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Sample records for plasmid transfer drives

  1. Plasmid transfer systems in the rhizobia.

    PubMed

    Ding, Hao; Hynes, Michael F

    2009-08-01

    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  2. Plasmid and clonal interference during post horizontal gene transfer evolution.

    PubMed

    Bedhomme, S; Perez Pantoja, D; Bravo, I G

    2017-04-01

    Plasmids are nucleic acid molecules that can drive their own replication in a living cell. They can be transmitted horizontally and can thrive in the host cell to high-copy numbers. Plasmid replication and gene expression consume cellular resources and cells carrying plasmids incur fitness costs. But many plasmids carry genes that can be beneficial under certain conditions, allowing the cell to endure in the presence of antibiotics, toxins, competitors or parasites. Horizontal transfer of plasmid-encoded genes can thus instantaneously confer differential adaptation to local or transient selection conditions. This conflict between cellular fitness and plasmid spread sets the scene for multilevel selection processes. We have engineered a system to study the short-term evolutionary impact of different synonymous versions of a plasmid-encoded antibiotic resistance gene. Applying experimental evolution under different selection conditions and deep sequencing allowed us to show rapid local adaptation to the presence of antibiotic and to the specific version of the resistance gene transferred. We describe the presence of clonal interference at two different levels: at the within-cell level, because a single cell can carry several plasmids, and at the between-cell level, because a bacterial population may contain several clones carrying different plasmids and displaying different fitness in the presence/absence of antibiotic. Understanding the within-cell and between-cell dynamics of plasmids after horizontal gene transfer is essential to unravel the dense network of mobile elements underlying the worldwide threat to public health of antibiotic resistance. © 2017 John Wiley & Sons Ltd.

  3. Camphor Plasmid-Mediated Chromosomal Transfer in Pseudomonas putida

    PubMed Central

    Shaham, M.; Chakrabarty, A. M.; Gunsalus, I. C.

    1973-01-01

    Camphor-utilizing strains of Pseudomonas putida have been shown to carry the genetic information required for camphor degradation on a plasmid. The plasmid-carrying strains can serve as donors of both plasmid-borne and chromosomal genes. As recipients, plasmid-deleted strains are much superior to those carrying the camphor pathway genes. The transfer frequency of chromosomal, but not plasmid-borne, genes is markedly enhanced if the donor cells are irradiated with ultraviolet light followed by 3-h of growth on a rich medium in the dark. Recombinants selected for prototrophy are stable and most acquire the camphor (CAM) plasmid concomitantly; only a few of the Cam+ recombinants inherit the donor's ability to transfer chromosomal genes at a high frequency. Transfer-defective mutations occur on the CAM plasmid, affecting both CAM and chromosomal gene transfer. PMID:4745436

  4. Therapeutic option of plasmid-DNA based gene transfer.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Kunugiza, Yasuo; Iekushi, Kazuma; Rakugi, Hiromi; Morishita, Ryuichi

    2012-01-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common method in clinical cases because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid deoxyribonucleic acid (DNA)-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a phase III clinical trial did not show sufficient efficiency. In this situation, more efficient plasmid DNA gene transfer is needed all over the world. This review focuses on plasmid DNA gene transfer and its enhancement, including ultrasound with microbubbles, electroporation, hydrodynamic method, gene gun, jet injection, cationic lipids and cationic polymers.

  5. Multiple Pathways of Plasmid DNA Transfer in Helicobacter pylori

    PubMed Central

    Rohrer, Stefanie; Holsten, Lea; Weiss, Evelyn; Benghezal, Mohammed; Fischer, Wolfgang; Haas, Rainer

    2012-01-01

    Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species. PMID:23029142

  6. Plasmid DNA-based gene transfer with ultrasound and microbubbles.

    PubMed

    Taniyama, Yoshiaki; Azuma, Junya; Rakugi, Hiromi; Morishita, Ryuichi

    2011-12-01

    Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.

  7. F'-plasmid transfer from Escherichia coli to Pseudomonas fluorescens.

    PubMed Central

    Mergeay, M; Gerits, J

    1978-01-01

    Various F' plasmids of Escherichia coli K-12 could be transferred into mutants of the soil strain 6.2, classified herein as a Pseudomonas fluorescens biotype IV. This strain was previously found to receive Flac plasmid (N. Datta and R.W. Hedges, J. Gen Microbiol. 70:453-460, 1972). ilv, leu, met, arg, and his auxotrophs were complemented by plasmids carrying isofunctional genes; trp mutants were not complemented or were very poorly complemented. The frequency of transfer was 10(-5). Subsequent transfer into other P. fluorescens recipients was of the same order of magnitude. Some transconjugants were unable to act as donors, and these did not lose the received information if subcultured on nonselective media. Use of F' plasmids helped to discriminate metabolic blocks in P. fluorescens. In particular, metA, metB, and argH mutants were so distinguished. In addition, F131 plasmid carrying the his operon and a supD mutation could partially relieve the auxotrophy of thr, ilv, and metA13 mutants, suggesting functional expression of E. coli tRNA in P. fluorescens. In P. fluorescens metA Rifr mutants carrying the F110 plasmid, which carried the E. coli metA gene and the E. coli rifs allele, sensitivity to rifampin was found to be dominant at least temporarily over resistance. This suggests interaction of E. coli and P. fluorescens subunits of RNA polymerase. his mutations were also complemented by composite P plasmids containing the his-nif region of Klebsiella pneumoniae (plasmids FN68 and RP41). nif expression could be detected by acetylene reduction in some his+ transconjugants. The frequency of transfer of these P plasmids was 5 X 10(-4). PMID:97267

  8. Regulation of conjugative transfer of plasmids and integrative conjugative elements.

    PubMed

    Bañuelos-Vazquez, Luis Alfredo; Torres Tejerizo, Gonzalo; Brom, Susana

    2017-05-01

    Horizontal gene transfer has been recognized as one of the principal contributors to bacterial evolution and diversification. One of the mechanisms involved in this process is conjugative transfer of plasmids and Integrative Conjugative Elements (ICEs). Plasmids and ICEs often encode traits beneficial for bacterial survival in specific environments, or for the establishment of symbiosis or pathogenesis, in addition to genes allowing conjugative transfer. In this review, we analyze the mechanisms that regulate the expression of conjugative transfer genes. For traits such as antibiotic or metal resistance, the compounds involved may induce conjugative transfer directly, while symbiosis and pathogenesis are modulated by quorum-sensing and/or signal molecules released by the host. However, multiple layers of regulation are usually involved in modulating transfer. In addition to the plasmid-encoded regulatory elements, conjugation seems to be regulated by what we have labeled as the "internal environment", defined by the interaction between the host chromosome and the plasmids or ICEs. Another regulatory level depends on the "external environment", which affects conjugative transfer due to the composition and conditions of the community. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Modelling the spatial dynamics of plasmid transfer and persistence.

    PubMed

    Krone, Stephen M; Lu, Ruinan; Fox, Randal; Suzuki, Haruo; Top, Eva M

    2007-08-01

    Bacterial plasmids are extra-chromosomal genetic elements that code for a wide variety of phenotypes in their bacterial hosts and are maintained in bacterial communities through both vertical and horizontal transfer. Current mathematical models of plasmid-bacteria dynamics, based almost exclusively on mass-action differential equations that describe these interactions in completely mixed environments, fail to adequately explain phenomena such as the long-term persistence of plasmids in natural and clinical bacterial communities. This failure is, at least in part, due to the absence of any spatial structure in these models, whereas most bacterial populations are spatially structured in microcolonies and biofilms. To help bridge the gap between theoretical predictions and observed patterns of plasmid spread and persistence, an individual-based lattice model (interacting particle system) that provides a predictive framework for understanding the dynamics of plasmid-bacteria interactions in spatially structured populations is presented here. To assess the accuracy and flexibility of the model, a series of experiments that monitored plasmid loss and horizontal transfer of the IncP-1beta plasmid pB10 : : rfp in Escherichia coli K12 and other bacterial populations grown on agar surfaces were performed. The model-based visual patterns of plasmid loss and spread, as well as quantitative predictions of the effects of different initial parental strain densities and incubation time on densities of transconjugants formed on a 2D grid, were in agreement with this and previously published empirical data. These results include features of spatially structured populations that are not predicted by mass-action differential equation models.

  10. Plasmid transfer via transduction from Streptococcus thermophilus to Lactococcus lactis.

    PubMed

    Ammann, Andreas; Neve, Horst; Geis, Arnold; Heller, Knut J

    2008-04-01

    Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.

  11. Conjugative transfer of cadmium resistance plasmids in Rhodococcus fascians strains.

    PubMed

    Desomer, J; Dhaese, P; Van Montagu, M

    1988-05-01

    The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.

  12. Modelling the spatial dynamics of plasmid transfer and persistence

    PubMed Central

    Krone, Stephen M.; Lu, Ruinan; Fox, Randal; Suzuki, Haruo; Top, Eva M.

    2008-01-01

    Bacterial plasmids are extra-chromosomal genetic elements that code for a wide variety of phenotypes in their bacterial hosts and are maintained in bacterial communities through both vertical and horizontal transfer. Current mathematical models of plasmid–bacteria dynamics, based almost exclusively on mass-action differential equations that describe these interactions in completely mixed environments, fail to adequately explain phenomena such as the long-term persistence of plasmids in natural and clinical bacterial communities. This failure is, at least in part, due to the absence of any spatial structure in these models, whereas most bacterial populations are spatially structured in microcolonies and biofilms. To help bridge the gap between theoretical predictions and observed patterns of plasmid spread and persistence, an individual-based lattice model (interacting particle system) that provides a predictive framework for understanding the dynamics of plasmid–bacteria interactions in spatially structured populations is presented here. To assess the accuracy and flexibility of the model, a series of experiments that monitored plasmid loss and horizontal transfer of the IncP-1 β plasmid pB10 ∷ rfp in Escherichia coli K12 and other bacterial populations grown on agar surfaces were performed. The model-based visual patterns of plasmid loss and spread, as well as quantitative predictions of the effects of different initial parental strain densities and incubation time on densities of transconjugants formed on a 2D grid, were in agreement with this and previously published empirical data. These results include features of spatially structured populations that are not predicted by mass-action differential equation models. PMID:17660444

  13. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer.

    PubMed

    Getino, María; Sanabria-Ríos, David J; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M; Fernández, Antonio; Carballeira, Néstor M; de la Cruz, Fernando

    2015-09-01

    Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. Diseases caused by multidrug-resistant bacteria are taking an important toll with respect to human morbidity and mortality. The most relevant antibiotic resistance genes come to human pathogens carried by plasmids, mainly using conjugation as a transmission mechanism. Here, we identified and characterized a series of compounds that were active against several plasmid groups of clinical relevance, in a wide variety of bacterial hosts. These inhibitors might be used for fighting antibiotic-resistance dissemination by inhibiting conjugation. Potential inhibitors could be used in specific settings (e.g., farm, fish factory, or even clinical settings) to

  14. Bacterial plasmid transfer under space flight conditions: The Mobilisatsia experience

    NASA Astrophysics Data System (ADS)

    de Boever, P.; Ilyin, V.; Mahillon, J.; Mergeay, M.

