349

Plasmids which make their host bacteria mutable as well as resistant to ultraviolet irradiation

International Nuclear Information System (INIS)

Arai, Toshihiko; Ando, Takao

1980-01-01

Some of the naturally occurring Iα, I zeta, M, N, O and T group plasmids increase both the mutability and UV resistance of their host bacteria, while group H and S plasmids only increase mutability. This suggests that these two plasmid-mediated repair functions are separable. The two functions have no direct relation to their restriction-modification systems and nitrofuran resistant functions. In addition, the close linking between the restriction-modification genes and these repair function genes was suggested in group N plasmids. (author)

Transformation of medicinal plants using Agrobacterium tumefaciens

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Katarzyna Bandurska

2016-12-01

Full Text Available For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall. This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.

Transformation of medicinal plants using Agrobacterium tumefaciens.

Science.gov (United States)

Bandurska, Katarzyna; Berdowska, Agnieszka; Król, Małgorzata

2016-12-20

For many years attempts are made to develop efficient methods for transformation of medicinal plants via Agrobacterium tumefaciens. It is a soil bacteria which possess a natural ability to infect plants in places of injures which results in arise of cancerous growths (crown gall). This is possible thanks a transfer of fragment of Ti plasmid into plant cells and stable integration with a plant genome. Efficiency of medicinal plant transformation depends on many factors for example: Agrobacterium strain, methods and procedures of transformation as well as on plant species, type and age of the explants and regeneration conditions. The main goal of plant transformation is to increase the amount of naturally occurring bioactive compounds and the production of biopharmaceuticals. Genetic plant transformation via bacteria of the genus Agrobacterium is a complex process which requires detailed analysis of incorporated transgene expression and occurs only in the case when the plant cell acquires the ability to regenerate. In many cases, the regeneration efficiency observed in medicinal plants are inefficient after applied transformation procedures. To date there have been attempts of genetic transformation by using A. tumefaciens of medicinal plants belonging to the families: Apocynaceae, Araceae, Araliaceae, Asphodelaceae, Asteraceae, Begoniaceae, Crassulaceae, Fabaceae, Lamiaceae, Linaceae, Papaveraceae, Plantaginaceae, Scrophulariaceae and Solanaceae.

Antibiotic resistance and plasmid carriage among Escherichia coli isolates from chicken meat in Malaysia

International Nuclear Information System (INIS)

Tin Tin Myaing; Saleha, A.A.; Arifah, A.K.; Raha, A.R.

2005-01-01

Escherichia coli isolates from 131 raw chicken meat samples were tested for susceptibility to 12 antibiotics. Plasmids were isolated from many samples and their DNA molecular weight calculated. An 81.7% plasmid occurrence rate was observed among the isolates, ranging from 0 to 8 in number and with sizes from 1.2 to 118.6 MDa. Plasmids were detected in 93.8% of E. coIi isolates resistant to all 12 antibiotics, and in 90.5% of E. coli isolates resistant to 11. Three (2.8%) isolates harboured 8 plasmids and were resistant to all 12 antibiotics. Antibiotic resistant genes in bacteria are usually carried in extrachromosomal DNA and it is postulated that E. coli with a high number of plasmids possesses wider resistance to antibiotics. (author)

Expansion of a plasmid classification system for Gram-positive bacteria and determination of the diversity of plasmids in Staphylococcus aureus strains of human, animal, and food origins

DEFF Research Database (Denmark)

Lozano, C.; Garcia-Migura, L.; Aspiroz, C.

2012-01-01

An expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92 Staphylococcus aureus strains of different origins. rep genes of other genera were detected in Staphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybrid...

Specific structural probing of plasmid-coded ribosomal RNAs from Escherichia coli

DEFF Research Database (Denmark)

Aagaard, C; Rosendahl, G; Dam, M

1991-01-01

The preferred method for construction and in vivo expression of mutagenised Escherichia coli ribosomal RNAs (rRNAs) is via high copy number plasmids. Transcription of wild-type rRNA from the seven chromosomal rrn operons in strains harbouring plasmid-coded mutant rRNAs leads to a heterogeneous...

Distribution of Plasmids in Distinct Leptospira Pathogenic Species.

Science.gov (United States)

Wang, Yanzhuo; Zhuang, Xuran; Zhong, Yi; Zhang, Cuicai; Zhang, Yan; Zeng, Lingbing; Zhu, Yongzhang; He, Ping; Dong, Ke; Pal, Utpal; Guo, Xiaokui; Qin, Jinhong

2015-11-01

Leptospirosis, caused by pathogenic Leptospira, is a worldwide zoonotic infection. The genus Leptospira includes at least 21 species clustered into three groups--pathogens, non-pathogens, and intermediates--based on 16S rRNA phylogeny. Research on Leptospira is difficult due to slow growth and poor transformability of the pathogens. Recent identification of extrachromosomal elements besides the two chromosomes in L. interrogans has provided new insight into genome complexity of the genus Leptospira. The large size, low copy number, and high similarity of the sequence of these extrachromosomal elements with the chromosomes present challenges in isolating and detecting them without careful genome assembly. In this study, two extrachromosomal elements were identified in L. borgpetersenii serovar Ballum strain 56604 through whole genome assembly combined with S1 nuclease digestion following pulsed-field gel electrophoresis (S1-PFGE) analysis. Further, extrachromosomal elements in additional 15 Chinese epidemic strains of Leptospira, comprising L. borgpetersenii, L. weilii, and L. interrogans, were successfully separated and identified, independent of genome sequence data. Southern blot hybridization with extrachromosomal element-specific probes, designated as lcp1, lcp2 and lcp3-rep, further confirmed their occurrences as extrachromosomal elements. In total, 24 plasmids were detected in 13 out of 15 tested strains, among which 11 can hybridize with the lcp1-rep probe and 11 with the lcp2-rep probe, whereas two can hybridize with the lcp3-rep probe. None of them are likely to be species-specific. Blastp search of the lcp1, lcp2, and lcp3-rep genes with a nonredundant protein database of Leptospira species genomes showed that their homologous sequences are widely distributed among clades of pathogens but not non-pathogens or intermediates. These results suggest that the plasmids are widely distributed in Leptospira species, and further elucidation of their biological

Radiation-induced irreparable heritable changes in cells promoting their tumoral transformation

International Nuclear Information System (INIS)

Kuzin, A.M.; Vagabova, M.Eh.; Yurov, S.S.

1988-01-01

In experiments with model plant tumors (Kalanchoe-ti plasmid Agrobat. tumefaciens C-58D) it was shown that exposure of the recepient plant to low-level γ-radiation of Gy induced changes in cells that were not repaired over two months promoting tumoral transformations in them. Those changes were shown to persist in the offspring of the exposed somatic cells

Plasmid Transfer in the Ocean – A Case Study from the Roseobacter Group

Directory of Open Access Journals (Sweden)

Jörn Petersen

2017-07-01

Full Text Available Plasmid mediated horizontal gene transfer (HGT has been speculated to be one of the prime mechanisms for the adaptation of roseobacters (Rhodobacteraceae to their ecological niches in the marine habitat. Their plasmids contain ecologically crucial functional modules of up to ∼40-kb in size, e.g., for aerobic anoxygenic photosynthesis, flagellar formation and the biosynthesis of the antibiotic tropodithietic acid. Furthermore, the widely present type four secretion system (T4SS of roseobacters has been shown to mediate conjugation across genus barriers, albeit in the laboratory. Here we discovered that Confluentimicrobium naphthalenivorans NS6T, a tidal flat bacterium isolated in Korea, carries a 185-kb plasmid, which exhibits a long-range synteny with the conjugative 126-kb plasmid of Dinoroseobacter shibae DFL12T. Both replicons are stably maintained by RepABC operons of the same compatibility group (-2 and they harbor a homologous T4SS. Principal component analysis of the codon usage shows a large similarity between the two plasmids, while the chromosomes are very distinct, showing that neither of the two bacterial species represents the original host of those RepABC-2 type plasmids. The two species do not share a common habitat today and they are phylogenetically only distantly related. Our finding demonstrates the first clear-cut evidence for conjugational plasmid transfer across biogeographical and phylogenetic barriers in Rhodobacteraceae and documents the importance of conjugative HGT in the ocean.

Plasmid mediated quinolone resistance in Enterobacteriaceae

NARCIS (Netherlands)

Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

2014-01-01

This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and

Plasmid and chromosome segregation in prokaryotes

DEFF Research Database (Denmark)

Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

2000-01-01

Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic...

Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host.

Science.gov (United States)

Heuer, Holger; Fox, Randal E; Top, Eva M

2007-03-01

IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.

Genetic engineering of microorganisms for biodiesel production

Science.gov (United States)

Lin, Hui; Wang, Qun; Shen, Qi; Zhan, Jumei; Zhao, Yuhua

2013-01-01

Biodiesel, as one type of renewable energy, is an ideal substitute for petroleum-based diesel fuel and is usually made from triacylglycerides by transesterification with alcohols. Biodiesel production based on microbial fermentation aiming to establish more efficient, less-cost and sustainable biodiesel production strategies is under current investigation by various start-up biotechnology companies and research centers. Genetic engineering plays a key role in the transformation of microbes into the desired cell factories with high efficiency of biodiesel production. Here, we present an overview of principal microorganisms used in the microbial biodiesel production and recent advances in metabolic engineering for the modification required. Overexpression or deletion of the related enzymes for de novo synthesis of biodiesel is highlighted with relevant examples. PMID:23222170

Degenerate primer MOB typing of multiresistant clinical isolates of E. coli uncovers new plasmid backbones.

Science.gov (United States)

Garcillán-Barcia, M Pilar; Ruiz del Castillo, Belén; Alvarado, Andrés; de la Cruz, Fernando; Martínez-Martínez, Luis

2015-01-01

Degenerate Primer MOB Typing is a PCR-based protocol for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families. It was applied to a multiresistant E. coli collection, previously characterized by PCR-based replicon-typing, in order to compare both methods. Plasmids from 32 clinical isolates of multiresistant E. coli (19 extended spectrum beta-lactamase producers and 13 non producers) and their transconjugants were analyzed. A total of 95 relaxases were detected, at least one per isolate, underscoring the high potential of these strains for antibiotic-resistance transmission. MOBP12 and MOBF12 plasmids were the most abundant. Most MOB subfamilies detected were present in both subsets of the collection, indicating a shared mobilome among multiresistant E. coli. The plasmid profile obtained by both methods was compared, which provided useful data upon which decisions related to the implementation of detection methods in the clinic could be based. The phylogenetic depth at which replicon and MOB-typing classify plasmids is different. While replicon-typing aims at plasmid replication regions with non-degenerate primers, MOB-typing classifies plasmids into relaxase subfamilies using degenerate primers. As a result, MOB-typing provides a deeper phylogenetic depth than replicon-typing and new plasmid groups are uncovered. Significantly, MOB typing identified 17 plasmids and an integrative and conjugative element, which were not detected by replicon-typing. Four of these backbones were different from previously reported elements. Copyright © 2014 Elsevier Inc. All rights reserved.

Structural analysis of the ParR/parC plasmid partition complex

DEFF Research Database (Denmark)

Møller-Jensen, Jakob; Ringgaard, Simon; Mercogliano, Christopher P

2007-01-01

Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA...

Cefotaxime resistant Escherichia coli collected from a healthy volunteer; characterisation and the effect of plasmid loss.

Directory of Open Access Journals (Sweden)

Miranda Kirchner

Full Text Available In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment.

Complete sequences of IncHI1 plasmids carrying bla_CTX-M-1 and qnrS1 in equine Escherichia coli provide new insights into plasmid evolution

DEFF Research Database (Denmark)

Dolejska, Monika; Villa, Laura; Minoia, Marco

2014-01-01

OBJECTIVES: To determine the structure of two multidrug-resistant IncHI1 plasmids carrying blaCTX-M-1 in Escherichia coli isolates disseminated in an equine clinic in the Czech Republic. METHODS: A complete nucleotide sequencing of 239 kb IncHI1 (pEQ1) and 287 kb IncHI1/X1 (pEQ2) plasmids was per...... highlight the structure and evolution of IncHI1 from equine E. coli. A plasmid-mediated sugar metabolic element could play a key role in strain fitness, contributing to the successful dissemination and maintenance of these plasmids in the intestinal microflora of horses....