    Background Microorganisms are subject to a genetic evolution which may lead to the capacity to colonize new environments and to cause infections Central players in this evolutionary process are mobile genetic elements phages plasmids and transposons The latter help to mobilize and reorganize genes be it within a given genome intragenomic mobility or between bacterial cells intercellular mobility Confined environment and space flight related factors such as microgravity and cosmic radiation may influence the frequency with which mobile genetic elements are exchanged between microorganisms Aim Within the frame of the Mobilisatsia experiment a triparental microbial plasmid transfer was promoted aboard the International Space Station ISS The efficiency of the plasmid exchange process was compared with a synchronously performed ground control experiment An experiment was carried out with well-characterized Gram-negative test strains and one experiment was done with Gram-positive test strains Results The experiment took place during the Soyouz Mission 8 to the ISS from April 19th until April 30th 2004 Liquid cultures of the bacterial strains Cupriavidus metallidurans AE815 final recipient Escherichia coli CM1962 carrying a mobilisable vector with a nickel-resistance marker and E coli CM140 carrying the Broad Host Range plasmid RP4 for the Gram-negative experiment and Bacillus thuringiensis Bti AND931 carrying the conjugative plasmid pXO16 Bti 4Q7 with mobilisable vector pC194 carrying a resistance to chloramphenicol and Bti GBJ002

  15. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    PubMed Central

    Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

    2015-01-01

    ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

  16. Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

    PubMed

    Götz, F; Ahrné, S; Lindberg, M

    1981-01-01

    The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters.

  17. Plasmid transfer and genetic recombination by protoplast fusion in staphylococci.

    PubMed Central

    Götz, F; Ahrné, S; Lindberg, M

    1981-01-01

    The experimental conditions for plasmid transfer and genetic recombination in Staphylococcus aureus and some coagulase-negative staphylococci by protoplast fusion are described. Protoplasts were prepared by treatment with lysostaphin and lysozyme in a buffered medium with 0.7 to 0.8 M sucrose. Regeneration of cell walls was accomplished on a hypertonic agar medium containing succinate and bovine serum albumin. Transfer of plasmids occurred after treatment of the protoplast mixtures with polyethylene glycol (molecular weight, 6,000) not only between strains of the same species but also between parents of different species, although at approximately 100 times lower frequency in the latter case. Recombination of the chromosomal genes in fused protoplasts required simultaneous treatment of the mixed protoplasts with polyethylene glycol and CaCl2. A method was developed for isolation of recombinants after fusion between mutants of S. areus carrying unselectable markers. Antibiotic resistance plasmids were introduced into the parental strains and used as primary markers to detect protoplast fusion. Chromosomal recombinants were found among the clones with both parental plasmids at a high frequency. The method appears to have simple applications in the construction of strains with multiple mutant characters. PMID:7007333

  18. Transfer of plasmid-mediated ampicillin resistance from Haemophilus to Neisseria gonorrhoeae requires an intervening organism.

    PubMed

    McNicol, P J; Albritton, W L; Ronald, A R

    1986-01-01

    Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids.

  19. Conjugative transfer of cadmium resistance plasmids in Rhodococcus fascians strains

    SciTech Connect

    Desomer, J.; Dhaese, P.; Montagu, M.V.

    1988-05-01

    The presence of a 138-kilobase plasmid (pD188) correlated with increased resistance to cadmium in Rhodococcus fascians D188. This plasmid could be transferred by a conjugation-like system in matings between R. fascians strains. To examine this correlation we used large /sup 32/P-labeled pD188 subclones as probes in hybridization analyses with Southern blots of restricted total DNAs of D188 and its derivative mutants. Transconjugants expressed the cadmium resistance and could be used as donors in subsequent matings. Four other R. fascians strains (NCPPB 1488, NCPPB 1675, NCPPB 2551, and ATCC 12974) could also be used as donors for cadmium resistance in matings. Strain NCPPB 1675 showed a 100% cotransfer of cadmium and chloramphenicol resistance markers.

  20. Conjugal Transfer of a Virulence Plasmid in the Opportunistic Intracellular Actinomycete Rhodococcus equi

    PubMed Central

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

    2012-01-01

    Rhodococcus equi is a facultative intracellular, Gram-positive, soilborne actinomycete which can cause severe pyogranulomatous pneumonia with abscessation in young horses (foals) and in immunocompromised people, such as persons with AIDS. All strains of R. equi isolated from foals and approximately a third isolated from humans contain a large, ∼81-kb plasmid which is essential for the intramacrophage growth of the organism and for virulence in foals and murine in vivo model systems. We found that the entire virulence plasmid could be transferred from plasmid-containing strains of R. equi (donor) to plasmid-free R. equi strains (recipient) at a high frequency and that plasmid transmission reestablished the capacity for intracellular growth in macrophages. Plasmid transfer required living cells and cell-to-cell contact and was unaffected by the presence of DNase, factors pointing to conjugation as the major means of genetic transfer. Deletion of a putative relaxase-encoding gene, traA, located in the proposed conjugative region of the plasmid, abolished plasmid transfer. Reversion of the traA mutation restored plasmid transmissibility. Finally, plasmid transmission to other Rhodococcus species and some additional related organisms was demonstrated. This is the first study showing a virulence plasmid transfer in R. equi, and it establishes a mechanism by which the virulence plasmid can move among bacteria in the soil. PMID:23042997

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

    PubMed

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

    2012-12-01

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

  2. In Rhizobium etli symbiotic plasmid transfer, nodulation competitivity and cellular growth require interaction among different replicons.

    PubMed

    Brom, S; García-de los Santos, A; Cervantes, L; Palacios, R; Romero, D

    2000-07-01

    Bacteria belonging to the genus Rhizobium are able to develop two different lifestyles, in symbiotic association with plant roots or through saprophytic growth. The genome of Rhizobium strains is constituted by a chromosome and several large plasmids, one of them containing most of the genes involved in symbiosis (symbiotic plasmid or pSym). Our model strain Rhizobium etli CFN42 contains six plasmids. We have constructed multiple plasmid-cured derivatives of this strain and used them to analyze the contribution of these plasmids to free-living cellular viability, competitivity for nodulation, plasmid transfer, and utilization of diverse carbon sources. Our results show that the transfer of the pSym is strictly dependent on the presence of another plasmid; consequently under conditions where pSym transfer is required, nodulation relies on the presence of a plasmid devoid of nodulation genes. We also found a drastic decrease in competitivity for nodulation in multiple plasmid-cured derivatives when compared with single plasmid-cured strains. Cellular growth and viability were greatly diminished in some multiple plasmid-cured strains. The utilization of a number of carbon sources depends on the presence of specific plasmids. The results presented in this work indicate that functional interactions among sequences scattered in the different plasmids are required for successful completion of both lifestyles. Copyright 2000 Academic Press.

  3. R-plasmid transfer in a wastewater treatment plant.

    PubMed Central

    Mach, P A; Grimes, D J

    1982-01-01

    Enteric bacteria have been examined for their ability to transfer antibiotic resistance in a wastewater treatment plant. Resistant Salmonella enteritidis, Proteus mirabilis, and Escherichia coli were isolated from clinical specimens and primary sewage effluent. Resistance to ampicillin, chloramphenicol, streptomycin, sulfadiazine, and tetracycline was demonstrated by spread plate and tube dilution techniques. Plasmid mediation of resistance was shown by ethidium bromide curing, agarose gel electrophoresis, and direct cell transfer. Each donor was mated with susceptible E. coli and Shigella sonnei. Mating pairs (and recipient controls) were suspended in unchlorinated primary effluent that had been filtered and autoclaved. Suspensions were added to membrane diffusion chambers which were then placed in the primary and secondary setting tanks of the wastewater treatment plant. Resistant recombinants were detected by replica plating nutrient agar master plates onto xylose lysine desoxycholate agar plates that contained per milliliter of medium 10 micrograms of ampicillin, 30 micrograms of chloramphenicol, 10 micrograms of streptomycin, 100 micrograms of sulfadiazine, or 30 micrograms of tetracycline. Mean transfer frequencies for laboratory matings were 2.1 X 10(-3). In situ matings for primary and secondary settling resulted in frequencies of 4.9 X 10(-5) and 7.5 X 10(-5), respectively. These values suggest that a significant level of resistance transfer occurs in wastewater treatment plants in the absence of antibiotics as selective agents. Images PMID:6760813

  4. Factors That Affect Transfer of the IncI1 β-Lactam Resistance Plasmid pESBL-283 between E. coli Strains

    PubMed Central

    Händel, Nadine; Otte, Sarah; Jonker, Martijs; Brul, Stanley; ter Kuile, Benno H.

    2015-01-01

    The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL) plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC) resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates and shows how these

  5. Scalable Plasmid Transfer using Engineered P1-based Phagemids

    PubMed Central

    Kittleson, Joshua T.; DeLoache, Will; Cheng, Hsiao-Ying; Anderson, J. Christopher

    2013-01-01

    Dramatic improvements to computational, robotic, and biological tools have enabled genetic engineers to conduct increasingly sophisticated experiments. Further development of biological tools offers a route to bypass complex or expensive mechanical operations, thereby reducing the time and cost of highly parallelized experiments. Here, we engineer a system based on bacteriophage P1 to transfer DNA from one E. coli cell to another, bypassing the need for intermediate DNA isolation (e.g., minipreps). To initiate plasmid transfer, we refactored a native phage element into a DNA module capable of heterologously inducing phage lysis. After incorporating known cis-acting elements, we identified a novel cis-acting element that further improves transduction efficiency, exemplifying the ability of synthetic systems to offer insight into native ones. The system transfers DNAs up to 25 kilobases, the maximum assayed size, and operates well at microliter volumes, enabling manipulation of most routinely used DNAs. The system’s large DNA capacity and physical coupling of phage particles to phagemid DNA suggest applicability to biosynthetic pathway evolution, functional proteomics, and ultimately, diverse molecular biology operations including DNA fabrication. PMID:23656280

  6. Serum resistance encoded by colicin V plasmids in Escherichia coli and its relationship to the plasmid transfer system.

    PubMed Central

    Nilius, A M; Savage, D C

    1984-01-01

    Eight colicin V plasmids were conjugated into a plasmidless Escherichia coli (K-12) strain that was susceptible to the bactericidal effects of normal rabbit serum. The resulting colicin V-positive strains were examined for their capacity to resist the lethal effects of serum. Serum resistance was assessed as growth of the bacterial strain in medium containing 5% normal rabbit serum inoculated from a culture in the early exponential phase. Only three of the eight colicin V plasmids were found to confer the serum resistance phenotype on the host strain. Derepression of the transfer system was associated with serum resistance in two of the plasmids. Two other derepressed plasmids did not confer serum resistance on the host bacterium. Therefore, such derepression alone was insufficient to produce serum resistance. The factor(s) encoded by colicin V plasmids and responsible for the serum resistance of the bacterial strains bearing the plasmids was shown to be a property associated with the cell and not an extracellular factor excreted into the growth medium. PMID:6365788

  7. Power transfer device for four wheel drive

    SciTech Connect

    Katayama, N.

    1987-02-17

    This patent describes a power transfer device for four-wheel drive in combination with a power transmission having a transmission casing secured at one side thereof to a cylinder block of an internal combustion engine, an input shaft rotatably mounted within the transmission casing and arranged coaxially with a crank-shaft of the engine, and an output shaft rotatably mounted within the transmission casing in parallel with the input shaft. A change-speed gearing is mounted on the input and output shafts, and an output gear is integral with the output shaft. The power transfer device comprises: a transfer casing detachably secured at one side thereof to the transmission casing; a first inner casing positioned within the transmission casing and rotatably supported by a pair of axially spaced bearings which are supported by the transmission casing; a second inner casing positioned within the transfer casing coaxially with the first inner casing and rotatably supported by a pair of axially spaced bearings which are supported by the transfer casing; an output gearing assembled within the transfer casing; a first ring gear mounted on the first inner casing for rotation therewith and in mesh with the output gear on the output shaft; and a second ring gear mounted on the second inner casing for rotation therewith and drivingly connected to the output gearing.

  8. Transfer of Plasmids to an Antibiotic-Sensitive Mutant of Zymomonas mobilis†

    PubMed Central

    Buchholz, Steven E.; Eveleigh, Douglas E.

    1986-01-01

    Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis. Images PMID:16347136

  9. Transfer of plasmids to an antibiotic-sensitive mutant of Zymomonas mobilis

    SciTech Connect

    Buchholz, S.E.; Eveleigh, D.E.

    1986-08-01

    Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis.

  10. Interspecific Transfer of Streptomyces Giant Linear Plasmids in Sterile Amended Soil Microcosms†

    PubMed Central

    Ravel, Jacques; Wellington, Elizabeth M. H.; Hill, Russell T.

    2000-01-01

    The interspecific transfer of two giant linear plasmids was investigated in sterile soil microcosms. Plasmids pRJ3L (322 kb) and pRJ28 (330 kb), both encoding mercury resistance, were successfully transferred in amended soil microcosms from their streptomycete hosts, the isolates CHR3 and CHR28, respectively, to a plasmidless and mercury-sensitive strain, Streptomyces lividans TK24. Transconjugants of S. lividans TK24 were first observed after 2 to 3 days of incubation at 30°C, which corresponded to the time taken for the formation of mycelia in soil. Transfer frequencies were 4.8 × 10−4 and 3.6 × 10−5 CFU/donor genome for pRJ3L and pRJ28, respectively. Transconjugants were analyzed by pulsed-field gel electrophoresis for the presence of plasmids, and plasmid identity was confirmed by restriction digests. Total genomic DNA digests confirmed that transconjugants were S. lividans TK24. The mercury resistance genes were shown to be on the plasmid in the transconjugants by hybridization analysis and were still functional. This is the first demonstration of transfer of giant linear plasmids in sterile soil microcosms. Giant linear plasmids were detected in many Streptomyces spp. isolated from mercury-contaminated sediments from Boston Harbor (United States), Townsville Harbor (Australia), and the Sali River (Tucuman, Argentina). Mercury resistance genes were shown to be present on some of these plasmids. Our findings that giant linear plasmids can be transferred between Streptomyces spp. and are common in environmental Streptomyces isolates suggest that these plasmids are important in gene transfer between streptomycetes in the environment. PMID:10653714

  11. Potential of predominant activated sludge bacteria as recipients in conjugative plasmid transfer.

    PubMed

    Inoue, Daisuke; Sei, Kazunari; Soda, Satoshi; Ike, Michihiko; Fujita, Masanori

    2005-12-01

    We investigated the possibility of conjugative plasmid transfer to the predominant bacteria in activated sludge and the factors influencing the transfer frequency in the activated sludge process. We performed conjugative transfers of a self-transmissible, broad-host-range plasmid RP4 from Escherichia coli C600 to activated sludge bacteria by broth mating. Most of the activated sludge bacteria tested could acquire plasmid RP4, although the transfer frequencies varied from 8.8 x 10(-7) to 1.3 x 10(-2) transconjugants per recipient. The transfer frequencies in several strains were similar to, or higher than, that in intraspecific transfer to E. coli HB101. Matings under various environmental conditions showed that factors relevant to physiological activity, such as temperature and nutrient conditions, seemed to affect the transfer frequency. In addition, conjugative transfer was detected even in filtered raw and treated wastewaters. Thus, the predominant activated sludge bacteria seem to have sufficient potential as recipients in conjugative plasmid transfer under the conditions likely to occur in the activated sludge process. Transfer frequency was reduced by agitation in the presence of suspended solid. This may suggest that conjugative plasmid transfer is physically inhibited in aeration tanks.

  12. Experimental and Mathematical Models of Escherichia coli Plasmid Transfer In Vitro and In Vivo

    PubMed Central

    Freter, Rolf; Freter, Rolf R.; Brickner, Howard

    1983-01-01

    Little is known about the factors that govern plasmid transfers in natural ecosystems such as the gut. The consistent finding by earlier workers that plasmid transfer in the normal gut can be detected only at very low rates, if at all, has given rise to numerous speculations concerning the presence in vivo of various inhibitors of plasmid transfer. Plasmids R1, R1drd-19, and pBR322 were studied in Escherichia coli K-12 and wild-type E. coli hosts in two experimental systems: (i) gnotobiotic mice carrying a synthetic indigenous microflora (F-strains) which resemble in their function the normal indigenous microflora of the mouse large intestine, and (ii) anaerobic continuous-flow cultures of indigenous large intestinal microflora of the mouse, which can simulate bacterial interactions observed in the mouse gut. Mathematical models were developed to estimate plasmid transfer rates as a measure of the “fertility,” i.e., of the intrinsic ability to transfer the plasmid under the environmental conditions of the gut. The models also evaluate the effects of plasmid segregation, reduction of the growth rates of plasmid-bearing bacterial hosts, repression of transfer functions, competition for nutrients, and bacterial attachment to the wall of the gut or culture vessel. Some confidence in the validity of these mathematical models was gained because they were able to reproduce a number of known phenomena such as the repression of fertility of the R1 plasmid, as well as known differences in the transmission and mobilization of the plasmids studied. Interpretation of the data obtained permitted a number of conclusions, some of which were rather unexpected. (i) Fertility of plasmid-bearing E. coli in the normal intestine was not impaired. The observed low rates of plasmid transfer in the normal gut can be explained on quantitative grounds alone and do not require hypothetical inhibitory mechanisms. (ii) Conditions for long-term spread and maintenance throughout human or

  13. Genomics of microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy

    PubMed Central

    Shintani, Masaki; Sanchez, Zoe K.; Kimbara, Kazuhide

    2015-01-01

    Plasmids are important “vehicles” for the communication of genetic information between bacteria. The exchange of plasmids transmits pathogenically and environmentally relevant traits to the host bacteria, promoting their rapid evolution and adaptation to various environments. Over the past six decades, a large number of plasmids have been identified and isolated from different microbes. With the revolution of sequencing technology, more than 4600 complete sequences of plasmids found in bacteria, archaea, and eukaryotes have been determined. The classification of a wide variety of plasmids is not only important to understand their features, host ranges, and microbial evolution but is also necessary to effectively use them as genetic tools for microbial engineering. This review summarizes the current situation of the classification of fully sequenced plasmids based on their host taxonomy and their features of replication and conjugative transfer. The majority of the fully sequenced plasmids are found in bacteria in the Proteobacteria, Firmicutes, Spirochaetes, Actinobacteria, Cyanobacteria and Euryarcheota phyla, and key features of each phylum are included. Recent advances in the identification of novel types of plasmids and plasmid transfer by culture-independent methods using samples from natural environments are also discussed. PMID:25873913

  14. Molecular Mechanisms That Contribute to Horizontal Transfer of Plasmids by the Bacteriophage SPP1.

    PubMed

    Valero-Rello, Ana; López-Sanz, María; Quevedo-Olmos, Alvaro; Sorokin, Alexei; Ayora, Silvia

    2017-01-01

    Natural transformation and viral-mediated transduction are the main avenues of horizontal gene transfer in Firmicutes. Bacillus subtilis SPP1 is a generalized transducing bacteriophage. Using this lytic phage as a model, we have analyzed how viral replication and recombination systems contribute to the transfer of plasmid-borne antibiotic resistances. Phage SPP1 DNA replication relies on essential phage-encoded replisome organizer (G38P), helicase loader (G39P), hexameric replicative helicase (G40P), recombinase (G35P) and in less extent on the partially dispensable 5'→3' exonuclease (G34.1P), the single-stranded DNA binding protein (G36P) and the Holliday junction resolvase (G44P). Correspondingly, the accumulation of linear concatemeric plasmid DNA, and the formation of transducing particles were blocked in the absence of G35P, G38P, G39P, and G40P, greatly reduced in the G34.1P, G36P mutants, and slightly reduced in G44P mutants. In contrast, establishment of injected linear plasmid DNA in the recipient host was independent of viral-encoded functions. DNA homology between SPP1 and the plasmid, rather than a viral packaging signal, enhanced the accumulation of packagable plasmid DNA. The transfer efficiency was also dependent on plasmid copy number, and rolling-circle plasmids were encapsidated at higher frequencies than theta-type replicating plasmids.

  15. Transfer case for multiple drive axle vehicle

    SciTech Connect

    Batchelor, R.B.

    1987-07-07

    This patent describes a transfer case for a vehicle drive train having a transmission comprising: a housing; input means for receiving the torque output from the vehicle transmission; a first output shaft and first means for rotatably supporting the first output shaft in the housing; a second output shaft and second means for rotatably supporting the second output shaft in the housing; and means for selectively driving the first and second output shafts in response to actuation of the input means. The means for selectively driving comprises a planetary gear set having an annulus, a first set of first planet gears a second set of second planet gears, engaged with one of the first planet gear. A sun gear engages with the second planet gears, third means for rotatably mounting the sun gear in the housing and a planet carrier having means for rotatably supporting the first and second planet gears and co-axially secured to the first output shaft for rotation. A clutch means for selectively engages the sun gear with the planet carrier and the second output shaft, and the input means comprises means for rotatably driving the annulus.

  16. Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil.

    PubMed

    Neilson, J W; Josephson, K L; Pepper, I L; Arnold, R B; Di Giovanni, G D; Sinclair, N A

    1994-11-01

    Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.

  17. Conjugative transferability of the A/C plasmids from Salmonella enterica isolates that possess or lack blaCMY in the A/C plasmid backbone

    USDA-ARS?s Scientific Manuscript database

    The objective of this study was to gain a better understanding of the conjugative transfer of antimicrobial resistance plasmids from 205 Salmonella enterica strains, isolated from cattle to E. coli or Salmonella recipients. PCR-based replicon typing (PBRT) was used to type incompatibility plasmid r...

  18. Plasmid coding for transferable drug resistance in bacteria isolated from cultured rainbow trout.

    PubMed Central

    Toranzo, A E; Combarro, P; Lemos, M L; Barja, J L

    1984-01-01

    The occurrence of drug resistance and plasmid-mediated transferability was investigated in 170 strains belonging to eight bacterial groups isolated from cultured rainbow trout. It was found that 87.6% of the strains were resistant to at least one drug, with the highest percentages of resistance being detected for ampicillin (54.7%), sulfadiazine (46.5%), nitrofurantoin (38.2%), and chloramphenicol (37.0%). Six enterobacteria, two Vibrio, and one Aeromonas isolate transferred resistance factors to Escherichia coli K-12. The most common transmissible R factor determined resistance to chloramphenicol and sulfadiazine, demonstrating an association between a specific plasmid and the resistance pattern transferred. The presence of chloramphenicol in fish food was detected by bioassay. In general, transfer frequencies were similar in primary and secondary matings, which indicate the potential water-borne dissemination of these R plasmids. Images PMID:6508296

  19. [Effect of exogenic proteolytic enzymes on transfer of plasmid genes in mixed bacterial biofilms].

    PubMed

    Tets, G V; Artemenko, N K; Zaslavskaia, N V; Artemenko, K L; Knorring, G Iu; Tets, V V; Sternin, Iu I

    2009-01-01

    The effect of the enzyme complex from Vobenzin on transfer of plasmid genes in biofilms of gramnegative bacteria was studied. The extracellular matrix of the bacterial biofilms was shown to contain extracellular DNA carrying the antibiotic resistance markers. The action of the enzyme complex resulted in lower frequency of the antibiotic resistance genes transfer in mixed bacterial biofilms.