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

Science.gov (United States)

Rogers, Elizabeth E; Stenger, Drake C

2012-01-01

A ≈ 38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th) century.

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

Directory of Open Access Journals (Sweden)

Elizabeth E Rogers

Full Text Available A ≈ 38kB plasmid (pXF-RIV5 was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51 sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th century.

Imipenem-resistance in Serratia marcescens is mediated by plasmid expression of KPC-2.

Science.gov (United States)

Su, W-Q; Zhu, Y-Q; Deng, N-M; Li, L

2017-04-01

Imipenem is a broad-spectrum carbapenem antibiotic with applications against severe bacterial infections. Here, we describe the identification of imipenem-resistant Serratia marcescens in our hospital and the role of plasmid-mediated KPC-2 expression in imipenem resistance. We used the modified Hodge test to detect carbapenemase produced in imipenem-resistant strains. His resistance can be transferred to E. coli in co-culture tests, which implicates the plasmid in imipenem resistance. PCR amplification from the plasmid identified two products consistent with KPC-2 of 583 and 1050 bp that were also present in E. coli after co-culture. The restriction pattern for both plasmids was identical, supporting the transfer from the S. marcescens isolate to E. coli. Finally, gene sequencing confirmed KPC-2 in the plasmid. Due to the presence of KPC-2 in the imipenem-resistant S. marcescens, we propose that KPC-2 mediates antibiotic resistance in the S. marcescens isolate.

Assessment of impact of culture conditions on capability of wastewater's microorganisms to flocculation

Directory of Open Access Journals (Sweden)

Il'inskiy V. V.

2017-06-01

Full Text Available Wastewater is one of the significant sources of pollution of the aquatic ecosystems of the Kola North. Sewage coming to the ground waters, surface waters and near shore marine basin have a complex negative impact on the biocenosis of water objects. Considering the fact that basin self-purification process in climatic environment of the Far North is slow, it seems to be current problem to research influence of external factors on the native microorganisms involved in the biological transformation of most pollutants. Along with oxygenizing activity microorganisms are able to accumulate pollutant in the cells and to form floccules. As a result, microorganisms fix the dissolved contaminants that may be mechanically derived from water. Using the data on the chemical makeup of some urban and domestic sewage, nutrient media have been developed where microorganisms isolated from effluents have been cultivated. As major characteristics of the cultivation media affecting the intensity and direction of metabolic processes in microorganisms, the ratios C / N, C / P and N / P have been chosen. Intensity growth of bacteria in experimental nutrient media has been studied and the flocculating activity of bacterial suspensions has been determined. The rate of these microorganisms (Pseudomonas spp. and cultures of bacteria of Enterobacteriaceae family average has been 70∙103 and 117∙103 cells/h respectively. The growth rate of each culture on different composition nutrient media has varied within three orders, and has correlated with the relative content of phosphates in the nutrient media. The flocculating potential does not depend on the ratio of biogenic elements C / N, C / P and N / P. Both cultures have shown the ability to precipitate suspended matter at the level of 50 % or more after cultivation on nutrient media similar in composition to habitat conditions.

DNA transformations of Candida tropicalis with replicating and integrative vectors.

Science.gov (United States)

Sanglard, D; Fiechter, A

1992-12-01

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

Science.gov (United States)

Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

2008-09-01

We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

Comparative Genomics of Rhodococcus equi Virulence Plasmids Indicates Host-Driven Evolution of the vap Pathogenicity Island.

Science.gov (United States)

MacArthur, Iain; Anastasi, Elisa; Alvarez, Sonsiray; Scortti, Mariela; Vázquez-Boland, José A

2017-05-01

The conjugative virulence plasmid is a key component of the Rhodococcus equi accessory genome essential for pathogenesis. Three host-associated virulence plasmid types have been identified the equine pVAPA and porcine pVAPB circular variants, and the linear pVAPN found in bovine (ruminant) isolates. We recently characterized the R. equi pangenome (Anastasi E, et al. 2016. Pangenome and phylogenomic analysis of the pathogenic actinobacterium Rhodococcus equi. Genome Biol Evol. 8:3140-3148.) and we report here the comparative analysis of the virulence plasmid genomes. Plasmids within each host-associated type were highly similar despite their diverse origins. Variation was accounted for by scattered single nucleotide polymorphisms and short nucleotide indels, while larger indels-mostly in the plasticity region near the vap pathogencity island (PAI)-defined plasmid genomic subtypes. Only one of the plasmids analyzed, of pVAPN type, was exceptionally divergent due to accumulation of indels in the housekeeping backbone. Each host-associated plasmid type carried a unique PAI differing in vap gene complement, suggesting animal host-specific evolution of the vap multigene family. Complete conservation of the vap PAI was observed within each host-associated plasmid type. Both diversity of host-associated plasmid types and clonality of specific chromosomal-plasmid genomic type combinations were observed within the same R. equi phylogenomic subclade. Our data indicate that the overall strong conservation of the R. equi host-associated virulence plasmids is the combined result of host-driven selection, lateral transfer between strains, and geographical spread due to international livestock exchanges. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

Science.gov (United States)

Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

2008-01-01

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli. PMID:18390685

Fermentation of starch by Klebsiella oxytoca P2, containing plasmids with {alpha}-amylase and pullulanase genes

Energy Technology Data Exchange (ETDEWEB)

Santos, V.L. dos; Araujo, E.F.; Barros, E.G. de; Guimaraes, W.V.

1999-12-20

Klebsiella oxytoca P2(pC46), an ethanol-producing recombinant, has been evaluated in fermentation of maltose and starch. The maximum ethanol produced by P2(pC46) was 0.34 g ethanol/g maltose and 0.38, 0.40, or 0.36 g ethanol/g starch in fermentation of 1, 2, or 4% starch, representing 68, 71, and 64% the theoretical yield. The pC46 plasmid transformed to cells of K. oxytoca P2 reduced the ethanol production from maltose and starch. In fermentation of starch after its digestion at 60 C for 24 h, in two-step fermentation, the time for maximum ethanol production was reduced to 12--24 h and the theoretical yield was around 90%. The increase in starch concentration resulted in lower {alpha}-amylase activity but in higher pullulanase activity. The high activity and thermostability of the amylolytic enzymes from this transformant suggest that it has a potential for amylolytic enzymes source.

Microorganisms involved in MIC

Energy Technology Data Exchange (ETDEWEB)

Sorensen, K. [Danish Technological Institute (Denmark)

2011-07-01

Microbiologically influenced corrosion (MIC) is a widespread problem that is difficult to detect and assess because of its complex mechanism. This paper presents the involvement of microorganisms in MIC. Some of the mechanisms that cause MIC include hydrogen consumption, production of acids, anode-cathode formation and electron shuttling. A classic bio-corrosive microorganism in the oil and gas industry is sulphate-reducing prokaryotes (SRP). Methanogens also increase corrosion rates in metals. Some of the phylogenetic orders detected while studying SRP and methanogens are archaeoglobales, clostridiales, methanosarcinales and methanothermococcus. There were some implications, such as growth of SRP not being correlated with growth of methanogens; methanogens were included in MIC risk assessment. A few examples are used to display how microorganisms are involved in topside corrosion and microbial community in producing wells. From the study, it can be concluded that, MIC risk assessment includes system data and empirical knowledge of the distribution and number of microorganisms in the system.

Plasmid fingerprinting and virulence gene detection among indigenous strains of salmonella enterica serovar enteritidis

International Nuclear Information System (INIS)

Sajid, S.U.; Schwarz, S.

2009-01-01

Salmonella enterica serovar Enteritidis is an important frequently reported zoonotic pathogen and a common cause of human gastroenteritis worldwide. The highly conserved Serospecific plasmids (SSPs) and Salmonella plasmid virulence (Spv) genes have been shown to mediate extra-intestinal colonization and systemic infection. The objective of current study was to document the presence of SSPs and SpvB/SpvC genes prevailing in the indigenous population of serovar Enteritidis. A total of 48 epidemiologically unrelated strains of Salmonella enteritidis were included in the study. Preparation of plasmids DNA suitable for endonuclease digestion and separation of respective fragments by agarose gel electrophoresis followed previously described protocols. The plasmids of Escherichia coli V517, 1-kbp ladder, and lambda DNA HindIII fragments served as DNA size standards. Transfer of DNA fragments from agarose gels to nitrocellulose membranes was achieved by capillary blot procedure. An ECL labeled 3.6 kbp HindIII fragment of plasmid PRQ 51 was used as probe for SpvB/SpvC gene detection. Plasmid DNA fingerprinting revealed the presence of two different profiles of approximately 55 kbp and 90 kbp and were identified as virulence plasmids by DNA hybridization. The SpvB/SpvC genes were located on HindIII fragments of 3.6 kbp in each of the two types of virulence plasmids. The study confirms the presence of SSPs and SpvB/SpvC genes in indigenous strains of S. enteritidis isolated from Northern Punjab area of Pakistan and substantiate the previous data on such findings from other parts of the world. (author)

Salmonella Typhimurium ST213 is associated with two types of IncA/C plasmids carrying multiple resistance determinants.

Science.gov (United States)

Wiesner, Magdalena; Calva, Edmundo; Fernández-Mora, Marcos; Cevallos, Miguel A; Campos, Freddy; Zaidi, Mussaret B; Silva, Claudia

2011-01-11

Salmonella Typhimurium ST213 was first detected in the Mexican Typhimurium population in 2001. It is associated with a multi-drug resistance phenotype and a plasmid-borne blaCMY-2 gene conferring resistance to extended-spectrum cephalosporins. The objective of the current study was to examine the association between the ST213 genotype and blaCMY-2 plasmids. The blaCMY-2 gene was carried by an IncA/C plasmid. ST213 strains lacking the blaCMY-2 gene carried a different IncA/C plasmid. PCR analysis of seven DNA regions distributed throughout the plasmids showed that these IncA/C plasmids were related, but the presence and absence of DNA stretches produced two divergent types I and II. A class 1 integron (dfrA12, orfF and aadA2) was detected in most of the type I plasmids. Type I contained all the plasmids carrying the blaCMY-2 gene and a subset of plasmids lacking blaCMY-2. Type II included all of the remaining blaCMY-2-negative plasmids. A sequence comparison of the seven DNA regions showed that both types were closely related to IncA/C plasmids found in Escherichia, Salmonella, Yersinia, Photobacterium, Vibrio and Aeromonas. Analysis of our Typhimurium strains showed that the region containing the blaCMY-2 gene is inserted between traA and traC as a single copy, like in the E. coli plasmid pAR060302. The floR allele was identical to that of Newport pSN254, suggesting a mosaic pattern of ancestry with plasmids from other Salmonella serovars and E. coli. Only one of the tested strains was able to conjugate the IncA/C plasmid at very low frequencies (10-7 to 10-9). The lack of conjugation ability of our IncA/C plasmids agrees with the clonal dissemination trend suggested by the chromosomal backgrounds and plasmid pattern associations. The ecological success of the newly emerging Typhimurium ST213 genotype in Mexico may be related to the carriage of IncA/C plasmids. We conclude that types I and II of IncA/C plasmids originated from a common ancestor and that the

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

Directory of Open Access Journals (Sweden)

TC Leal-Balbino

2004-11-01

Full Text Available Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.

Studies on the expression of plasmid-borne genes in the endosymbiotic state of Rhizobium leguminosarum

NARCIS (Netherlands)

Krol, A.J.M.

1982-01-01

The subject matter of the research reported in this thesis is the role of plasmid-borne genes of Rhizobium in symbiosis and nitrogen fixation. Plasmid DNA was isolated from Rhizobium leguminosarum strain PRE and the expression of plasmid DNA in nitrogen

Identification of a Novel Conjugative Plasmid in Mycobacteria That Requires Both Type IV and Type VII Secretion

KAUST Repository

Ummels, R.