  20. Plasmid Transfer between Bacillus thuringiensis subsp. israelensis Strains in Laboratory Culture, River Water, and Dipteran Larvae

    PubMed Central

    Thomas, D. John I.; Morgan, J. Alun W.; Whipps, John M.; Saunders, Jon R.

    2001-01-01

    Plasmid transfer between strains of Bacillus thuringiensis subsp. israelensis was studied under a range of environmentally relevant laboratory conditions in vitro, in river water, and in mosquito larvae. Mobilization of pBC16 was detected in vitro at a range of temperatures, pH values, and available water conditions, and the maximum transfer ratio was 10−3 transconjugant per recipient under optimal conditions. Transfer of conjugative plasmid pXO16∷Tn5401 was also detected under this range of conditions. However, a maximum transfer ratio of 1.0 transconjugant per recipient was attained, and every recipient became a transconjugant. In river water, transfer of pBC16 was not detected, probably as a result of the low transfer frequency for this plasmid and the formation of spores by the introduced donor and recipient strains. In contrast, transfer of plasmid pXO16∷Tn5401 was detected in water, but at a lower transfer ratio (ca. 10−2 transconjugant per donor). The number of transconjugants increased over the first 7 days, probably as a result of new transfer events between cells, since growth of both donor and recipient cells in water was not detected. Mobilization of pBC16 was not detected in killed mosquito larvae, but transfer of plasmid pXO16::Tn5401 was evident, with a maximum rate of 10−3 transconjugant per donor. The reduced transfer rate in insects compared to broth cultures may be accounted for by competition from the background bacterial population present in the mosquito gut and diet or by the maintenance of a large population of B. thuringiensis spores in the insects. PMID:11133463

  1. Biofilm models for the food industry: hot spots for plasmid transfer?

    PubMed

    Van Meervenne, Eva; De Weirdt, Rosemarie; Van Coillie, Els; Devlieghere, Frank; Herman, Lieve; Boon, Nico

    2014-04-01

    Biofilms represent a substantial problem in the food industry, with food spoilage, equipment failure, and public health aspects to consider. Besides, biofilms may be a hot spot for plasmid transfer, by which antibiotic resistance can be disseminated to potential foodborne pathogens. This study investigated biomass and plasmid transfer in dual-species (Pseudomonas putida and Escherichia coli) biofilm models relevant to the food industry. Two different configurations (flow-through and drip-flow) and two different inoculation procedures (donor-recipient and recipient-donor) were tested. The drip-flow configuration integrated stainless steel coupons in the setup while the flow-through configuration included a glass flow cell and silicone tubing. The highest biomass density [10 log (cells cm-²)] was obtained in the silicone tubing when first the recipient strain was inoculated. High plasmid transfer ratios, up to 1/10 (transconjugants/total bacteria), were found. Depending on the order of inoculation, a difference in transfer efficiency between the biofilm models could be found. The ease by which the multiresistance plasmid was transferred highlights the importance of biofilms in the food industry as hot spots for the acquisition of multiresistance plasmids. This can impede the treatment of foodborne illnesses if pathogens acquire this multiresistance in or from the biofilm.

  2. 13. DETAIL OF BEVEL GEAR TRANSFERRING HORIZONTAL DRIVE FROM MAIN ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. DETAIL OF BEVEL GEAR TRANSFERRING HORIZONTAL DRIVE FROM MAIN WATERWHEEL SHAFT TO VERTICAL SHAFT DRIVING COFFEE HUSKING MILL ON SECOND FLOOR - Hacienda Cafetalera Santa Clara, Coffee Mill, KM 19, PR Route 372, Hacienda La Juanita, Yauco Municipio, PR

  3. Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins

    PubMed Central

    Gruber, Christian J.; Lang, Silvia; Rajendra, Vinod K. H.; Nuk, Monika; Raffl, Sandra; Schildbach, Joel F.; Zechner, Ellen L.

    2016-01-01

    Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination. PMID:27486582

  4. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  5. A New and Improved Host-Independent Plasmid System for RK2-Based Conjugal Transfer

    PubMed Central

    Strand, Trine Aakvik; Lale, Rahmi; Degnes, Kristin Fløgstad; Lando, Malin; Valla, Svein

    2014-01-01

    Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT) are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system. PMID:24595202

  6. A new and improved host-independent plasmid system for RK2-based conjugal transfer.

    PubMed

    Strand, Trine Aakvik; Lale, Rahmi; Degnes, Kristin Fløgstad; Lando, Malin; Valla, Svein

    2014-01-01

    Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT) are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.

  7. Transfer of plasmid-mediated resistance to tetracycline in pathogenic bacteria from fish and aquaculture environments.

    PubMed

    Guglielmetti, Elena; Korhonen, Jenni M; Heikkinen, Jouni; Morelli, Lorenzo; von Wright, Atte

    2009-04-01

    The transferability of a large plasmid that harbors a tetracycline resistance gene tet(S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients (Lactococcus garvieae and Listeria monocytogenes), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L. garvieae. A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes, even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.

  8. A genetic study of a Staphylococus aureus plasmid involving cure and transference.

    PubMed

    Darini, A L

    1996-01-01

    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 (55)Tet 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.

  9. Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer.

    PubMed

    Kwong, S M; Yeo, C C; Suwanto, A; Poh, C L

    2000-01-01

    The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

  10. Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens.

    PubMed

    Pankhurst, C E; Broughton, W J; Wieneke, U

    1983-08-01

    Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.

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

    PubMed

    Jussila, Minna M; Zhao, Ji; Suominen, Leena; Lindström, Kristina

    2007-03-01

    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.

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

    PubMed Central

    Van Horn, Christopher R.

    2017-01-01

    ABSTRACT 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

  13. Conjugative plasmid transfer in Xylella fastidiosa is dependent on tra and trb operon functions.

    PubMed

    Burbank, Lindsey P; Van Horn, Christopher R

    2017-08-14

    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 strains M23 (subspecies fastidiosa) or Dixon (subspecies multiplex) as the donor strain and Temecula (subspecies 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.IMPORTANCEXylella fastidiosa is an important plant pathogen world-wide, infecting a wide range of different plant species. 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 and adaptation, and disease emergence in this diverse pathogen. This is a work

  14. 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

    PubMed Central

    Madsen, Jonas Stenløkke; Riber, Leise; Kot, Witold; Basfeld, Alrun; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    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 to have originated. We find that mrkABCDFs of plasmids are highly expressed via a unique promoter that differs from the original Klebsiella promoter resulting in fundamental behavioral consequences. Plasmid associated mrkABCDFs did not influence the swimming behavior of the host, that hereby acquired an exceptional phenotype being able to both actively swim (planktonic behavior) and express biofilm associated fimbriae (sessile behavior). We show that this exceptional phenotype enhances the conjugal transfer of the plasmid. PMID:27627107

  15. Construction of disarmed Ti plasmids transferable between Escherichia coli and Agrobacterium species.

    PubMed

    Kiyokawa, Kazuya; Yamamoto, Shinji; Sakuma, Kei; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2009-04-01

    Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1lambdapir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

  16. TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens.

    PubMed

    Wisniewski, Jessica A; Traore, Daouda A; Bannam, Trudi L; Lyras, Dena; Whisstock, James C; Rood, Julian I

    2016-03-01

    Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.

  17. Effect of growth rate and selection pressure on rates of transfer of an antibiotic resistance plasmid between E. coli strains.

    PubMed

    Schuurmans, Jasper M; van Hijum, Sacha A F T; Piet, Jurgen R; Händel, Nadine; Smelt, Jan; Brul, Stanley; ter Kuile, Benno H

    2014-03-01

    Antibiotic resistance increases costs for health care and causes therapy failure. An important mechanism for spreading resistance is transfer of plasmids containing resistance genes and subsequent selection. Yet the factors that influence the rate of transfer are poorly known. Rates of plasmid transfer were measured in co-cultures in chemostats of a donor and a acceptor strain under various selective pressures. To document whether specific mutations in either plasmid or acceptor genome are associated with the plasmid transfer, whole genome sequencing was performed. The DM0133 TetR tetracycline resistance plasmid was transferred between Escherichia coli K-12 strains during co-culture at frequencies that seemed higher at increased growth rate. Modeling of the take-over of the culture by the transformed strain suggests that in reality more transfer events occurred at low growth rates. At moderate selection pressure due to an antibiotic concentration that still allowed growth, a maximum transfer frequency was determined of once per 10(11) cell divisions. In the absence of tetracycline or in the presence of high concentrations the frequency of transfer was sometimes zero, but otherwise reduced by at least a factor of 5. Whole genome sequencing showed that the plasmid was transferred without mutations, but two functional mutations in the genome of the recipient strain accompanied this transfer. Exposure to concentrations of antibiotics that fall within the mutant selection window stimulated transfer of the resistance plasmid most. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Horizontal gene transfer and antibiotic resistance plasmids in multi-drug resistant Salmonella enterica serovars

    USDA-ARS?s Scientific Manuscript database

    Antibiotic resistant foodborne pathogens pose serious public health concerns and increase the burden of disease treatment. Antibiotic resistance genes can reside on the bacterial chromosome or on other self-replicating DNA molecules such as plasmids. The resistance genes/DNA can be transferred int...

  19. Mobilizable Rolling-Circle Replicating Plasmids from Gram-Positive Bacteria: A Low-Cost Conjugative Transfer.

    PubMed

    Fernández-López, Cris; Bravo, Alicia; Ruiz-Cruz, Sofía; Solano-Collado, Virtu; Garsin, Danielle A; Lorenzo-Díaz, Fabián; Espinosa, Manuel

    2014-10-01

    Conjugation is a key mechanism for horizontal gene transfer in bacteria. Some plasmids are not self-transmissible but can be mobilized by functions encoded in trans provided by other auxiliary conjugative elements. Although the transfer efficiency of mobilizable plasmids is usually lower than that of conjugative elements, mobilizable plasmids are more frequently found in nature. In this sense, replication and mobilization can be considered important mechanisms influencing plasmid promiscuity. Here we review the currently available information on two families of small mobilizable plasmids from Gram-positive bacteria that replicate via the rolling-circle mechanism. One of these families, represented by the streptococcal plasmid pMV158, is an interesting model since it contains a specific mobilization module (MOBV) that is widely distributed among mobilizable plasmids. We discuss a mechanism in which the promiscuity of the pMV158 replicon is based on the presence of two origins of lagging strand synthesis. The current strategies to assess plasmid transfer efficiency as well as to inhibit conjugative plasmid transfer are presented. Some applications of these plasmids as biotechnological tools are also reviewed.

  20. Plasmid-mediated transfer of the bla(NDM-1) gene in Gram-negative rods.

    PubMed

    Potron, Anaïs; Poirel, Laurent; Nordmann, Patrice

    2011-11-01

    The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase NDM-1 (New Delhi metallo-β-lactamase) producers, mostly in Enterobacteriacae. Five bla(NDM) (-1) -positive plasmids of different incompatibility groups (IncL/M, FII, A/C and two untypeable plasmids) from clinical Enterobacteriaceae were evaluated for conjugation properties and host specificity. Successful conjugative transfers were obtained using all tested enterobacterial species as recipients (Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Proteus mirabilis) and all plasmid types. Conjugation frequencies varied from 1 × 10(-4) to 6 × 10(-8) transconjugants per donor. Higher conjugation rates were obtained for two plasmids at 30 °C compared with that observed at 25 and 37 °C. Carbapenems used as selector did not lead to higher conjugation frequencies. None of the five plasmids was transferable to Acinetobacter baumannii or Pseudomonas aeruginosa by conjugation. This work underlines how efficient the spread of the carbapenemase bla(NDM) (-1) gene could be among Enterobacteriaceae.

  1. Conjugative transfer of staphylococcal antibiotic resistance markers in the absence of detectable plasmid DNA.

    PubMed Central

    el Solh, N; Allignet, J; Bismuth, R; Buret, B; Fouace, J M

    1986-01-01

    Eleven Staphylococcus aureus clinical isolates were tested for transfer of resistance markers by transduction and filter mating. The resistance markers of six of the strains could be transferred only by transduction; however, the five remaining strains transferred their resistance both by transduction and filter mating. The resistance markers that were cotransferred in filter matings (transfer of resistance to penicillin and streptogramin A was accompanied, in each case, by the transfer of one or more markers, i.e., resistance to aminoglycosides, cadmium, or tetracycline, depending on the donor) were not cotransduced. The filter mating transfers were recA independent and were observed with both Staphylococcus aureus and Staphylococcus epidermidis recipients. Experiments to elucidate the mechanism of transfer by filter mating suggested that conjugation requiring cell-to-cell contact may have been involved. These transfers occurred in the absence of detectable plasmid DNA. PMID:2944478

  2. Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome

    PubMed Central

    Reeves, Analise Z; Lesser, Cammie F

    2017-01-01

    Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives. PMID:28203614

  3. Strain-Specific Transfer of Antibiotic Resistance from an Environmental Plasmid to Foodborne Pathogens

    PubMed Central

    Van Meervenne, Eva; Van Coillie, Els; Kerckhof, Frederiek-Maarten; Devlieghere, Frank; Herman, Lieve; De Gelder, Leen S. P.; Top, Eva M.; Boon, Nico

    2012-01-01

    Pathogens resistant to multiple antibiotics are rapidly emerging, entailing important consequences for human health. This study investigated if the broad-host-range multiresistance plasmid pB10, isolated from a wastewater treatment plant, harbouring amoxicillin, streptomycin, sulfonamide, and tetracycline resistance genes, was transferable to the foodborne pathogens Salmonella spp. or E. coli O157:H7 and how this transfer alters the phenotype of the recipients. The transfer ratio was determined by both plating and flow cytometry. Antibiotic resistance profiles were determined for both recipients and transconjugants using the disk diffusion method. For 14 of the 15 recipient strains, transconjugants were detected. Based on plating, transfer ratios were between 6.8 × 10−9 and 3.0 × 10−2 while using flow cytometry, transfer ratios were between <1.0 × 10−5 and 1.9 × 10−2. With a few exceptions, the transconjugants showed phenotypically increased resistance, indicating that most of the transferred resistance genes were expressed. In summary, we showed that an environmental plasmid can be transferred into foodborne pathogenic bacteria at high transfer ratios. However, the transfer ratio seemed to be recipient strain dependent. Moreover, the newly acquired resistance genes could turn antibiotic susceptible strains into resistant ones, paving the way to compromise human health. PMID:22791963

  4. Increased transfer of a multidrug resistance plasmid in Escherichia coli biofilms at the air-liquid interface.

    PubMed

    Król, Jaroslaw E; Nguyen, Hung Duc; Rogers, Linda M; Beyenal, Haluk; Krone, Stephen M; Top, Eva M

    2011-08-01

    Although biofilms represent a common bacterial lifestyle in clinically and environmentally important habitats, there is scant information on the extent of gene transfer in these spatially structured populations. The objective of this study was to gain insight into factors that affect transfer of the promiscuous multidrug resistance plasmid pB10 in Escherichia coli biofilms. Biofilms were grown in different experimental settings, and plasmid transfer was monitored using laser scanning confocal microscopy and plate counting. In closed flow cells, plasmid transfer in surface-attached submerged biofilms was negligible. In contrast, a high plasmid transfer efficiency was observed in a biofilm floating at the air-liquid interface in an open flow cell with low flow rates. A vertical flow cell and a batch culture biofilm reactor were then used to detect plasmid transfer at different depths away from the air-liquid interface. Extensive plasmid transfer occurred only in a narrow zone near that interface. The much lower transfer frequencies in the lower zones coincided with rapidly decreasing oxygen concentrations. However, when an E. coli csrA mutant was used as the recipient, a thick biofilm was obtained at all depths, and plasmid transfer occurred at similar frequencies throughout. These results and data from separate aerobic and anaerobic matings suggest that oxygen can affect IncP-1 plasmid transfer efficiency, not only directly but also indirectly, through influencing population densities and therefore colocalization of donors and recipients. In conclusion, the air-liquid interface can be a hot spot for plasmid-mediated gene transfer due to high densities of juxtaposed donor and recipient cells.

  5. Kinetics of plasmid transfer among Bacillus cereus group strains within lepidopteran larvae.

    PubMed

    Yuan, Y M; Hu, X M; Liu, H Z; Hansen, Bjarne Munk; Yan, J P; Yuan, Z M

    2007-06-01

    The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44-56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 x 10(-6) CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.

  6. Minimal and Contributing Sequence Determinants of the cis-Acting Locus of Transfer (clt) of Streptomycete Plasmid pIJ101 Occur within an Intrinsically Curved Plasmid Region

    PubMed Central

    Ducote, Matthew J.; Prakash, Shubha; Pettis, Gregg S.

    2000-01-01

    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. PMID:11073933

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

    PubMed

    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.

  8. Mobilizable Rolling-Circle Replicating Plasmids from Gram-Positive Bacteria: A Low-Cost Conjugative Transfer

    PubMed Central

    Fernández-López, Cris; Bravo, Alicia; Ruiz-Cruz, Sofía; Solano-Collado, Virtu; Garsin, Danielle A.; Lorenzo-Díaz, Fabián; Espinosa, Manuel

    2014-01-01

    Chapter summary Conjugation is a key mechanism for horizontal gene transfer in bacteria. Some plasmids are not self-transmissible but can be mobilized by functions encoded in trans provided by other auxiliary conjugative elements. Although the transfer efficiency of mobilizable plasmids is usually lower than that of conjugative elements, mobilizable plasmidsare more frequently found in nature. In this sense, replication and mobilization can be considered as important mechanisms influencing plasmid promiscuity. Here we review the present available information on two families of small mobilizable plasmids from Gram-positive bacteria that replicate via the rolling-circle mechanism. One of these families, represented by the streptococcal plasmid pMV158, is an interesting model since it contains a specific mobilization module (MOBV) that is widely distributed among mobilizable plasmids. We discuss a mechanism in which the promiscuity of the pMV158 replicon is based on the presence of two origins of lagging strand synthesis. The current strategies to assess plasmid transfer efficiency as well as to inhibit conjugative plasmid transfer are presented. Some applications of these plasmids as biotechnological tools are also reviewed. PMID:25606350

  9. Root growth and exudate production define the frequency of horizontal plasmid transfer in the Rhizosphere.

    PubMed

    Mølbak, Lars; Molin, Søren; Kroer, Niels

    2007-01-01

    To identify the main drivers of plasmid transfer in the rhizosphere, conjugal transfer was studied in the rhizospheres of pea and barley. The donor Pseudomonas putida KT2442, containing plasmid pKJK5::gfp, was coated onto the seeds, while the recipient P. putida LM24, having a chromosomal insertion of dsRed, was inoculated into the growth medium. Mean transconjugant-to-donor ratios in vermiculite were 4.0+/-0.8 x 10(-2) in the pea and 5.9+/-1.4 x 10(-3) in the barley rhizospheres. In soil, transfer ratios were about 10 times lower. As a result of a 2-times higher root exudation rate in pea, donor densities in pea (1 x 10(6)-2 x 10(9) CFU g(-1) root) were about 10 times higher than in barley. No difference in recipient densities was observed. In situ visualization of single cells on the rhizoplane and macroscopic visualization of the colonization pattern showed that donors and transconjugants were ubiquitously distributed in the pea rhizosphere, while they were only located on the upper parts of the barley roots. Because the barley root elongated about 10 times faster than the pea root, donors were probably outgrown by the elongating barley root. Thus by affecting the cell density and distribution, exudation and root growth appear to be key parameters controlling plasmid transfer in the rhizosphere.

  10. The mechanism and control of DNA transfer by the conjugative relaxase of resistance plasmid pCU1

    SciTech Connect

    Nash, Rebekah Potts; Habibi, Sohrab; Cheng, Yuan; Lujan, Scott A.; Redinbo, Matthew

    2010-11-15

    Bacteria expand their genetic diversity, spread antibiotic resistance genes, and obtain virulence factors through the highly coordinated process of conjugative plasmid transfer (CPT). A plasmid-encoded relaxase enzyme initiates and terminates CPT by nicking and religating the transferred plasmid in a sequence-specific manner. We solved the 2.3 {angstrom} crystal structure of the relaxase responsible for the spread of the resistance plasmid pCU1 and determined its DNA binding and nicking capabilities. The overall fold of the pCU1 relaxase is similar to that of the F plasmid and plasmid R388 relaxases. However, in the pCU1 structure, the conserved tyrosine residues (Y18,19,26,27) that are required for DNA nicking and religation were displaced up to 14 {angstrom} out of the relaxase active site, revealing a high degree of mobility in this region of the enzyme. In spite of this flexibility, the tyrosines still cleaved the nic site of the plasmid's origin of transfer, and did so in a sequence-specific, metal-dependent manner. Unexpectedly, the pCU1 relaxase lacked the sequence-specific DNA binding previously reported for the homologous F and R388 relaxase enzymes, despite its high sequence and structural similarity with both proteins. In summary, our work outlines novel structural and functional aspects of the relaxase-mediated conjugative transfer of plasmid pCU1.

  11. Conjugal Transfer of Broad-Host-Range Plasmid pAMβ1 into Enteric Species of Lactic Acid Bacteria

    PubMed Central

    Vescovo, M.; Morelli, L.; Bottazzi, V.; Gasson, M. J.

    1983-01-01

    The broad-host-range plasmid pAMβ1, which codes for erythromycin and lincomycin resistance, was transferred by conjugation into Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius. A novel 17-megadalton plasmid molecule was detected in the transconjugants, confirming the introduction of pAMβ1 into each species. Images PMID:16346389

  12. Origin-of-transfer sequences facilitate mobilisation of non-conjugative antimicrobial-resistance plasmids in Staphylococcus aureus

    PubMed Central

    O'Brien, Frances G.; Yui Eto, Karina; Murphy, Riley J. T.; Fairhurst, Heather M.; Coombs, Geoffrey W.; Grubb, Warren B.; Ramsay, Joshua P.