2014-09-23

Conjugative plasmids have been identified in a wide variety of different bacteria, ranging from proteobacteria to firmicutes, and conjugation is one of the most efficient routes for horizontal gene transfer. The most widespread mechanism of plasmid conjugation relies on different variants of the type IV secretion pathway. Here, we describe the identification of a novel type of conjugative plasmid that seems to be unique for mycobacteria. Interestingly, while this plasmid is efficiently exchanged between different species of slow-growing mycobacteria, including Mycobacterium tuberculosis, it could not be transferred to any of the fast-growing mycobacteria tested. Genetic analysis of the conjugative plasmid showed the presence of a locus containing homologues of three type IV secretion system components and a relaxase. In addition, a new type VII secretion locus was present. Using transposon insertion mutagenesis, we show that in fact both these secretion systems are essential for conjugation, indicating that this plasmid represents a new class of conjugative plasmids requiring two secretion machineries. This plasmid could form a useful new tool to exchange or introduce DNA in slow-growing mycobacteria. IMPORTANCE: Conjugative plasmids play an important role in horizontal gene transfer between different bacteria and, as such, in their adaptation and evolution. This effect is most obvious in the spread of antibiotic resistance genes. Thus far, conjugation of natural plasmids has been described only rarely for mycobacterial species. In fact, it is generally accepted that M. tuberculosis does not show any recent sign of horizontal gene transfer. In this study, we describe the identification of a new widespread conjugative plasmid that can also be efficiently transferred to M. tuberculosis. This plasmid therefore poses both a threat and an opportunity. The threat is that, through the acquisition of antibiotic resistance markers, this plasmid could start a rapid spread of

DNA damage produced by exposure of supercoiled plasmid DNA to high- and low-LET ionizing radiation: Effects of hydroxyl radical quenchers. DNA breakage, neutrons, OH radicals

International Nuclear Information System (INIS)

Peak, J.G.; Ito, T.; Peak, M.J.; Robb, F.T.

1994-01-01

A supercoiled plasmid of 7300 base pairs was isolated and exposed in an aqueous environment to 60 Co γ rays and JANUS 0.85 MeV fission-spectrum neutrons. Dose responses for the production of single-strand breaks (SSBs), double-strand breaks (DSBs) and alkali-labile sites (ALSs) were compared with computations made from the conversion of the supercoil to its relaxed and linear forms. The relative biological effectiveness (RBE) for production of SSBs and DSBs was similar to that previously measured in the cellular environment. The RBE for destruction of genetic transforming activity of M13 viral DNA followed that for DNA damage. This is in contrast to the situation for biological effects such as lethality, mutagenesis, and cellular transformation measured in mammalian cells, where the RBE values are reversed. The role of hydroxyl (OH) radical in DNA damage induction by neutrons was investigated by exposure of plasmid in the presence of known quenchers of this species. Of four quenchers tested, all were able to reduce the yields of both SSBs and DSBs. These findings are consistent with a model for SSB and DSB induction by high linear energy transfer that involves OH radical mediation

TOL plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo

International Nuclear Information System (INIS)

Jussila, Minna M.; Zhao, Ji; Suominen, Leena; Lindstroem, Kristina

2007-01-01

Molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pWW0 was conjugated from Pseudomonas to Rhizobium. The xylE gene was detected both in Rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. TOL plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-contaminated rhizosphere of Galega orientalis. A new finding was that some Pseudomonas migulae and Pseudomonas oryzihabitans strains harbored a TOL plasmid with both pWW0- and pDK1-type xylE gene. P. oryzihabitans 29 had received the archetypal TOL plasmid pWW0 from Pseudomonas putida PaW85. As an application for environmental biotechnology, the biodegradation potential of oil-polluted soil and the success of bioremediation could be estimated by monitoring changes not only in the type and amount but also in transfer of degradation plasmids. - Horizontal transfer of degradation plasmids in the oil-contaminated rhizosphere reveals the dynamic nature of the intrinsic biodegradation potential

Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

DEFF Research Database (Denmark)

Reisner, A.; Molin, Søren; Zechner, E.L.

2002-01-01

An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination...... resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E coli and is not restricted...

The key microorganisms for anaerobic degradation of pentachlorophenol in paddy soil as revealed by stable isotope probing

Energy Technology Data Exchange (ETDEWEB)

Tong, Hui [Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650 (China); Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Liu, Chengshuai [State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang 550009 (China); Li, Fangbai, E-mail: cefbli@soil.gd.cn [Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650 (China); Luo, Chunling [Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640 (China); Chen, Manjia; Hu, Min [Guangdong Key Laboratory of Agricultural Environment Pollution Integrated Control, Guangdong Institute of Eco-Environmental and Soil Sciences, Guangzhou 510650 (China)

2015-11-15

Highlights: • SIP suggested that Dechloromonas can mineralize PCP in soil. • Methanosaeta and Methanocella acquired PCP-derived carbon. • Lactate enhanced microbial degradation of PCP in soil. - Abstract: Pentachlorophenol (PCP) is a common residual persistent pesticide in paddy soil and has resulted in harmful effect on soil ecosystem. The anaerobic microbial transformation of PCP, therefore, has been received much attentions, especially the functional microbial communities for the reductive transformation. However, the key functional microorganisms for PCP mineralization in the paddy soil still remain unknown. In this work, DNA-based stable isotope probing (SIP) was applied to explore the key microorganisms responsible for PCP mineralization in paddy soil. The SIP results indicated that the dominant bacteria responsible for PCP biodegradation belonged to the genus Dechloromonas of the class β-Proteobacteria. In addition, the increased production of {sup 13}CH{sub 4} and {sup 13}CO{sub 2} indicated that the addition of lactate enhanced the rate of biodegradation and mineralization of PCP. Two archaea classified as the genera of Methanosaeta and Methanocella of class Methanobacteria were enriched in the heavy fraction when with lactate, whereas no archaea was detected in the absence of lactate. These findings provide direct evidence for the species of bacteria and archaea responsible for anaerobic PCP or its breakdown products mineralization and reveal a new insight into the microorganisms linked with PCP degradation in paddy soil.

The key microorganisms for anaerobic degradation of pentachlorophenol in paddy soil as revealed by stable isotope probing

International Nuclear Information System (INIS)

Tong, Hui; Liu, Chengshuai; Li, Fangbai; Luo, Chunling; Chen, Manjia; Hu, Min

2015-01-01

Highlights: • SIP suggested that Dechloromonas can mineralize PCP in soil. • Methanosaeta and Methanocella acquired PCP-derived carbon. • Lactate enhanced microbial degradation of PCP in soil. - Abstract: Pentachlorophenol (PCP) is a common residual persistent pesticide in paddy soil and has resulted in harmful effect on soil ecosystem. The anaerobic microbial transformation of PCP, therefore, has been received much attentions, especially the functional microbial communities for the reductive transformation. However, the key functional microorganisms for PCP mineralization in the paddy soil still remain unknown. In this work, DNA-based stable isotope probing (SIP) was applied to explore the key microorganisms responsible for PCP mineralization in paddy soil. The SIP results indicated that the dominant bacteria responsible for PCP biodegradation belonged to the genus Dechloromonas of the class β-Proteobacteria. In addition, the increased production of 13 CH 4 and 13 CO 2 indicated that the addition of lactate enhanced the rate of biodegradation and mineralization of PCP. Two archaea classified as the genera of Methanosaeta and Methanocella of class Methanobacteria were enriched in the heavy fraction when with lactate, whereas no archaea was detected in the absence of lactate. These findings provide direct evidence for the species of bacteria and archaea responsible for anaerobic PCP or its breakdown products mineralization and reveal a new insight into the microorganisms linked with PCP degradation in paddy soil

Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes.

Science.gov (United States)

Rudder, Steven; Doohan, Fiona; Creevey, Christopher J; Wendt, Toni; Mullins, Ewen

2014-04-07

Recently it has been shown that Ensifer adhaerens can be used as a plant transformation technology, transferring genes into several plant genomes when equipped with a Ti plasmid. For this study, we have sequenced the genome of Ensifer adhaerens OV14 (OV14) and compared it with those of Agrobacterium tumefaciens C58 (C58) and Sinorhizobium meliloti 1021 (1021); the latter of which has also demonstrated a capacity to genetically transform crop genomes, albeit at significantly reduced frequencies. The 7.7 Mb OV14 genome comprises two chromosomes and two plasmids. All protein coding regions in the OV14 genome were functionally grouped based on an eggNOG database. No genes homologous to the A. tumefaciens Ti plasmid vir genes appeared to be present in the OV14 genome. Unexpectedly, OV14 and 1021 were found to possess homologs to chromosomal based genes cited as essential to A. tumefaciens T-DNA transfer. Of significance, genes that are non-essential but exert a positive influence on virulence and the ability to genetically transform host genomes were identified in OV14 but were absent from the 1021 genome. This study reveals the presence of homologs to chromosomally based Agrobacterium genes that support T-DNA transfer within the genome of OV14 and other alphaproteobacteria. The sequencing and analysis of the OV14 genome increases our understanding of T-DNA transfer by non-Agrobacterium species and creates a platform for the continued improvement of Ensifer-mediated transformation (EMT).

RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon.

Science.gov (United States)

Pérez-Oseguera, Angeles; Cevallos, Miguel A

2013-11-01

Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process. Copyright © 2013 Elsevier Inc. All rights reserved.

Radioresistant microorganisms and food irradiation

Energy Technology Data Exchange (ETDEWEB)

Ito, H [Japan Atomic Energy Research Inst., Takasaki, Gunma. Takasaki Radiation Chemistry Research Establishment

1976-01-01

This paper deals with Micrococcus radiodurans, Arthrobacter radiotolerance, etc., which were isolated and discovered as radioresistant microorganisms. As for the explanation of the mechanism of radioresistance of these microorganisms, the consideration that these organisms have marked repair power of the damaged DNA and have many opportunity to repair the damaged DNA because of their long fission term were cited. The relationship between the radioresistance of microorganisms and food irradiation was also mentioned.

Transformation and Stability of Cloned Polysaccharidase Genes in Gram-Positive Bacteria

OpenAIRE

EKİNCİ, Mehmet Sait

2014-01-01

Polysaccharidase genes from rumen bacteria were transferred to and expressed in ruminal and non-ruminal Gram-positive bacteria. The transformation efficiency and genetic stability of polysaccharidase genes in bacteria from different habitats were investigated. PCR amplification of cloned polysaccharidase genes from Escherichia coli, Lactococcus lactis, Enterococcus faecalis, Streptococcus sanguis and S. bovis strain 26 showed that rearrangement of plasmid and the gene fragment did not occur. ...

New plasmid-mediated aminoglycoside 6'-N-acetyltransferase, AAC(6')-Ian, and ESBL, TLA-3, from a Serratia marcescens clinical isolate.

Science.gov (United States)

Jin, Wanchun; Wachino, Jun-Ichi; Kimura, Kouji; Yamada, Keiko; Arakawa, Yoshichika

2015-05-01

Enterobacteriaceae clinical isolates showing amikacin resistance (MIC 64 to >256 mg/L) in the absence of 16S rRNA methyltransferase (MTase) genes were found. The aim of this study was to clarify the molecular mechanisms underlying amikacin resistance in Enterobacteriaceae clinical isolates that do not produce 16S rRNA MTases. PCR was performed to detect already-known amikacin resistance determinants. Cloning experiments and sequence analyses were performed to characterize unknown amikacin resistance determinants. Transfer of amikacin resistance determinants was performed by conjugation and transformation. The complete nucleotide sequence of the plasmids was determined by next-generation sequencing technology. Amikacin resistance enzymes were purified with a column chromatography system. The enzymatic function of the purified protein was investigated by thin-layer chromatography (TLC) and HPLC. Among the 14 isolates, 9 were found to carry already-known amikacin resistance determinants such as aac(6')-Ia and aac(6')-Ib. Genetic analyses revealed the presence of a new amikacin acetyltransferase gene, named aac(6')-Ian, located on a 169 829 bp transferable plasmid (p11663) of the Serratia marcescens strain NUBL-11663, one of the five strains negative for known aac(6') genes by PCR. Plasmid p11663 also carried a novel ESBL gene, named blaTLA-3. HPLC and TLC analyses demonstrated that AAC(6')-Ian catalysed the transfer of an acetyl group from acetyl coenzyme A onto an amine at the 6'-position of various aminoglycosides. We identified aac(6')-Ian as a novel amikacin resistance determinant together with a new ESBL gene, blaTLA-3, on a transferable plasmid of a S. marcescens clinical isolate. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Enterococcus faecalis Sex Pheromone cCF10 Enhances Conjugative Plasmid Transfer In Vivo.