    2015-01-01

    Staphylococcus aureus is a common cause of hospital, community and livestock-associated infections and is increasingly resistant to multiple antimicrobials. A significant proportion of antimicrobial-resistance genes are plasmid-borne, but only a minority of S. aureus plasmids encode proteins required for conjugative transfer or Mob relaxase proteins required for mobilisation. The pWBG749 family of S. aureus conjugative plasmids can facilitate the horizontal transfer of diverse antimicrobial-resistance plasmids that lack Mob genes. Here we reveal that these mobilisable plasmids carry copies of the pWBG749 origin-of-transfer (oriT) sequence and that these oriT sequences facilitate mobilisation by pWBG749. Sequences resembling the pWBG749 oriT were identified on half of all sequenced S. aureus plasmids, including the most prevalent large antimicrobial-resistance/virulence-gene plasmids, pIB485, pMW2 and pUSA300HOUMR. oriT sequences formed five subfamilies with distinct inverted-repeat-2 (IR2) sequences. pWBG749-family plasmids encoding each IR2 were identified and pWBG749 mobilisation was found to be specific for plasmids carrying matching IR2 sequences. Specificity of mobilisation was conferred by a putative ribbon-helix-helix-protein gene smpO. Several plasmids carried 2–3 oriT variants and pWBG749-mediated recombination occurred between distinct oriT sites during mobilisation. These observations suggest this relaxase-in trans mechanism of mobilisation by pWBG749-family plasmids is a common mechanism of plasmid dissemination in S. aureus. PMID:26243776

  13. Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

    PubMed

    Wiesner, Magdalena; Fernández-Mora, Marcos; Cevallos, Miguel A; Zavala-Alvarado, Crispín; Zaidi, Mussaret B; Calva, Edmundo; Silva, Claudia

    2013-11-21

    Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the "genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid. We showed that the transfer of the YU39 blaCMY-2 gene

  14. A safe packaging line for gene transfer: separating viral genes on two different plasmids.

    PubMed Central

    Markowitz, D; Goff, S; Bank, A

    1988-01-01

    A retrovirus packaging cell line was constructed by using portions of the Moloney murine leukemia virus in which the gag, pol, and env genes of the helper virus were separated onto two different plasmids and in which the psi packaging signal and 3' long terminal repeat were removed. The plasmid containing the gag and pol genes and the plasmid containing the env gene were cotransfected into NIH 3T3 cells. Clones that produced high levels of reverse transcriptase and env protein were tested for their ability to package the replication-defective retrovirus vectors delta neo and N2. One of the gag-pol and env clones (GP+E-86) was able to transfer G418 resistance to recipient cells at a titer of as high as 1.7 X 10(5) when it was used to package delta neo and as high as 4 X 10(6) when it was used to package N2. Supernatants of clones transfected with the intact parent gag-pol-env plasmid 3P0 had comparable titers (as high as 6.5 X 10(4) with delta neo; as high as 1.7 X 10(5) with N2). Tests for recombination events that might result in intact retrovirus showed no evidence for the generation of replication-competent virus. These results suggest that gag, pol, and env, when present on different plasmids, may provide an efficient and safe packaging line for use in retroviral gene transfer. Images PMID:2831375

  15. Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance

    SciTech Connect

    Bender, C.L.; Cooksey, D.A.

    1986-02-01

    Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO/sub 4/): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS 61 were inhibited by 0.1 mM CuSO/sub 4/, whereas the CuSO/sub 4/ MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT 17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb).

  16. MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation.

    PubMed

    Puttini, Stefania; van Zwieten, Ruthger W; Saugy, Damien; Lekka, Małgorzata; Hogger, Florence; Ley, Deborah; Kulik, Andrzej J; Mermod, Nicolas

    2013-12-02

    The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression. This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.

  17. MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation

    PubMed Central

    2013-01-01

    Background The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. Results In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression. Conclusions This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo. PMID:24295286

  18. Superinfection drives virulence evolution in experimental populations of bacteria and plasmids

    PubMed Central

    smith, jeff

    2010-01-01

    A prominent hypothesis proposes that pathogen virulence evolves in large part due to a trade-off between infectiousness and damage to hosts. Other explanations emphasize how virulence evolves in response to competition among pathogens within hosts. Given the proliferation of theoretical possibilities, what best predicts how virulence evolves in real biological systems? Here I show that virulence evolution in experimental populations of bacteria and self-transmissible plasmids is best explained by within-host competition. Plasmids evolved to severely reduce the fitness of their hosts even in the absence of uninfected cells. This result is inconsistent with the trade-off hypothesis, which predicts that under these conditions vertically transmitted pathogens would evolve to be less virulent. Plasmid virulence was strongly correlated with the ability to superinfect cells containing competing plasmid genotypes, suggesting a key role for within-host competition. When virulent genotypes became common, hosts evolved resistance to plasmid infection. These results show that the trade-off hypothesis can incorrectly predict virulence evolution when within-host interactions are neglected. They also show that symbioses between bacteria and plasmids can evolve to be surprisingly antagonistic. PMID:21054359

  19. Superinfection drives virulence evolution in experimental populations of bacteria and plasmids.

    PubMed

    Smith, Jeff

    2011-03-01

    A prominent hypothesis proposes that pathogen virulence evolves in large part due to a trade-off between infectiousness and damage to hosts. Other explanations emphasize how virulence evolves in response to competition among pathogens within hosts. Given the proliferation of theoretical possibilities, what best predicts how virulence evolves in real biological systems? Here, I show that virulence evolution in experimental populations of bacteria and self-transmissible plasmids is best explained by within-host competition. Plasmids evolved to severely reduce the fitness of their hosts even in the absence of uninfected cells. This result is inconsistent with the trade-off hypothesis, which predicts that under these conditions vertically transmitted pathogens would evolve to be less virulent. Plasmid virulence was strongly correlated with the ability to superinfect cells containing competing plasmid genotypes, suggesting a key role for within-host competition. When virulent genotypes became common, hosts evolved resistance to plasmid infection. These results show that the trade-off hypothesis can incorrectly predict virulence evolution when within-host interactions are neglected. They also show that symbioses between bacteria and plasmids can evolve to be surprisingly antagonistic.

  20. Reduction in horizontal transfer of conjugative plasmid by UV irradiation and low-level chlorination.

    PubMed

    Lin, Wenfang; Li, Shuai; Zhang, Shuting; Yu, Xin

    2016-03-15

    The widespread presence of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) in the drinking water system facilitates their horizontal gene transfer among microbiota. In this study, the conjugative gene transfer of RP4 plasmid after disinfection including ultraviolet (UV) irradiation and low-level chlorine treatment was investigated. It was found that both UV irradiation and low-level chlorine treatment reduced the conjugative gene transfer frequency. The transfer frequency gradually decreased from 2.75 × 10(-3) to 2.44 × 10(-5) after exposure to UV doses ranging from 5 to 20 mJ/cm(2). With higher UV dose of 50 and 100 mJ/cm(2), the transfer frequency was reduced to 1.77 × 10(-6) and 2.44 × 10(-8). The RP4 plasmid transfer frequency was not significantly affected by chlorine treatment at dosages ranging from 0.05 to 0.2 mg/l, but treatment with 0.3-0.5 mg/l chlorine induced a decrease in conjugative transfer to 4.40 × 10(-5) or below the detection limit. The mechanisms underlying these phenomena were also explored, and the results demonstrated that UV irradiation and chlorine treatment (0.3 and 0.5 mg/l) significantly reduced the viability of bacteria, thereby lowering the conjugative transfer frequency. Although the lower chlorine concentrations tested (0.05-0.2 mg/l) were not sufficient to damage the cells, exposure to these concentrations may still depress the expression of a flagellar gene (FlgC), an outer membrane porin gene (ompF), and a DNA transport-related gene (TraG). Additionally, fewer pili were scattered on the bacteria after chlorine treatment. These findings are important in assessing and controlling the risk of ARG transfer and dissemination in the drinking water system.

  1. Power transfer device for four-wheel drive

    SciTech Connect

    Sasaki, K.; Takimura, K.; Katayama, N.

    1987-10-06

    A power transfer device is described for four-wheel drive in combination with a power transmission having a transmission casing, an input shaft rotatably mounted, an output shaft rotatably mounted, a change-speed gearing mounted on the input and output shafts, and an output gear mounted on the output shaft for rotation therewith. The power transfer device comprises: a transfer casing detachably secured at one side to the transmission casing; a first differential gear unit including an input element in the form of a first gear casing rotatably mounted within the transmission casing and drivingly connected to the output gear, and an air of first output elements rotatably mounted within the first gear casing respectively for front-wheel drive and rear-wheel drive; a second differential gear unit arranged coaxially with the first differential gear unit and including a second input member in the form of a second gear casing; a carrier member rotatably mounted within the transfer casing and arranged coaxially with the first and second differential gear units; a first wheel axle drivingly connected at an inner end; a second wheel axle drivingly connected at an inner end with the other second output element of the second differential gear unit and extending outwardly from the transfer casing through the carrier member; a hollow shaft arranged with the second wheel axle; and a clutch sleeve; an output gearing assembled within the transfer casing and arranged to be driven by the carrier member; and means for axially slidably coupling an end of the clutch sleeve with one end of the carrier member in such a manner that the clutch sleeve is positioned relative to the carrier member in a predetermined radial and circumferential direction.

  2. Power transfer device for four-wheel drive

    SciTech Connect

    Katayama, N.; Sasaki, K.; Koide, T.; Takimura, K.

    1989-02-07

    A power transfer device is described for four-wheel drive in combination with a power transmission, comprising: a first differential of the bevel gear type arranged to split the two driving torque from the power transmission into two torque delivery paths respectively for front-wheel drive and rear-wheel drive; a second differential of the bevel gear type arranged coaxially with the first differential at one side thereof to transfer the split driving torque from a first side gear of the first differential to one of front and rear-wheel axles; an output gear arranged coaxially with the first differential at the other side thereof to transfer the split driving torque from a second side gear of the first differential to the other of the rear and front-wheel axles; and a limited-slip differential assembled within a mounting case for the output gear to effect limited slip torque proportional to the difference in rotational speed between the side gears.

  3. Power transfer device for four-wheel drive vehicle

    SciTech Connect

    Hayashi, H.

    1986-10-21

    A power transfer device is described for a four-wheel drive vehicle having a transmission drivingly connected to a prime mover of the vehicle and having a set of front road wheels and a set of rear road wheels adapted to be driven from the transmission. The transfer device comprises: a first differential of the bevel gear type including a differential case arranged to be applied with drive torque from the transmission, a pinion gear rotatably mounted within the differential case, and a pair or side gears rotatably mounted within the differential case and in mesh with the pinion gear; a first output shaft connected to one of the side gears for driving the front or rear road wheels; a second output shaft connected to the other side gear for driving the rear or front road wheels; a second differential of the planetary gear type including a differential carrier formed to contain the differential case therein; and a selector mechanism arranged to selectively provide a drive connection between the second output shaft and the rear or front road wheels or a drive connection between the differential carrier and the rear or front road wheels.

  4. Demonstrating plasmid-based horizontal gene transfer in complex environmental matrices: a practical approach for a critical review.

    PubMed

    Bellanger, Xavier; Guilloteau, Hélène; Bonot, Sébastien; Merlin, Christophe

    2014-09-15

    Plasmid-based dissemination of antibiotic resistance genes in environmental microbial communities is a matter of concern for public health, but it remains difficult to study for methodological reasons. In this study, we used the broad host range plasmid pB10 to compare and to point out the main drawbacks of the three different approaches currently used to evaluate plasmid transfer in natural communities. Culture-based selection of transconjugants appeared to be compromised by high prevalence of antibiotic resistances among natural communities, unless high loads of initial pB10-donor inocula were used. Fluorescence-based detection of transconjugants reached a dead-end consequently to the narrow host range of bacteria expressing fluorescent proteins from a genetically modified pB10 plasmid, in addition to the relatively high background level of fluorescence exhibited by some environmental matrices. The molecular-based approach was the only one to provide a mean to detect rare plasmid transfer events following a low but realistic initial pB10-donor inoculation. Whatever the method, culture-based or molecular-based, the detection of successful transfer events in a given environmental matrix seemed to be linked to the initial stability of the donor inoculum. Depending on the matrix considered, eukaryotic predation plays a significant role in either limiting or promoting the plasmid transfer events.

  5. Plasmid Transfer between Spatially Separated Donor and Recipient Bacteria in Earthworm-Containing Soil Microcosms

    PubMed Central

    Daane, L. L.; Molina, J.; Sadowsky, M. J.

    1997-01-01

    Most gene transfer studies have been performed with relatively homogeneous soil systems in the absence of soil macrobiota, including invertebrates. In this study we examined the influence of earthworm activity (burrowing, casting, and feeding) on transfer of plasmid pJP4 between spatially separated donor (Alcaligenes eutrophus) and recipient (Pseudomonas fluorescens) bacteria in nonsterile soil columns. A model system was designed such that the activity of earthworms would act to mediate cell contact and gene transfer. Three different earthworm species (Aporrectodea trapezoides, Lumbricus rubellus, and Lumbricus terrestris), representing each of the major ecological categories (endogeic, epigeic, and anecic), were evaluated. Inoculated soil microcosms, with and without added earthworms, were analyzed for donor, recipient, and transconjugant bacteria at 5-cm-depth intervals by using selective plating techniques. Transconjugants were confirmed by colony hybridization with a mer gene probe. The presence of earthworms significantly increased dispersal of the donor and recipient strains. In situ gene transfer of plasmid pJP4 from A. eutrophus to P. fluorescens was detected only in earthworm-containing microcosms, at a frequency of (symbl)10(sup2) transconjugants per g of soil. The depth of recovery was dependent on the burrowing behavior of each earthworm species; however, there was no significant difference in the total number of transconjugants among the earthworm species. Donor and recipient bacteria were recovered from earthworm feces (casts) of all three earthworm species, with numbers up to 10(sup6) and 10(sup4) bacteria per g of cast, respectively. A. trapezoides egg capsules (cocoons) formed in the inoculated soil microcosms contained up to 10(sup7) donor and 10(sup6) recipient bacteria per g of cocoon. No transconjugant bacteria, however, were recovered from these microhabitats. To our knowledge, this is the first report of gene transfer between physically

  6. A comparison of the kinetics of plasmid transfer in the conjugation systems encoded by the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis.

    PubMed

    Andrup, L; Andersen, K

    1999-08-01

    Quantitative measurements of horizontal DNA transfer are critical if one wishes to address questions relating to ecology, evolution and the safe use of recombinant bacteria. Traditionally, the efficiency of a conjugation system has been described by its transfer frequency. However, transfer frequencies can be determined in many ways and may be sensitive to physical, chemical and biological conditions. In this study the authors have used the mechanistic similarity between bacterial conjugation and simple enzyme catalysis in order to calculate the maximal conjugation rate (Vmax) and the recipient concentration (K(m)) at which the conjugation rate is half its maximal value, for two different conjugation systems: the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis. The results are compared with the data obtained from the aggregation-mediated conjugation system encoded on pXO16 from Bacillus thuringiensis. The conjugation systems analysed are fundamentally different; however, they have some characteristics in common: they are able to sustain conjugative transfer in liquid medium and the transfer efficiencies are very high. Conjugation encoded by the F plasmid in E. coli involves the formation of small aggregates (2-20 cells), established by sex pili, and the plasmid's maximal conjugation rate was estimated to be approximately 0.15 transconjugants per donor per minute. Pheromone-induced conjugation in Ent. faecalis, which involves the formation of large aggregates, was found to proceed at a maximal conjugation rate of 0.29 transconjugants per donor per minute. Also, the K(m) value differed significantly between these conjugation systems; this may reflect the inherent differences in mating pair formation and transfer mechanisms. In these conjugation systems, the donors underwent a 'recovery period' between rounds of conjugative transfer and newly formed transconjugants required a period of about 40-80 min to mature into proficient donors.

  7. 1. TRANSFER CONVEYOR AND DRIVE MECHANISM ON SOUTH SIDE OF ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. TRANSFER CONVEYOR AND DRIVE MECHANISM ON SOUTH SIDE OF LEVEL 4; LOOKING SOUTH; CONVEYORS WERE USED TO MOVE CARCASSES BETWEEN FLOORS FOR FURTHER PROCESSING - Rath Packing Company, Cooler Building-1929 Beef House, Sycamore Street between Elm & Eighteenth Streets, Waterloo, Black Hawk County, IA

  8. Measuring the Rate of Conjugal Plasmid Transfer and Phage Infection in a Bacterial Population Using Quantitative PCR

    NASA Astrophysics Data System (ADS)

    Wan, Zhenmao; Goddard, Noel

    2012-02-01

    Horizontal gene transfer between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor(F^+) to a recipient(F^-) cell through cell-to-cell contact. We demonstrate a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. It also allows us to investigate the inhibition of conjugation in the presence of filamentous bacteriophages M13. Experimental data is then compared with numerical simulation using a mass action, resource limited model.

  9. Interference of Lactobacillus plantarum strains in the in vitro conjugative transfer of R-plasmids.

    PubMed

    Sabia, Carla; de Niederhäusern, Simona; Guerrieri, Elisa; Bondi, Moreno; Anacarso, Immacolata; Iseppi, Ramona; Messi, Patrizia

    2009-02-01

    Probiotic compounds, which are often constituted of lactobacilli, exert a number of health benefits through maintenance of the intestinal ecosystem balance. Among the important interactions that occur in the gut microbiota, plasmid transfer by mating is an increasing cause of concern, particularly when antibiotic-resistant genes are involved. Because lactobacilli seem to be able to influence this mechanism, the aim of the present work was to investigate the in vitro capability of two Lactobacillus plantarum strains (one bacteriocin producer and one nonproducer) to interfere with the conjugation processes. For this purpose different matings were performed adding to the donor and recipient cells L. plantarum 35d bac+ and L. plantarum 396/1 bac- as agents of interference. Conjugations added with a Staphylococcus aureus strain or without any agent of interference were used as controls. The results of our experiments demonstrated that both lactobacillus strains were able to decrease mating frequency. Statistically significant differences in the viable transconjugants were obtained in the presence and in the absence of the lactobacilli. The effect was almost the same with the two L. plantarum independent of bacteriocin production. In the trial performed with S. aureus, no decrease in mating frequency was observed, confirming that the capability to interfere with R-plasmid transfer ability could be a property of the tested L. plantarum strains.

  10. Transfer of IncP plasmids to extremely acidophilic Thiobacillus thiooxidans

    SciTech Connect

    Jin, S.M.; Yan, W.M.; Wang, Z.N. )

    1992-01-01

    Thiobacillus thiooxidans is an acidophilic, obligately autotrophic bacterium which derives its energy by oxidizing reduced or partially reduced sulfur compounds and obtains its carbon by fixing carbon dioxide from the atmosphere. The strain is able to live in inorganic, acidic environments and is present in large numbers in coal mine drainage and in mineral ores. T. thiooxidans has been used industrially in metal leaching from mineral ores and in the microbial desulfurization of coal in combination with Thiobacillus ferrooxidans. Although T. thiooxidans has been well studied physiologically, very little is known about it genetics. The broad-host-range IncP plasmids RP4, R68.45, RP1::Tn501, and pUB307 were transferred directly to extremely acidophilic Thiobacillus thiooxidans from Escherichia coli by conjugation at frequencies of 10{sup {minus}5} to 10{sup {minus}7} per recipient. The ability of T. thiooxidans to receive and express the antibiotic resistance markers was examined. The plasmid RP4 was transferred back to E. coli from T. thiooxidans at a frequency of 1.0 {times} 10{sup {minus}3} per recipient.

  11. Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

    PubMed Central

    Reid, Robert J.D.; González-Barrera, Sergio; Sunjevaric, Ivana; Alvaro, David; Ciccone, Samantha; Wagner, Marisa; Rothstein, Rodney

    2011-01-01

    We have streamlined the process of transferring plasmids into any yeast strain library by developing a novel mating-based, high-throughput method called selective ploidy ablation (SPA). SPA uses a universal plasmid donor strain that contains conditional centromeres on every chromosome. The plasmid-bearing donor is mated to a recipient, followed by removal of all donor-strain chromosomes, producing a haploid strain containing the transferred plasmid. As proof of principle, we used SPA to transfer plasmids containing wild-type and mutant alleles of DNA topoisomerase I (TOP1) into the haploid yeast gene-disruption library. Overexpression of Top1 identified only one sensitive mutation, rpa34, while overexpression of top1-T722A allele, a camptothecin mimetic, identified 190 sensitive gene-disruption strains along with rpa34. In addition to known camptothecin-sensitive strains, this set contained mutations in genes involved in the Rpd3 histone deacetylase complex, the kinetochore, and vesicle trafficking. We further show that mutations in several ESCRT vesicle trafficking components increase Top1 levels, which is dependent on SUMO modification. These findings demonstrate the utility of the SPA technique to introduce plasmids into the haploid gene-disruption library to discover new interacting pathways. PMID:21173034

  12. Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid

    PubMed Central

    2013-01-01

    Background Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. Results YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the “genetic load” operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid. Conclusions We showed that the

  13. Transferable antibiotic resistance plasmids from biogas plant digestates often belong to the IncP-1ε subgroup.

    PubMed

    Wolters, Birgit; Kyselková, Martina; Krögerrecklenfort, Ellen; Kreuzig, Robert; Smalla, Kornelia

    2014-01-01

    Manure is known to contain residues of antibiotics administered to farm animals as well as bacteria carrying antibiotic resistance genes (ARGs). These genes are often located on mobile genetic elements. In biogas plants (BGPs), organic substrates such as manure and plant material are mixed and fermented in order to provide energy, and resulting digestates are used for soil fertilization. The fate of plasmid carrying bacteria from manure during the fermentation process is unknown. The present study focused on transferable antibiotic resistance plasmids from digestates of seven BGPs, using manure as a co-substrate, and their phenotypic and genotypic characterization. Plasmids conferring resistance to either tetracycline or sulfadiazine were captured by means of exogenous plasmid isolation from digestates into Pseudomonas putida KT2442 and Escherichia coli CV601 recipients, at transfer frequencies ranging from 10(-5) to 10(-7). Transconjugants (n = 101) were screened by PCR-Southern blot hybridization and real-time PCR for the presence of IncP-1, IncP-1ε, IncW, IncN, IncP-7, IncP-9, LowGC, and IncQ plasmids. While 61 plasmids remained unassigned, 40 plasmids belonged to the IncP-1ε subgroup. All these IncP-1ε plasmids were shown to harbor the genes tet(A), sul1, qacEΔ1, intI1, and integron gene cassette amplicons of different size. Further analysis of 16 representative IncP-1ε plasmids showed that they conferred six different multiple antibiotic resistance patterns and their diversity seemed to be driven by the gene cassette arrays. IncP-1ε plasmids displaying similar restriction and antibiotic resistance patterns were captured from different BGPs, suggesting that they may be typical of this environment. Our study showed that BGP digestates are a potential source of transferable antibiotic resistance plasmids, and in particular the broad host range IncP-1ε plasmids might contribute to the spread of ARGs when digestates are used as fertilizer.