Science.gov (United States)

Hirt, Helmut; Greenwood-Quaintance, Kerryl E; Karau, Melissa J; Till, Lisa M; Kashyap, Purna C; Patel, Robin; Dunny, Gary M

2018-02-13

Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer in vitro in Enterococcus faecalis To examine the role of pheromone signaling in vivo , we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF- recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF- recipient. While rescue of the CF- mating defect by coculture with CF+ recipients is easily accomplished in vitro , no extracellular complementation occurred in vivo This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo In the absence of CF+ recipients, a low level of transfer to CF- recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis , an important nosocomial pathogen. Previous studies showing efficient conjugative plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did

Standardized Cloning and Curing of Plasmids

DEFF Research Database (Denmark)

Lauritsen, Ida; Kim, Se Hyeuk; Porse, Andreas

2018-01-01

and exchange of genetic parts in the Standard European Vectors Architecture (SEVA) vector system. Additionally, to facilitate rapid testing and iterative bioengineering using different vector designs, we provide a one-step protocol for a universal CRISPR-Cas9-based plasmid curing system (pFREE) and demonstrate...

Characterization of a plasmid carrying cat, ermB and tetS genes in a foodborne Listeria monocytogenes strain and uptake of the plasmid by cariogenic Streptococcus mutans

DEFF Research Database (Denmark)

Li, Lili; Olsen, Rikke Heidemann; Shi, Lei

2016-01-01

A multi-drug resistant (MDR) Listeria monocytogenes isolate (serotype 1/2c) was recovered from a quick-frozen rice flour product collected from Langfang city in northern China. PCR screening identified the presence of cat, ermB and tetS genes. The plasmid profile of the strain showed the presence...... of an approximately 22.4-kb plasmid. Curing of this plasmid resulted in the loss of cat, ermB and tetS genes and increased susceptibility to several antibiotics, suggesting the involvement of the plasmid in multiple antibiotic resistances. Moreover, the plasmid was able to be uptaken by human oral pathogen...

Functionalized tetrapod-like ZnO nanostructures for plasmid DNA purification, polymerase chain reaction and delivery

International Nuclear Information System (INIS)

Nie Leng; Gao Lizeng; Yan Xiyun; Wang Taihong

2007-01-01

Functionalized tetrapodal ZnO nanostructures are tested in plasmid DNA experiments (1) as a solid-phase adsorbent for plasmid DNA purification (2) as improving reagents in a polymerase chain reaction (PCR) and (3) as novel carriers for gene delivery. The amino-modification, the tetrapod-like shape of the nanostructure and its high biocompatibility all contribute to measurements showing promise for applications. A sol-gel method is used for silica coating and amino-modification. Plasmid DNA is purified through reversible conjugations of amino-modified ZnO tetrapods with DNA. Also, as additional reagents, functionalized tetrapods are shown to improve the amount of PCR product. For transfection, ZnO tetrapods provide some protection against deoxyribonuclease cleavage of plasmid DNA and deliver plasmid DNA into cells with little cytotoxicity

A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

Directory of Open Access Journals (Sweden)

George Lezin

Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences

International Nuclear Information System (INIS)

Frappier, L.; Zannis-Hadjopoulos, M.

1987-01-01

Twelve clones of origin-enriched sequences (ORS) isolated from early replicating monkey (CV-1) DNA were examined for transient episomal replication in transfected CV-1, COS-7, and HeLa cells. Plasmid DNA was isolated at time intervals after transfection and screened by the Dpn I resistance assay or by the bromodeoxyuridine substitution assay to differentiate between input and replicated DNA. The authors have identified four monkey ORS (ORS3, -8, -9, and -12) that can support plasmid replication in mammalian cells. This replication is carried out in a controlled and semiconservative manner characteristic of mammalian replicons. ORS replication was most efficient in HeLa cells. Electron microscopy showed ORS8 and ORS12 plasmids of the correct size with replication bubbles. Using a unique restriction site in ORS12, we have mapped the replication bubble within the monkey DNA sequence

Microorganisms associated to tomato seedlings growing in saline culture act as osmoprotectant

Directory of Open Access Journals (Sweden)

Daniel Cortés-Jiménez

2014-06-01

Full Text Available Less than 0.5% of total water in the world is available for human consumption and agriculture. The major part of the world's water is saline and salinity in soils interferes in germination of seeds and the posterior development of the plant. In order to increase the osmotolerance of tomato, seedlings were associated with Azospirillum brasilense Cd, Azospirillum brasilense Cd transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion or Chlorella vulgaris. Two plant culture media: Hydroponic and Murashige and Skoog were tested. In the first set of studies seedlings were associated to single free cells meanwhile in a second set single and combined free cells were studied. A positive interaction between transformed Azospirillum and Chlorella vulagris and tomato plants was observed. Seedlings showed a salt concentration tolerance, as sodium chloride, up to 200 mM. According to our results, the association of plants with A. brasilense Cd-BIF and C. vulgaris is a viable approach to increase their salt tolerance and biomass, as consequence the possible use of sea water to irrigate horticultural plants.

Plasmids replicatable in Bacillus subtilis, E. coli and lactic acid streptococcus bacteria

NARCIS (Netherlands)

Kok, Jan; Maat, Jan; van der Vossen, Josephus Mauritius; Venema, Gerard

1997-01-01

The claimed invention is drawn to a recombinant plasmid which can replicate in Bacillus subtilis, Escherichia coli, and lactic acid Streptococcus bacteria comprising the replication of origin from Streptococcus cremoris plasmid pWV01 as its origin of replication, in addition to coding marker genes

Construction of pTM series plasmids for gene expression in Brucella species.

Science.gov (United States)

Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

2016-04-01

Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp. Copyright © 2016 Elsevier B.V. All rights reserved.

40 CFR 725.85 - Microorganism identity.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Microorganism identity. 725.85 Section... to Information § 725.85 Microorganism identity. (a) Claims applicable to the period prior to... specific microorganism identity at the time of submission of the information. This claim will apply only to...

Structural and functional analysis of the kid toxin protein from E. coli Plasmid R1

NARCIS (Netherlands)

Hargreaves, D.; Santos-Sierra, S.; Giraldo, R.; Sabariegos-Jareño, R.; de la Cueva-Méndez, G.; Boelens, R.|info:eu-repo/dai/nl/070151407; Díaz-Orejas, R.; Rafferty, J.B.

2002-01-01

We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 Å crystal structure of Kid reveals a 2-fold

Evaluation of the effect of non-B DNA structures on plasmid integrity via accelerated stability studies.

Science.gov (United States)

Ribeiro, S C; Monteiro, G A; Prazeres, D M F

2009-04-01

Plasmid biopharmaceuticals are a new class of medicines with an enormous potential. Attempts to increase the physical stability of highly purified supercoiled (SC) plasmid DNA in pharmaceutical aqueous solutions have relied on: (i) changing the DNA sequence, (ii) improving manufacturing to reduce deleterious impurities and initial DNA damage, and (iii) controlling the storage medium characteristics. In this work we analyzed the role of secondary structures on the degradation of plasmid molecules. Accelerated stability experiments were performed with SC, open circular (OC) and linear (L) isoforms of three plasmids which differed only in the "single-strandlike" content of their polyadenylation (poly A) signals. We have proved that the presence of more altered or interrupted (non-B) DNA secondary structures did not directly translate into an easier strand scission of the SC isoforms. Rather, those unusual structures imposed a lower degree of SC in the plasmids, leading to an increase in their resistance to thermal degradation. However, this behavior was reversed when the relaxed or L isoforms were tested, in which case the absence of SC rendered the plasmids essentially double-stranded. Overall, this work suggests that plasmid DNA sequence and secondary structures should be taken into account in future investigations of plasmid stability during prolonged storage.

trans-Acting Virulence Functions of the Octopine Ti Plasmid from Agrobacterium tumefaciens

NARCIS (Netherlands)

Hille, Jacques; Kan, Jan van; Schilperoort, Rob

1984-01-01

All Ti plasmid-encoded virulence functions that were studied act in trans. An octopine Ti plasmid-specific vir operon, called vir-O, located on an EcoRI restriction fragment has been characterized. Sequences with promoter activity in Escherichia coli were identified for a second vir operon, called

piggybac- and PhiC31-mediated genetic transformation of the Asian tiger mosquito, Aedes albopictus (Skuse.

Directory of Open Access Journals (Sweden)

Geneviève M C Labbé

Full Text Available BACKGROUND: The Asian tiger mosquito, Aedes albopictus (Skuse, is a vector of several arboviruses including dengue and chikungunya. This highly invasive species originating from Southeast Asia has travelled the world in the last 30 years and is now established in Europe, North and South America, Africa, the Middle East and the Caribbean. In the absence of vaccine or antiviral drugs, efficient mosquito control strategies are crucial. Conventional control methods have so far failed to control Ae. albopictus adequately. METHODOLOGY/PRINCIPAL FINDINGS: Germline transformation of Aedes albopictus was achieved by micro-injection of embryos with a piggyBac-based transgene carrying a 3xP3-ECFP marker and an attP site, combined with piggyBac transposase mRNA and piggyBac helper plasmid. Five independent transgenic lines were established, corresponding to an estimated transformation efficiency of 2-3%. Three lines were re-injected with a second-phase plasmid carrying an attB site and a 3xP3-DsRed2 marker, combined with PhiC31 integrase mRNA. Successful site-specific integration was observed in all three lines with an estimated transformation efficiency of 2-6%. CONCLUSIONS/SIGNIFICANCE: Both piggybac- and site-specific PhiC31-mediated germline transformation of Aedes albopictus were successfully achieved. This is the first report of Ae. albopictus germline transformation and engineering, a key step towards studying and controlling this species using novel molecular techniques and genetic control strategies.

Tn5-induced pBS286 plasmid mutations blocking early stages of napthalene oxidation

International Nuclear Information System (INIS)

Kosheleva, I.A.; Tsoi, T.V.; Ivashina, T.V.; Selifonov, S.A.; Starovoitov, I.I.; Boronin, A.M.

1988-01-01

The authors present data on the further analysis of the structural and functional organization of the nah region of plasmid pBS286 controlling the constitutive oxidation of naphthalene by Pseudomonas putida cells. They have studied Tn5-induced mutations blocking early stages of naphthalene oxidation. They present and discuss data providing evidence that, in contrast to plasmid NAH7, the mechanism of regulation of the nahl operon of plasmid NPL-1, the parent plasmid of plasmid pBS286, with inducible synthesis of naphthalene dioxygenase can include elements of a negative control with participation of the regulatory locus R, located proximal to the structural nah genes and closely linked to or overlapped by the inverted control DNA segment (4.2 kb). They also present data on the possibility of regulation of the activity of the catechol-splitting meta-pathway genes with the participation of products of early stages of naphthalene oxidation

Minimal and contributing sequence determinants of the cis-acting locus of transfer (clt) of streptomycete plasmid pIJ101 occur within an intrinsically curved plasmid region.

Science.gov (United States)

Ducote, M J; Prakash, S; Pettis, G S

2000-12-01

Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.

Microbial transformation and degradation of polychlorinated biphenyls

Energy Technology Data Exchange (ETDEWEB)

Field, Jim A. [Department of Chemical and Environmental Engineering, University of Arizona, PO Box 210011, Tucson, AZ 85721 (United States)], E-mail: jimfield@email.arizona.edu; Sierra-Alvarez, Reyes [Department of Chemical and Environmental Engineering, University of Arizona, PO Box 210011, Tucson, AZ 85721 (United States)

2008-09-15

This paper reviews the potential of microorganisms to transform polychlorinated biphenyls (PCBs). In anaerobic environments, higher chlorinated biphenyls can undergo reductive dehalogenation. Meta- and para-chlorines in PCB congeners are more susceptible to dechlorination than ortho-chlorines. Anaerobes catalyzing PCB dechlorination have not been isolated in pure culture but there is strong evidence from enrichment cultures that some Dehalococcoides spp. and other microorganisms within the Chloroflexi phylum can grow by linking the oxidation of H{sub 2} to the reductive dechlorination of PCBs. Lower chlorinated biphenyls can be co-metabolized aerobically. Some aerobes can also grow by utilizing PCB congeners containing only one or two chlorines as sole carbon/energy source. An example is the growth of Burkholderia cepacia by transformation of 4-chlorobiphenyl to chlorobenzoates. The latter compounds are susceptible to aerobic mineralization. Higher chlorinated biphenyls therefore are potentially fully biodegradable in a sequence of reductive dechlorination followed by aerobic mineralization of the lower chlorinated products. - Higher chlorinated biphenyls are potentially fully biodegradable in a sequence of anaerobic reductive dechlorination followed by aerobic mineralization of the lower chlorinated products.

Microbial transformation and degradation of polychlorinated biphenyls

International Nuclear Information System (INIS)

Field, Jim A.; Sierra-Alvarez, Reyes

2008-01-01

This paper reviews the potential of microorganisms to transform polychlorinated biphenyls (PCBs). In anaerobic environments, higher chlorinated biphenyls can undergo reductive dehalogenation. Meta- and para-chlorines in PCB congeners are more susceptible to dechlorination than ortho-chlorines. Anaerobes catalyzing PCB dechlorination have not been isolated in pure culture but there is strong evidence from enrichment cultures that some Dehalococcoides spp. and other microorganisms within the Chloroflexi phylum can grow by linking the oxidation of H 2 to the reductive dechlorination of PCBs. Lower chlorinated biphenyls can be co-metabolized aerobically. Some aerobes can also grow by utilizing PCB congeners containing only one or two chlorines as sole carbon/energy source. An example is the growth of Burkholderia cepacia by transformation of 4-chlorobiphenyl to chlorobenzoates. The latter compounds are susceptible to aerobic mineralization. Higher chlorinated biphenyls therefore are potentially fully biodegradable in a sequence of reductive dechlorination followed by aerobic mineralization of the lower chlorinated products. - Higher chlorinated biphenyls are potentially fully biodegradable in a sequence of anaerobic reductive dechlorination followed by aerobic mineralization of the lower chlorinated products

Plasmid DNA damage by heavy ions at spread-out Bragg peak energies

NARCIS (Netherlands)

Dang, H. M.; van Goethem, M. J.; van der Graaf, E. R.; Brandenburg, S.; Hoekstra, R.; Schlatholter, T.

2010-01-01

Interaction of ionizing radiation with plasmid DNA can lead to formation of single strand breaks, double strand breaks and clustered lesions. We have investigated the response of the synthetic plasmid pBR322 in aqueous solution upon irradiation with (12)C ions under spread-out Bragg peak conditions

Presence and analysis of plasmids in human and animal associated Arcobacter species

DEFF Research Database (Denmark)

Douidah, Laid; De Zutter, Lieven; Van Nieuwerburgh, Filip

2014-01-01

coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried...

The Genetic Intractability Of Symbiodinium microadriaticum To Standard Algal Transformation Methods

KAUST Repository

Chen, Jit Ern

2017-05-23

Modern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, in order to perform CRISPR-Ca9 mediated genome editing. Plasmid vectors containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation, silica whiskers and glass bead agitation. We report that we have been unable to confer chloramphenicol resistance to our specific Symbiodinium strain. These results are intended to provide other researchers with an overview of previously attempted techniques and sequences in order to support efficient planning of future experiments in this important field.

Sequence analysis and characterization of rolling-circle replicating plasmid pVCM01 from Salmonella enterica

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Penido, A. F. B.

2013-12-01

Full Text Available Aims: Characterization of cryptic plasmid pVCM01 (accession number JX133088 isolated from Salmonella enterica Enteritidis. Methodology and results: The complete sequence of pVCM01 was obtained. This plasmid possesses 1981 bp, with G+C content of 57% in agreement of the range of Salmonella genomic DNA. pVCM01 has a high degree of similarity to pB and pJ plasmids. It possesses six main open reading frames, only one have a very high degree of amino acid identity with protein involved in the rolling-circle-like replication (RCR. Based on the sequence similarities, pVCM01 plasmid belonged to the pC194/pUB110 rolling-circle replicating plasmid family. The Rep pVCM01 possesses the motifs: FLTLTVRN, HPHFHTL, SGDGYVKHERW, which were present in all Rep proteins. Conclusion, significance and impact of study: The small size of pVCM01 plasmid and its stability in E. coli cells, make it an attractive candidate to develop new vectors, such as cloning and/or expression vector.

Framing the impact of external magnetic field on bioconvection of a nanofluid flow containing gyrotactic microorganisms with convective boundary conditions

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Tanmoy Chakraborty

2018-03-01

Full Text Available The intention of this study is to examine the combined impacts of magnetic field and convective boundary state on bioconvection of a nanofluid flow along an expanding sheet co-existed with gyrotactic microorganisms. The fundamental partial differential equations are reduced to a set of nonlinear ordinary differential equations taking a guide of some appropriate similarity transformations. The numerical fallouts are calculated by considering the bvp4c function of Matlab. The impacts of magnetic field parameter, surface convection parameter, Eckert number and Peclet number on non-dimensional velocity, nanoparticle concentration, temperature and density of self-moving microorganisms are interpreted through graphs and charts. The fluid velocity near the surface and the Nusselt number is lessen with magnetic field. Surface convection parameter enhances the self-moving microorganism flux but a reverse result is noticed for Peclet number. Also, the contrast between the present results with formerly visited outcomes is in excellent harmony. Keywords: Nanofluid, Bioconvection, Gyrotactic microorganisms, Magnetic field, Convective boundary condition

The large universal Pantoea plasmid LPP-1 plays a major role in biological and ecological diversification

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De Maayer Pieter

2012-11-01

Full Text Available Abstract Background Pantoea spp. are frequently isolated from a wide range of ecological niches and have various biological roles, as plant epi- or endophytes, biocontrol agents, plant-growth promoters or as pathogens of both plant and animal hosts. This suggests that members of this genus have undergone extensive genotypic diversification. One means by which this occurs among bacteria is through the acquisition and maintenance of plasmids. Here, we have analyzed and compared the sequences of a large plasmid common to all sequenced Pantoea spp. Results and discussion The Large PantoeaPlasmids (LPP-1 of twenty strains encompassing seven different Pantoea species, including pathogens and endo-/epiphytes of a wide range of plant hosts as well as insect-associated strains, were compared. The LPP-1 plasmid sequences range in size from ~281 to 794 kb and carry between 238 and 750 protein coding sequences (CDS. A core set of 46 proteins, encompassing 2.2% of the total pan-plasmid (2,095 CDS, conserved among all LPP-1 plasmid sequences, includes those required for thiamine and pigment biosynthesis. Phylogenetic analysis reveals that these plasmids have arisen from an ancestral plasmid, which has undergone extensive diversification. Analysis of the proteins encoded on LPP-1 also showed that these plasmids contribute to a wide range of Pantoea phenotypes, including the transport and catabolism of various substrates, inorganic ion assimilation, resistance to antibiotics and heavy metals, colonization and persistence in the host and environment, pathogenesis and antibiosis. Conclusions LPP-1 is universal to all Pantoea spp. whose genomes have been sequenced to date and is derived from an ancestral plasmid. LPP-1 encodes a large array of proteins that have played a major role in the adaptation of the different Pantoea spp. to their various ecological niches and their specialization as pathogens, biocontrol agents or benign saprophytes found in many diverse

[Isolation and characterization of petroleum catabolic broad-host-range plasmids from Shen-Fu wastewater irrigation zone].

Science.gov (United States)

Wang, Ya-Fei; Wang, Ya-Fei; Li, Hui; Li, Xiao-Bin

2013-11-01

Based on triparental mating, we isolated a total of eight broad host range (BHR) petroleum hydrocarbon catabolic plasmids from the soils, sediments, and wastewater samples in the Shen-Fu irrigation zone. The antibiotic resistance of the plasmids was tested, and then, the plasmids were transferred to Escherichia coli EC100. The plasmids carrying no antibiotic resistance were tagged by miniTn5 transposon consisting of antibiotic resistant genes. The PCR-based incompatibility test revealed that the pS3-2C and pS4-6G belonged to Inc P group, the pS3-2G, pW22-3G, and pA15-7G belonged to Inc N group, the pS7-2G was identified as Inc W plasmid, and the pA23-1G and pA10-1C were placed into Inc Q group. By adopting the reported PCR amplification methods of petroleum hydrocarbon-degrading catabolic genes, the petroleum-degrading capability of these BHR plasmids were preliminarily analyzed. The plasmids pS3-2G, pS7-2G, pA23-1G, pW22-3G, and pA10-1C carried aromatic ring- hydroxylating dioxygenase gene phdA and toluene monooxygenase gene touA; the plasmid pA15-7G carried touA and toluene dioxygenase gene tod; the plasmid pS3-2C carried ben, phdA, and tod; whereas the pS4-6G only carried ben. The host range test showed that all the isolated plasmids except pS3-2C could be transferred and maintained stably in the representative strains Agrobacterium tumefaciens C58, Cupriavidus necator JMP228, and E. coli EC100 of the alpha-, beta-, and gamma-Proteobacteria, respectively.

Microbe and Mineral Mediated Transformation of Heavy Metals, Radionuclides, and Organic Contaminants

Science.gov (United States)

Gerlach, R.

2011-12-01

Microorganisms influence their surroundings in many ways and humans have utilized microbially catalyzed reactions for benefit for centuries. Over the past few decades, microorganisms have been used for the control of contaminant transport in subsurface environments where many microbe mineral interactions occur. This presentation will discuss microbially influenced mineral formation and transformation as well as their influence on the fate of organic contaminants such as chlorinated aliphatics & 2,4,6-trinitrotoluene (TNT), heavy metals such as chromium, and radionuclides such as uranium & strontium. Both, batch and flow experiments have been performed, which monitor the net effect of microbe mineral interactions on the fate of these contaminants. This invited presentation will place an emphasis on the relative importance of direct microbial (i.e. biotic) transformations, mineral-mediated transformations as well as other abiotic reactions influencing the fate of environmental contaminants. Experiments will be summarized and placed in context of past and future engineered applications for the control of subsurface contaminants.

Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.

Science.gov (United States)

Bharat, Tanmay A M; Murshudov, Garib N; Sachse, Carsten; Löwe, Jan

2015-07-02

Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

Two highly divergent lineages of exfoliative toxin B-encoding plasmids revealed in impetigo strains of Staphylococcus aureus.

Science.gov (United States)

Botka, Tibor; Růžičková, Vladislava; Svobodová, Karla; Pantůček, Roman; Petráš, Petr; Čejková, Darina; Doškař, Jiří

2017-09-01

Exfoliative toxin B (ETB) encoded by some large plasmids plays a crucial role in epidermolytic diseases caused by Staphylococcus aureus. We have found as yet unknown types of etb gene-positive plasmids isolated from a set of impetigo strains implicated in outbreaks of pemphigus neonatorum in Czech maternity hospitals. Plasmids from the strains of clonal complex CC121 were related to archetypal plasmid pETB TY4 . Sharing a 33-kb core sequence including virulence genes for ETB, EDIN C, and lantibiotics, they were assigned to a stand-alone lineage, named pETB TY4 -based plasmids. Differing from each other in the content of variable DNA regions, they formed four sequence types. In addition to them, a novel unique plasmid pETB608 isolated from a strain of ST130 was described. Carrying conjugative cluster genes, as well as new variants of etb and edinA genes, pETB608 could be regarded as a source of a new lineage of ETB plasmids. We have designed a helpful detection assay, which facilitates the precise identification of the all described types of ETB plasmids. Copyright © 2017 Elsevier GmbH. All rights reserved.

Expansion of plasmid mediated blaACT-2 among Pseudomonas aeruginosa associated with postoperative infection and its transcriptional response under cephalosporin stress.

Directory of Open Access Journals (Sweden)

Birson Ingti, Deepjyoti Paul, Anand Prakash Maurya

2017-06-01

Full Text Available Objectives: Organisms harboring multiple plasmid mediated β-lactamases are major concerns in nosocomial infections. Among these plasmid mediated β-lactamases, ACT (EBC family is a clinically important enzyme capable of hydrolyzing broad spectrum cephalosporins. Therefore, the present study was undertaken to determine the prevalence of ACT determinant along with other co-existing β-lactamase genes in P. aeruginosa strains. Methods: A total of 176 Pseudomonas isolates were phenotypically screened for the presence of AmpC β-lactamase by M3DET Method followed by Molecular detection using PCR assay. Transcriptional evaluation of blaACT-2 gene was analyzed by RT-PCR and its transferability was performed by transformation and conjugation. Results: Present study demonstrates the presence of ACT-2 allele among 12 strains of P. aeruginosa. Co-existence of other β-lactamase genes were encountered among ACT-2 harboring strains which includes CTX-M (n=2, SHV (n=3, TEM (n=2, VEB (n=2, OXA-10 (n=1, CIT (n=2 and DHA (n=3. Fingerprinting by REP PCR revealed the isolates harboring ACT-2 to be distinct and these isolates showed high resistance to expanded-spectrum cephalosporins and even to carbapenem group of drugs. This ACT-2 allele was encoded in the plasmid (L/M, FIA, FIB Inc. Group and conjugatively transferable. Transcriptional analysis revealed a significant increase in ACT-2 expression (483 fold when induced by ceftriaxone at 4 µg/ml followed by ceftazidime at 8 µg/ml (31 fold and cefotaxime 4 µg/ml (8 fold. Conclusion: In this study detection of ACT-2 plasmid mediated AmpC β-lactamase along with other β-lactamase genes in clinical isolates of P. aeruginosa represents a serious therapeutic challenge. Therefore, revision in antimicrobial policy is required for effective treatment of patients infected with pathogen expressing this mechanism. J Microbiol Infect Dis 2017; 7(2: 75-82

Growth dependence of conjugation explains limited plasmid invasion in biofilms: an individual‐based modelling study

DEFF Research Database (Denmark)

Merkey, Brian; Lardon, Laurent; Seoane, Jose Miguel

2011-01-01

Plasmid invasion in biofilms is often surprisingly limited in spite of the close contact of cells in a biofilm. We hypothesized that this poor plasmid spread into deeper biofilm layers is caused by a dependence of conjugation on the growth rate (relative to the maximum growth rate) of the donor......, we find that invasion of a resident biofilm is indeed limited when plasmid transfer depends on growth, but not so in the absence of growth dependence. Using sensitivity analysis we also find that parameters related to timing (i.e. a lag before the transconjugant can transfer, transfer proficiency...... and scan speed) and spatial reach (EPS yield, conjugal pilus length) are more important for successful plasmid invasion than the recipients' growth rate or the probability of segregational loss. While this study identifies one factor that can limit plasmid invasion in biofilms, the new individual...

Development and application of a general plasmid reference material for GMO screening.

Science.gov (United States)

Wu, Yuhua; Li, Jun; Wang, Yulei; Li, Xiaofei; Li, Yunjing; Zhu, Li; Li, Jun; Wu, Gang

The use of analytical controls is essential when performing GMO detection through screening tests. Additionally, the presence of taxon-specific sequences is analyzed mostly for quality control during GMO detection. In this study, 11 commonly used genetic elements involving three promoters (P-35S, P-FMV35S and P-NOS), four marker genes (Bar, NPTII, HPT and Pmi), and four terminators (T-NOS, T-35S, T-g7 and T-e9), together with the reference gene fragments from six major crops of maize, soybean, rapeseed, rice, cotton and wheat, were co-integrated into the same single plasmid to construct a general reference plasmid pBI121-Screening. The suitability test of pBI121-Screening plasmid as reference material indicated that the non-target sequence on the pBI121-Screening plasmid did not affect the PCR amplification efficiencies of screening methods and taxon-specific methods. The sensitivity of screening and taxon-specific assays ranged from 5 to 10 copies of pBI121-Screening plasmid, meeting the sensitivity requirement of GMO detection. The construction of pBI121-Screening solves the lack of a general positive control for screening tests, thereby reducing the workload and cost of preparing a plurality of the positive control. Copyright © 2016 Elsevier B.V. All rights reserved.

[Construction and identification of eukaryotic plasmid pGC-silencer-U6/Neo/GFP/ABCG2].

Science.gov (United States)

Yu, Yanping; Zhang, Song; Kong, Weijia

2010-09-01

To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human ABCG2 gene, to assay the expression of ABCG2 in a human nasopharyngeal carcinoma (NPC) cell line, CEN-2 cell line, and to detect the RNAi effect of shRNA. Targeting ABCG2 gene sequence, three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and then identified by restriction digestion, PCR and sequencing. The recombinant plasmids were transfected into CEN-2 cells. ABCG2 expression was assayed by real-time quantitative PCR and Western blot. The construction of pGC-silencer-U6/Neo/GFP/ABCG2 was succeed. The shRNA plasmids significantly down-regulated the ABCG2 expression in CEN-2 cells, at both mRNA level and protein level. Recombinant plasmid 1 had the strongest effect compared with plasmids 2 and 3 (P < 0.05), with an inhibition ratio of 75% at the mRNA level and 68% at the protein level. pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells, which could help further studies of ABCG2 functions CEN-2 cell line and contribute to the NPC gene therapy.

Bacterial mitosis: Partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

DEFF Research Database (Denmark)

Ebersbach, G.; Gerdes, Kenn

2004-01-01

The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells......A-GFP oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid...

Production of bioplastics and hydrogen gas by photosynthetic microorganisms

Science.gov (United States)

Yasuo, Asada; Masato, Miyake; Jun, Miyake

1998-03-01

Our efforts have been aimed at the technological basis of photosynthetic-microbial production of materials and an energy carrier. We report here accumulation of poly-(3-hydroxybutyrate) (PHB), a raw material of biodegradable plastics and for production of hydrogen gas, and a renewable energy carrier by photosynthetic microorganisms (tentatively defined as cyanobacteria plus photosynthetic bateria, in this report). A thermophilic cyanobacterium, Synechococcus sp. MA19 that accumulates PHB at more than 20% of cell dry wt under nitrogen-starved conditions was isolated and microbiologically identified. The mechanism of PHB accumulation was studied. A mesophilic Synechococcus PCC7942 was transformed with the genes encoding PHB-synthesizing enzymes from Alcaligenes eutrophus. The transformant accumulated PHB under nitrogen-starved conditions. The optimal conditions for PHB accumulation by a photosynthetic bacterium grown on acetate were studied. Hydrogen production by photosynthetic microorganisms was studied. Cyanobacteria can produce hydrogen gas by nitrogenase or hydrogenase. Hydrogen production mediated by native hydrogenase in cyanobacteria was revealed to be in the dark anaerobic degradation of intracellular glycogen. A new system for light-dependent hydrogen production was targeted. In vitro and in vivo coupling of cyanobacterial ferredoxin with a heterologous hydrogenase was shown to produce hydrogen under light conditions. A trial for genetic trasformation of Synechococcus PCC7942 with the hydrogenase gene from Clostridium pasteurianum is going on. The strong hydrogen producers among photosynthetic bacteria were isolated and characterized. Co-culture of Rhodobacter and Clostriumdium was applied to produce hydrogen from glucose. Conversely in the case of cyanobacteria, genetic regulation of photosynthetic proteins was intended to improve conversion efficiency in hydrogen production by the photosynthetic bacterium, Rhodobacter sphaeroides RV. A mutant acquired by

Crocodylians evolved scattered multi-sensory micro-organs

Science.gov (United States)

2013-01-01

Background During their evolution towards a complete life cycle on land, stem reptiles developed both an impermeable multi-layered keratinized epidermis and skin appendages (scales) providing mechanical, thermal, and chemical protection. Previous studies have demonstrated that, despite the presence of a particularly armored skin, crocodylians have exquisite mechanosensory abilities thanks to the presence of small integumentary sensory organs (ISOs) distributed on postcranial and/or cranial scales. Results Here, we analyze and compare the structure, innervation, embryonic morphogenesis and sensory functions of postcranial, cranial, and lingual sensory organs of the Nile crocodile (Crocodylus niloticus) and the spectacled caiman (Caiman crocodilus). Our molecular analyses indicate that sensory neurons of crocodylian ISOs express a large repertoire of transduction channels involved in mechano-, thermo-, and chemosensory functions, and our electrophysiological analyses confirm that each ISO exhibits a combined sensitivity to mechanical, thermal and pH stimuli (but not hyper-osmotic salinity), making them remarkable multi-sensorial micro-organs with no equivalent in the sensory systems of other vertebrate lineages. We also show that ISOs all exhibit similar morphologies and modes of development, despite forming at different stages of scale morphogenesis across the body. Conclusions The ancestral vertebrate diffused sensory system of the skin was transformed in the crocodylian lineages into an array of discrete multi-sensory micro-organs innervated by multiple pools of sensory neurons. This discretization of skin sensory expression sites is unique among vertebrates and allowed crocodylians to develop a highly-armored, but very sensitive, skin. PMID:23819918

Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster.

Science.gov (United States)

Tarazanova, Mariya; Beerthuyzen, Marke; Siezen, Roland; Fernandez-Gutierrez, Marcela M; de Jong, Anne; van der Meulen, Sjoerd; Kok, Jan; Bachmann, Herwig

2016-01-01

Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.

Characterization and plasmid elimination of NDM-1-producing Acinetobacter calcoaceticus from China.

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Yang Sun

Full Text Available The presence of multidrug-resistant bacterial pathogens in the environment poses a serious threat to public health. The opportunistic Acinetobacter spp. are among the most prevalent causes of nosocomial infections. Here, we performed complete genome sequencing of the Acinetobacter calcoaceticus strain XM1570, which was originally cultivated from the sputum of a patient diagnosed with pneumonia in Xiamen in 2010. We identified carbapenem resistance associated gene bla(NDM-1 located on a 47.3-kb plasmid. Three methods--natural reproduction, sodium dodecyl sulfate treatment and nalidixic acid treatment--were used to eliminate the bla(NDM-1-encoding plasmid, which achieved elimination rates of 3.32% (10/301, 83.78% (278/332, and 84.17% (298/354, respectively. Plasmid elimination dramatically increased antibiotic sensitivity, reducing the minimum bacteriostatic concentration of meropenem from 256 µg/ml in the clinical strain to 0.125 µg/ml in the plasmid-eliminated strain. Conjugation transfer assays showed that the bla(NDM-1-containing plasmid could be transferred into Escherichia coli DH5α:pBR322 in vitro as well as in vivo in mice. The bla(NDM-1 genetic environment was in accordance with that of other bla(NDM-1 genes identified from India, Japan, and Hong-Kong. The multilocus sequence type of the isolate was identified as ST-70. Two novel genes encoding intrinsic OXA and ADC were identified and named as OXA-417 and ADC-72. The finding of bla(NDM-1 in species like A. calcoaceticus demonstrates the wide spread of this gene in gram-negative bacteria which is possible by conjugative plasmid transfer. The results of this study may help in the development of a treatment strategy for controlling NDM-1 bacterial infection and transmission.

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

Science.gov (United States)

Bossé, Janine T; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Oshota, Olusegun; Holden, Matt T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

2015-08-01

The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Anaerobic microbial transformations of radioactive wastes in subsurface environments

International Nuclear Information System (INIS)

Francis, A.J.

1984-01-01

Radioactive wastes disposed of in subsurface environments contain a variety of radionuclides and organic compounds. Microorganisms play a major role in the transformation of organic and inorganic constituents of the waste and are partly responsible for the problems encountered at the waste disposal sites. These include microbial degradation of waste forms resulting in trench cover subsidence, migration of radionuclides, and production of radioactive gases such as 14 CO 2 , 14 CH 4 , HT, and CH 3 T. Microbial processes involved in solubilization, mobilization, and immobilization of toxic metals under aerobic and anaerobic conditions are reviewed. Complexing agents and several organic acids produced by microbial action affect mobilization of radionuclides and heavy metals from the wastes. Microorganisms play a significant role in the transformation and cycling of tritium in the environment by (i) oxidation of tritium and tritiated methane under aerobic conditions and (ii) production of tritium and tritiated methane from wastes containing tritiated water and organic compounds under anaerobic conditions. 23 references, 2 figures, 2 tables

Effects of Wheat and Faba Bean Intercropping on Microorganism Involved in Nitrogen Transformation in the Rhizosphere Soils

Directory of Open Access Journals (Sweden)

TANG Yan-fen

2016-09-01

Full Text Available Soil microorganism is one of the key factors that affects soil ecological activity. It is an important symbol of soil health, and the soil nitrogen cycle is closely related to the microorganisms. The relationship between nitrogen and microorganisms under the intercropping is im-portant for the farmland ecosystem. In this paper, phospholipid fatty acids(PLFA analysis was used to determine soil microbial communi-ties, e.g., biomasses of anaerobic bacteria, aerobic bacteria, bacteria, fungi and actinobacteria. The abundance of nitrifying genes(AOB, AOA and three denitrifying genes (nirK, norB, nosZ were measured using real-time quantitative polymerase chain reaction. The enzymes, nitrate and ammonium concentrations were measured using conventional methods. The results showed that along with the growth period, the TPLFAs(total phospholipid fatty acids increased and the bacterias, fungus, actinomyces and aerobic bacterias significantly(P<0.05 dif-fered between intercropping and monoculture. The greater abundance of AOB than AOA and the variation range of 105~106 were observed in all samples. The gene copies of norB and nosZ were pronounced by intercropping in the rhizosphere of faba bean at elongation and heading stages, respectively. The abundance of nirK remarkably(P<0.05differed between intercropping and monoculture. In intercropping rhizo-sphere, the contents of NO3--N were lower than monoculture, while the NH4+-N contents were converse (P<0.05. Conclusively, wheat and fa-ba bean intercropping system could change rhizosphere microenvironment, and then the microbial community structure in the soils, which would facilitate the conservation and supplying of soil nitrogen and reduce the nitrogen loss and pollution under the intercropping conditions to some extent. This might be the nitrogen nutrition mechanism for the overyielding of wheat and faba bean intercropping system.

Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

Energy Technology Data Exchange (ETDEWEB)

Choi, Charina L.; Claridge, Shelley A.; Garner, Ethan C.; Alivisatos, A. Paul; Mullins, R. Dyche

2008-07-15

To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. Wefound that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmidssegregating as a unit.

Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community

Science.gov (United States)

Cormier, Catherine Y.; Mohr, Stephanie E.; Zuo, Dongmei; Hu, Yanhui; Rolfs, Andreas; Kramer, Jason; Taycher, Elena; Kelley, Fontina; Fiacco, Michael; Turnbull, Greggory; LaBaer, Joshua

2010-01-01

The Protein Structure Initiative Material Repository (PSI-MR; http://psimr.asu.edu) provides centralized storage and distribution for the protein expression plasmids created by PSI researchers. These plasmids are a resource that allows the research community to dissect the biological function of proteins whose structures have been identified by the PSI. The plasmid annotation, which includes the full length sequence, vector information and associated publications, is stored in a freely available, searchable database called DNASU (http://dnasu.asu.edu). Each PSI plasmid is also linked to a variety of additional resources, which facilitates cross-referencing of a particular plasmid to protein annotations and experimental data. Plasmid samples can be requested directly through the website. We have also developed a novel strategy to avoid the most common concern encountered when distributing plasmids namely, the complexity of material transfer agreement (MTA) processing and the resulting delays this causes. The Expedited Process MTA, in which we created a network of institutions that agree to the terms of transfer in advance of a material request, eliminates these delays. Our hope is that by creating a repository of expression-ready plasmids and expediting the process for receiving these plasmids, we will help accelerate the accessibility and pace of scientific discovery. PMID:19906724

Spontaneous mutability and light-induced mutagenesis in Salmonella typhimurium: effects of an R-plasmid

International Nuclear Information System (INIS)

Valdivia, L.

1979-01-01

The UV-protecting plasmid R46 was transferred by conjugation to a genetically marked mouse-virulent Salmonella typhimurium strain, not derived from LT2; in this host the plasmid conferred UV protection and enhanced UV mutagenesis just as it does in LT2 lines. Tra - derivatives of R46 encountered during transduction retained UV-protecting and mutagenesis-enhancing ability. Stored strains carrying the R46-derived plasmids with strong mutator effect but not UV-protecting had lost most of their original streptomycin resistance but were slightly resistant to spectinomycin; attempts to transfer such plasmids failed. R46 enhanced the weak mutagenic effect of visible light on several his and trp mutants of strain LT2, including some whose frequency of spontaneous reversion was not increased by the plasmid. A mutagenic effect was produced by visible-light irradiation of hisG46(R46), either growing cells or nonmultiplying (histidine-deprived cells at 10 0 C). Presence of catalase or cyanide during irradiation did not prevent mutagenesis, which excludes some hypothetical mechanisms. Visible-light irradiation of hisG46 or hisG46(R46) under strict anaerobiosis had little or no mutagenic effect (controls showed that revertants if produced would have been detected). This is as expected if visible-light irradiation in air causes photodynamic damage to DNA and mutations are produced during error-prone, plasmid-enhanced repair

Isolation of a minireplicon of the plasmid pG6303 of Lactobacillus ...

Indian Academy of Sciences (India)

is a new mode of plasmid replication. [Fan J., Xi X., ... coli using the BioTeKe plasmid extraction kit (BioTeKe, Beijing, China) according .... media and incubated at 37◦C for three days. The methods of ..... Each experiment was repeated five times. Journal of ..... Cold Spring Harbor Laboratory Press, New York, USA. Soler N.

Bioplastics from microorganisms.

Science.gov (United States)

Luengo, José M; García, Belén; Sandoval, Angel; Naharro, Germán; Olivera, Elías R

2003-06-01

The term 'biomaterials' includes chemically unrelated products that are synthesised by microorganisms (or part of them) under different environmental conditions. One important family of biomaterials is bioplastics. These are polyesters that are widely distributed in nature and accumulate intracellularly in microorganisms in the form of storage granules, with physico-chemical properties resembling petrochemical plastics. These polymers are usually built from hydroxy-acyl-CoA derivatives via different metabolic pathways. Depending on their microbial origin, bioplastics differ in their monomer composition, macromolecular structure and physical properties. Most of them are biodegradable and biocompatible, which makes them extremely interesting from the biotechnological point of view.

Unraveling the regulatory network of IncA/C plasmid mobilization: When genomic islands hijack conjugative elements.

Science.gov (United States)

Carraro, Nicolas; Matteau, Dominick; Burrus, Vincent; Rodrigue, Sébastien

2015-01-01

Conjugative plasmids of the A/C incompatibility group (IncA/C) have become substantial players in the dissemination of multidrug resistance. These large conjugative plasmids are characterized by their broad host-range, extended spectrum of antimicrobials resistance, and prevalence in enteric bacteria recovered from both environmental and clinical settings. Until recently, relatively little was known about the basic biology of IncA/C plasmids, mostly because of the hindrance of multidrug resistance for molecular biology experiments. To circumvent this issue, we previously developed pVCR94ΔX, a convenient prototype that codes for a reduced set of antibiotic resistances. Using pVCR94ΔX, we then characterized the regulatory pathway governing IncA/C plasmid dissemination. We found that the expression of roughly 2 thirds of the genes encoded by this plasmid, including large operons involved in the conjugation process, depends on an FlhCD-like master activator called AcaCD. Beyond the mobility of IncA/C plasmids, AcaCD was also shown to play a key role in the mobilization of different classes of genomic islands (GIs) identified in various pathogenic bacteria. By doing so, IncA/C plasmids can have a considerable impact on bacterial genomes plasticity and evolution.

Characterization of Plasmid DNA Location within Chitosan/PLGA/pDNA Nanoparticle Complexes Designed for Gene Delivery

Directory of Open Access Journals (Sweden)

Hali Bordelon

2011-01-01

Full Text Available Poly(D,L-lactide-co-glycolide- (PLGA-chitosan nanoparticles are becoming an increasingly common choice for the delivery of nucleic acids to cells for various genetic manipulation techniques. These particles are biocompatible, with tunable size and surface properties, possessing an overall positive charge that promotes complex formation with negatively charged nucleic acids. This study examines properties of the PLGA-chitosan nanoparticle/plasmid DNA complex after formation. Specifically, the study aims to determine the optimal ratio of plasmid DNA:nanoparticles for nucleic acid delivery purposes and to elucidate the location of the pDNA within these complexes. Such characterization will be necessary for the adoption of these formulations in a clinical setting. The ability of PLGA-chitosan nanoparticles to form complexes with pDNA was evaluated by using the fluorescent intercalating due OliGreen to label free plasmid DNA. By monitoring the fluorescence at different plasmid: nanoparticle ratios, the ideal plasmid:nanoparticle ration for complete complexation of plasmid was determined to be 1:50. Surface-Enhanced Raman Spectroscopy and gel digest studies suggested that even at these optimal complexation ratios, a portion of the plasmid DNA was located on the outer complex surface. This knowledge will facilitate future investigations into the functionality of the system in vitro and in vivo.

Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

Science.gov (United States)

van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

2018-03-23

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L

Role of the adenovirus early region 1B tumor antigens in transformation and lytic infection

NARCIS (Netherlands)

Bernards, R.A.; Leeuw, M.G.W. de; Houweling, A.; Eb, A.J. van der

1986-01-01

We have investigated the contribution of each of the two adenovirus type 5 (Ad5) major early region lb (Elb) proteins in cell transformation and in lytic infection. An Ad5 El plasmid, in which the reading frame for the 19-kDa Elb protein was abolished by a stop codon close to the initiation codon,

Microbial transformations of radionuclides released from nuclear fuel reprocessing plants

International Nuclear Information System (INIS)

Francis, A.J.

2007-01-01

Microorganisms can affect the stability and mobility of the actinides U, Pu, Cm, Am, Np, and the fission products Tc, I, Cs, Sr, released from nuclear fuel reprocessing plants. Under appropriate conditions, microorganisms can alter the chemical speciation, solubility and sorption properties and thus could increase or decrease the concentrations of radionuclides in solution and the bioavailability. Dissolution or immobilization of radionuclides is brought about by direct enzymatic action or indirect non-enzymatic action of microorganisms. Although the physical, chemical, and geochemical processes affecting dissolution, precipitation, and mobilization of radionuclides have been investigated, we have only limited information on the effects of microbial processes. The mechanisms of microbial transformations of the major and minor actinides and the fission products under aerobic and anaerobic conditions in the presence of electron donors and acceptors are reviewed. (author)

Plasmid transfer by conjugation in Xylella fastidiosa.

Science.gov (United States)

Recombination and horizontal gene transfer have been implicated in the adaption of Xylella fastidiosa (Xf) to infect a wide variety of different plant species. There is evidence that certain strains of Xf carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as ...

Resveratrol production in bioreactor: Assessment of cell physiological states and plasmid segregational stability

Directory of Open Access Journals (Sweden)

Margarida S. Afonso

2015-03-01

Full Text Available Resveratrol is a plant secondary metabolite commonly found in peanuts and grapevines with significant health benefits. Recombinant organisms can produce large amounts of resveratrol and, in this work, Escherichia coli BW27784 was used to produce resveratrol in bioreactors while monitoring cell physiology and plasmid stability through flow cytometry and real-time qPCR, respectively. Initially, the influence of culture conditions and precursor addition was evaluated in screening assays and the data gathered was used to perform the bioreactor assays, allowing the production of 160 μg/mL of resveratrol. Cellular physiology and plasmid instability affected the final resveratrol production, with lower viability and plasmid copy numbers associated with lower yields. In sum, this study describes new tools to monitor the bioprocess, evaluating the effect of culture conditions, and its correlation with cell physiology and plasmid segregational stability, in order to define a viable and scalable bioprocess to fulfill the need for larger quantities of resveratrol.

Identification of a Novel Conjugative Plasmid in Mycobacteria That Requires Both Type IV and Type VII Secretion

KAUST Repository

Ummels, R.; Abdallah, A. M.; Kuiper, V.; Aajoud, A.; Sparrius, M.; Naeem, R.; Spaink, H. P.; van Soolingen, D.; Pain, Arnab; Bitter, W.

2014-01-01

Conjugative plasmids play an important role in horizontal gene transfer between different bacteria and, as such, in their adaptation and evolution. This effect is most obvious in the spread of antibiotic resistance genes. Thus far, conjugation of natural plasmids has been described only rarely for mycobacterial species. In fact, it is generally accepted that M. tuberculosis does not show any recent sign of horizontal gene transfer. In this study, we describe the identification of a new widespread conjugative plasmid that can also be efficiently transferred to M. tuberculosis. This plasmid therefore poses both a threat and an opportunity. The threat is that, through the acquisition of antibiotic resistance markers, this plasmid could start a rapid spread of antibiotic resistance genes between pathogenic mycobacteria. The opportunity is that we could use this plasmid to generate new tools for the efficient introduction of foreign DNA in slow-growing mycobacteria.

Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain

Directory of Open Access Journals (Sweden)

Skilton Rachel J

2009-05-01

Full Text Available Abstract Background Chlamydia trachomatis is the most common cause of sexually transmitted infections globally and the leading cause of preventable blindness in the developing world. There are two biovariants of C. trachomatis: 'trachoma', causing ocular and genital tract infections, and the invasive 'lymphogranuloma venereum' strains. Recently, a new variant of the genital tract C. trachomatis emerged in Sweden. This variant escaped routine diagnostic tests because it carries a plasmid with a deletion. Failure to detect this strain has meant it has spread rapidly across the country provoking a worldwide alert. In addition to being a key diagnostic target, the plasmid has been linked to chlamydial virulence. Analysis of chlamydial plasmids and their cognate chromosomes was undertaken to provide insights into the evolutionary relationship between chromosome and plasmid. This is essential knowledge if the plasmid is to be continued to be relied on as a key diagnostic marker, and for an understanding of the evolution of Chlamydia trachomatis. Results The genomes of two new C. trachomatis strains were sequenced, together with plasmids from six C. trachomatis isolates, including the new variant strain from Sweden. The plasmid from the new Swedish variant has a 377 bp deletion in the first predicted coding sequence, abolishing the site used for PCR detection, resulting in negative diagnosis. In addition, the variant plasmid has a 44 bp duplication downstream of the deletion. The region containing the second predicted coding sequence is the most highly conserved region of the plasmids investigated. Phylogenetic analysis of the plasmids and chromosomes are fully congruent. Moreover this analysis also shows that ocular and genital strains diverged from a common C. trachomatis progenitor. Conclusion The evolutionary pathways of the chlamydial genome and plasmid imply that inheritance of the plasmid is tightly linked with its cognate chromosome. These data

Microbial transformation of highly persistent chlorinated pesticides and industrial chemicals

NARCIS (Netherlands)

Middeldorp, P.J.M.

1997-01-01

Organic pollutants can be transformed, both in unsaturated and saturated areas of the soil, by means of biologically mediated reactions. The potential of soil microorganisms to clean up polluted soils is enormous. However, soil systems are highly heterogeneous with respect to the spatial

Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

DEFF Research Database (Denmark)

Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

2011-01-01

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

Microorganism identification technique using radioactive and fluorescent agent

International Nuclear Information System (INIS)

Silman, R.E.

1983-01-01

A method for identifying microorganisms is claimed. An emissive agent is added to a specimen of microorganisms to produce a mix of emissive products. These products are detected and characteristic pattern functioning as an identifier for the microorganisms is derived. The identifier is then compared with identifiers representing known microorganisms

Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

Science.gov (United States)

Burbank, Lindsey P; Stenger, Drake C

2016-08-01

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

A genetic study of a Staphylococus aureus plasmid involving cure and transference

Directory of Open Access Journals (Sweden)

Ana Lúcia Costa Darini

Full Text Available High frequency transfer and elimination of drug resistance may indicate an extrachromosomal inheritance of genetic determinants. This study shows the cure and transfer of a small plasmid and tetracycline resistance in Staphylococcus aureus 1030 (55TetR strains. Several methods are available for plasmid elimination. We used ethidium bromide, an agent that binds to DNA, and thus inhibits DNA polymerase. This caused a high frequency of loss of the small plasmid and resistance to tetracycline. Transfer of tetracycline resistance was done in a mixed culture at a frequency of 10-6. This type of study is very important to physicians and epidemiology investigators and provides better knowledge on antibiotic-resistance mechanisms that may occur in vivo in a hospital environment.

Regular cellular distribution of plasmids by oscillating and filament-forming ParA ATPase of plasmid pB171

DEFF Research Database (Denmark)

Ebersbach, Gitte; Ringgaard, Simon; Møller-Jensen, Jakob

2006-01-01

with each other in a bacterial two-hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions...

Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

Directory of Open Access Journals (Sweden)

Mette Burmølle

Full Text Available Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids into a laboratory strain (Escherichia coli Genehogs® for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54 and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33 were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

Science.gov (United States)

Burmølle, Mette; Norman, Anders; Sørensen, Søren J; Hansen, Lars Hestbjerg

2012-01-01

Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

Ordering the mob: Insights into replicon and MOB typing schemes from analysis of a curated dataset of publicly available plasmids.

Science.gov (United States)

Orlek, Alex; Phan, Hang; Sheppard, Anna E; Doumith, Michel; Ellington, Matthew; Peto, Tim; Crook, Derrick; Walker, A Sarah; Woodford, Neil; Anjum, Muna F; Stoesser, Nicole

2017-05-01

Plasmid typing can provide insights into the epidemiology and transmission of plasmid-mediated antibiotic resistance. The principal plasmid typing schemes are replicon typing and MOB typing, which utilize variation in replication loci and relaxase proteins respectively. Previous studies investigating the proportion of plasmids assigned a type by these schemes ('typeability') have yielded conflicting results; moreover, thousands of plasmid sequences have been added to NCBI in recent years, without consistent annotation to indicate which sequences represent complete plasmids. Here, a curated dataset of complete Enterobacteriaceae plasmids from NCBI was compiled, and used to assess the typeability and concordance of in silico replicon and MOB typing schemes. Concordance was assessed at hierarchical replicon type resolutions, from replicon family-level to plasmid multilocus sequence type (pMLST)-level, where available. We found that 85% and 65% of the curated plasmids could be replicon and MOB typed, respectively. Overall, plasmid size and the number of resistance genes were significant independent predictors of replicon and MOB typing success. We found some degree of non-concordance between replicon families and MOB types, which was only partly resolved when partitioning plasmids into finer-resolution groups (replicon and pMLST types). In some cases, non-concordance was attributed to ambiguous boundaries between MOBP and MOBQ types; in other cases, backbone mosaicism was considered a more plausible explanation. β-lactamase resistance genes tended not to show fidelity to a particular plasmid type, though some previously reported associations were supported. Overall, replicon and MOB typing schemes are likely to continue playing an important role in plasmid analysis, but their performance is constrained by the diverse and dynamic nature of plasmid genomes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Comparative assessment of plasmid DNA delivery by encapsulation ...

African Journals Online (AJOL)

Tropical Journal of Pharmaceutical Research January 2018; 17 (1): 1-10 ... Purpose: To compare the gene delivery effectiveness of plasmid DNA (pDNA) ..... Intramuscular delivery of DNA ... copolymeric system for gene delivery in complete.

Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system.

Science.gov (United States)

Tang, Yao Liang; Tang, Yi; Zhang, Y Clare; Qian, Keping; Shen, Leping; Phillips, M Ian

2004-04-01

Although human heme oxygenase-1 (hHO-1) could provide a useful approach for cellular protection in the ischemic heart, constitutive overexpression of hHO-1 may lead to unwanted side effects. To avoid this, we designed a hypoxia-regulated hHO-1 gene therapy system that can be switched on and off. This vigilant plasmid system is composed of myosin light chain-2v promoter and a gene switch that is based on an oxygen-dependent degradation domain from the hypoxia inducible factor-1-alpha. The vector can sense ischemia and switch on the hHO-1 gene system, specifically in the heart. In an in vivo experiment, the vigilant hHO-1 plasmid or saline was injected intramyocardially into myocardial infarction mice or sham operation mice. After gene transfer, expression of hHO-1 was only detected in the ischemic heart treated with vigilant hHO-1 plasmids. Masson trichrome staining showed significantly fewer fibrotic areas in vigilant hHO-1 plasmids-treated mice compared with saline control (43.0%+/-4.8% versus 62.5%+/-3.3%, PhHO-1 expression in peri-infarct border areas, concomitant with higher Bcl-2 levels and lower Bax, Bak, and caspase 3 levels in the ischemic myocardium compared with saline control. By use of a cardiac catheter, heart from vigilant hHO-1 plasmids-treated mice showed improved recovery of contractile and diastolic performance after myocardial infarction compared with saline control. This study documents the beneficial regulation and therapeutic potential of vigilant plasmid-mediated hHO-1 gene transfer. This novel gene transfer strategy can provide cardiac-specific protection from future repeated bouts of ischemic injury.

Plasmid vectors for Xylella fastidiosa utilizing a toxin-antitoxin system for plasmid stability in the absence of antibiotic selection

Science.gov (United States)

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacte...

Optimization of plasmid electrotransformation into Escherichia coli ...

African Journals Online (AJOL)

In order to improve electroporation, optical density of bacteria, recovery time and electrical parameter (field strength and capacitance) were optimized using the Taguchi statistical method. ANOVA of obtained data indicated that the optimal conditions of electrotransformation of pET-28a (+) plasmid into Escherichia coli ...

Antimicrobial resistance patterns and plasmid profiles of ...

African Journals Online (AJOL)

Objectives: To determine the frequency of resistance of Staphylococcus aureus to various antimicrobial agents, and the relationship between antimicrobial resistance of the isolates and carriage of plasmids. Design: A random sampling of milk and meat samples was carried out. Setting: Milk was collected from various dairy ...

A degenerate primer MOB typing (DPMT method to classify gamma-proteobacterial plasmids in clinical and environmental settings.

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Andrés Alvarado

Full Text Available Transmissible plasmids are responsible for the spread of genetic determinants, such as antibiotic resistance or virulence traits, causing a large ecological and epidemiological impact. Transmissible plasmids, either conjugative or mobilizable, have in common the presence of a relaxase gene. Relaxases were previously classified in six protein families according to their phylogeny. Degenerate primers hybridizing to coding sequences of conserved amino acid motifs were designed to amplify related relaxase genes from γ-Proteobacterial plasmids. Specificity and sensitivity of a selected set of 19 primer pairs were first tested using a collection of 33 reference relaxases, representing the diversity of γ-Proteobacterial plasmids. The validated set was then applied to the analysis of two plasmid collections obtained from clinical isolates. The relaxase screening method, which we call "Degenerate Primer MOB Typing" or DPMT, detected not only most known Inc/Rep groups, but also a plethora of plasmids not previously assigned to any Inc group or Rep-type.

Protocol for Evaluating the Permissiveness of Bacterial Communities Toward Conjugal Plasmids by Quantification and Isolation of Transconjugants

DEFF Research Database (Denmark)

Klümper, Uli; Dechesne, Arnaud; Smets, Barth F.

2014-01-01

may encode catabolic pathways, virulence factors, and antibiotic or metal resistances, it is of environmental, evolutionary, and medical relevance to track and monitor the fate of plasmids in mixed microbial community. When assessing the short-term and long-term implications of conjugal plasmid...... a gfp-tagged plasmid in a mCherry red fluorescently tagged donor strain repressing gfp expression. We take advantage of fluorescent marker genes to microscopically detect plasmid transfer events and use subsequent high-throughput fluorescence-activated cell sorting (FACS) to isolate...

Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts

DEFF Research Database (Denmark)

Porse, Andreas; Schønning, Kristian; Munck, Christian

2016-01-01

Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution...

A novel pAA virulence plasmid encoding toxins and two distinct variants of the fimbriae of enteroaggregative Escherichia coli

DEFF Research Database (Denmark)

Jønsson, Rie; Struve, Carsten; Boll, Erik J.

2017-01-01

phylogenetically distinct, strains harboring the major pilin subunits from both AAF/III and AAF/V. Whole-genome and plasmid sequencing revealed that in these six strains the agg3A and agg5A genes were located on a novel pAA plasmid variant. Moreover, the plasmid also encoded several other virulence genes including...... some not previously found on pAA plasmids. Thus, this plasmid endows the host strains with a remarkably high number of EAEC associated virulence genes hereby likely promoting strain pathogenicity....

The sudden dominance of blaCTX-M harbouring plasmids in Shigella spp. Circulating in Southern Vietnam.

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Nhu Thi Khanh Nguyen

2010-06-01

Full Text Available Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla(CTX-M encoding plasmid.We show that two different bla(CTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla(CTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356 carried the bla(CTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion.

Science.gov (United States)

Santillán, Orlando; Ramírez-Romero, Miguel A; Dávila, Guillermo

2017-06-25

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.