  14. Transferable antibiotic resistance plasmids from biogas plant digestates often belong to the IncP-1ε subgroup

    PubMed Central

    Wolters, Birgit; Kyselková, Martina; Krögerrecklenfort, Ellen; Kreuzig, Robert; Smalla, Kornelia

    2015-01-01

    Manure is known to contain residues of antibiotics administered to farm animals as well as bacteria carrying antibiotic resistance genes (ARGs). These genes are often located on mobile genetic elements. In biogas plants (BGPs), organic substrates such as manure and plant material are mixed and fermented in order to provide energy, and resulting digestates are used for soil fertilization. The fate of plasmid carrying bacteria from manure during the fermentation process is unknown. The present study focused on transferable antibiotic resistance plasmids from digestates of seven BGPs, using manure as a co-substrate, and their phenotypic and genotypic characterization. Plasmids conferring resistance to either tetracycline or sulfadiazine were captured by means of exogenous plasmid isolation from digestates into Pseudomonas putida KT2442 and Escherichia coli CV601 recipients, at transfer frequencies ranging from 10-5 to 10-7. Transconjugants (n = 101) were screened by PCR-Southern blot hybridization and real-time PCR for the presence of IncP-1, IncP-1ε, IncW, IncN, IncP-7, IncP-9, LowGC, and IncQ plasmids. While 61 plasmids remained unassigned, 40 plasmids belonged to the IncP-1ε subgroup. All these IncP-1ε plasmids were shown to harbor the genes tet(A), sul1, qacEΔ1, intI1, and integron gene cassette amplicons of different size. Further analysis of 16 representative IncP-1ε plasmids showed that they conferred six different multiple antibiotic resistance patterns and their diversity seemed to be driven by the gene cassette arrays. IncP-1ε plasmids displaying similar restriction and antibiotic resistance patterns were captured from different BGPs, suggesting that they may be typical of this environment. Our study showed that BGP digestates are a potential source of transferable antibiotic resistance plasmids, and in particular the broad host range IncP-1ε plasmids might contribute to the spread of ARGs when digestates are used as fertilizer. PMID:25653641

  15. Power transfer device for four-wheel drive vehicles

    SciTech Connect

    Ida, S.

    1989-02-21

    A power transfer device is described for four-wheel drive vehicles, comprising: a housing; an input shaft rotatable mounted within the housing for drive connection to an output shaft of a power transmission; a first output shaft rotatably mounted within the housing in a coaxial relationship with the input shaft; a second output shaft rotatable mounted within the housing in a concentric relationship with the input shaft; and a planetary gear unit including a sun gear, a carrier, a ring gear, and a plurality of planet gears journaled on the carrier and in mesh with the sun and ring gears and being axially slidably mounted on the input and output shafts.

  16. Transfer of mupirocin resistance from Staphylococcus haemolyticus clinical strains to Staphylococcus aureus through conjugative and mobilizable plasmids.

    PubMed

    Rossi, Ciro C; Ferreira, Natália C; Coelho, Marcus L V; Schuenck, Ricardo P; Bastos, Maria do Carmo de F; Giambiagi-deMarval, Marcia

    2016-07-01

    Coagulase-negative staphylococci are thought to act as reservoirs of antibiotic resistance genes that can be transferred to Staphylococcus aureus, thus hindering the combat of this bacterium. In this work, we analyzed the presence of plasmids conferring resistance to the antibiotic mupirocin-widely used to treat and prevent S. aureus infections in hospital environments-in nosocomial S. haemolyticus strains. About 12% of the 75 strains tested were resistant to mupirocin, and this phenotype was correlated with the presence of plasmids. These plasmids were shown to be diverse, being either conjugative or mobilizable, and capable of transferring mupirocin resistance to S. aureus Our findings reinforce that S. haemolyticus, historically and mistakenly considered as a less important pathogen, is a reservoir of resistance genes which can be transferred to other bacteria, such as S. aureus, emphasizing the necessity of more effective strategies to detect and combat this emergent opportunistic pathogen.

  17. Effects of nano-TiO2 on antibiotic resistance transfer mediated by RP4 plasmid.

    PubMed

    Qiu, Zhigang; Shen, Zhiqiang; Qian, Di; Jin, Min; Yang, Dong; Wang, Jingfeng; Zhang, Bin; Yang, Zhongwei; Chen, Zhaoli; Wang, Xinwei; Ding, Chengshi; Wang, Daning; Li, Jun-Wen

    2015-01-01

    The potential risks of nano-materials and the spread of antibiotic resistance genes (ARGs) have become two major global public concerns. Studies have confirmed that nano-alumina can promote the spread of ARGs mediated by plasmids. Nano-titanium dioxide (TiO(2)), an excellent photocatalytic nano-material, has been widely used and is often present in aqueous environments. At various nano-material concentrations, bacterial density, matting time, and matting temperature, nano-TiO(2) can significantly promote the conjugation of RP4 plasmid in Escherichia coli. We developed a mathematical model to quantitatively describe the conjugation process and used this model to evaluate the effects of nano-TiO(2) on the spread of ARGs. We obtained analytical solutions for total and resistant bacteria, which were enumerated by the abundance of genetic loci unique to the plasmid and the chromosome using qPCR. Our results showed that the mathematic model was able to fit the experimental data well and can be used to quantitatively evaluate the effects of nano-TiO(2). According to our model, the presence of nano-TiO(2) decreased the bacterial growth rate from 0.0360 to 0.0323 min(-1) and increased the conjugative transfer rate from 6.69 × 10(-12) to 3.93 × 10(-10 )mL cell(-1) min(-1). These results indicate that nano-TiO(2) inhibited bacterial growth and promoted conjugation simultaneously. The data for morphology and mRNA expression also demonstrated this phenomenon. Our results confirm that environmental nano-TiO(2) may cause the spread of ARGs and thus poses an environmental risk. In addition, we provide a potential method for monitoring changes in ARGs that result from conjugation and evaluating the effects of antimicrobial substances on ARG expression.

  18. Power transfer mechanism for four-wheel drive

    SciTech Connect

    Hiraiwa, K.

    1989-02-21

    A power transfer mechanism is described comprising: an input member; an first output member; a second output member; a planetary gear assembly including a ring gear, a carrier, a plurality of first planet gears in mesh with aid ring gear and journalled on the carrier, a plurality of second planet gears, each in mesh with one of the first planet gears, journalled on the carrier, and a sun gear in mesh with the second planet gears; the ring gear being drivingly connected to the input member; the carrier being drivingly connected to the first output member; and means for shifting the power transfer mechanism to a plurality of operating modes, aid shifting means including first clutch means for selectively engaging the first output member with the second output member and second clutch means for selectively engaging the second output member with the sun gear.

  19. The tra locus of streptomycete plasmid pIJ101 mediates efficient transfer of a circular but not a linear version of the same replicon.

    PubMed

    Wang, Jing; Pettis, Gregg S

    2010-09-01

    Conjugal transfer of circular plasmids in Streptomyces involves a unique mechanism employing few plasmid-encoded loci and the transfer of double-stranded DNA by an as yet uncharacterized intercellular route. Efficient transfer of the circular streptomycete plasmid pIJ101 requires only two plasmid loci: the pIJ101 tra gene, and as a cis-acting function known as clt. Here, we compared the ability of the pIJ101 transfer apparatus to promote conjugal transfer of circular versus linear versions of the same replicon. While the pIJ101 tra locus readily transferred the circular form of the replicon, the linear version was transferred orders of magnitude less efficiently and all plasmids isolated from the transconjugants were circular, regardless of their original configuration in the donor. Additionally, relatively rare circularization of linear plasmids was detectable in the donor cells, which is consistent with the notion that this event was a prerequisite for transfer by TraB(pIJ101). Linear versions of this same replicon did transfer efficiently, in that configuration, from strains containing the conjugative linear plasmid SLP2. Our data indicate that functions necessary and sufficient for transfer of circular DNA were insufficient for transfer of a related linear DNA molecule. The results here suggest that the conjugation mechanisms of linear versus circular DNA in Streptomyces spp. are inherently different and/or that efficient transfer of linear DNA requires additional components.

  20. Transfer of the virulence-associated protein a-bearing plasmid between field strains of virulent and avirulent Rhodococcus equi.

    PubMed

    Stoughton, W; Poole, T; Kuskie, K; Liu, M; Bishop, K; Morrissey, A; Takai, S; Cohen, N

    2013-01-01

    Virulent and avirulent isolates of Rhodococcus equi coexist in equine feces and the environment and are a source of infection for foals. The extent to which plasmid transfer occurs among field strains is ill-defined and this information is important for understanding the epidemiology of R. equi infections of foals. To estimate the frequency of transfer of the virulence plasmid between virulent and avirulent strains of R. equi derived from foals and their environment. None. In vitro study; 5 rifampin-susceptible, virulent R. equi isolates obtained from clinically affected foals or air samples from a farm with a history of recurrent R. equi foal pneumonia were each mixed with 5 rifampin-resistant, avirulent isolates derived from soil samples, using solid medium, at a ratio of 10 donor cells (virulent) per recipient cell. Presumed transconjugates were detected by plating on media with rifampin and colony immunoblotting to detect the presence of the virulence-associated protein A. Three presumed transconjugates were detected among 2,037 recipient colonies, indicating an overall estimated transfer frequency of 0.15% (95% CI, 0.03–0.43%). All 3 transconjugates were associated with a single donor and 2 recipient strains. Genotyping and multiplex PCR of presumed transconjugates demonstrated transfer of the virulence-associated protein A-bearing plasmid between virulent and avirulent R. equi. Transfer of the virulence plasmid occurs with relatively high frequency. These findings could impact strategies to control or prevent R. equi through environmental management.

  1. Gene electro transfer of plasmid encoding vascular endothelial growth factor for enhanced expression and perfusion in the ischemic swine heart.

    PubMed

    Hargrave, Barbara; Strange, Robert; Navare, Sagar; Stratton, Michael; Burcus, Nina; Murray, Len; Lundberg, Cathryn; Bulysheva, Anna; Li, Fanying; Heller, Richard

    2014-01-01

    Myocardial ischemia can damage heart muscle and reduce the heart's pumping efficiency. This study used an ischemic swine heart model to investigate the potential for gene electro transfer of a plasmid encoding vascular endothelial growth factor for improving perfusion and, thus, for reducing cardiomyopathy following acute coronary syndrome. Plasmid expression was significantly greater in gene electro transfer treated tissue compared to injection of plasmid encoding vascular endothelial growth factor alone. Higher gene expression was also seen in ischemic versus non-ischemic groups with parameters 20 Volts (p<0.03), 40 Volts (p<0.05), and 90 Volts (p<0.05), but not with 60 Volts (p<0.09) while maintaining a pulse width of 20 milliseconds. The group with gene electro transfer of plasmid encoding vascular endothelial growth factor had increased perfusion in the area at risk compared to control groups. Troponin and creatine kinase increased across all groups, suggesting equivalent ischemia in all groups prior to treatment. Echocardiography was used to assess ejection fraction, cardiac output, stroke volume, left ventricular end diastolic volume, and left ventricular end systolic volume. No statistically significant differences in these parameters were detected during a 2-week time period. However, directional trends of these variables were interesting and offer valuable information about the feasibility of gene electro transfer of vascular endothelial growth factor in the ischemic heart. The results demonstrate that gene electro transfer can be applied safely and can increase perfusion in an ischemic area. Additional study is needed to evaluate potential efficacy.

  2. Genomic Sequence and Transcriptional Analysis of a 23-Kilobase Mycobacterial Linear Plasmid: Evidence for Horizontal Transfer and Identification of Plasmid Maintenance Systems

    PubMed Central

    Le Dantec, Corinne; Winter, Nathalie; Gicquel, Brigitte; Vincent, Véronique; Picardeau, Mathieu

    2001-01-01

    Linear plasmids were unknown in mycobacteria until recently. Here, we report the complete nucleotide sequence of 23-kb linear plasmid pCLP from Mycobacterium celatum, an opportunistic pathogen. The sequence of pCLP revealed at least 19 putative open reading frames (ORFs). Expression of pCLP genes in exponential-phase cultures was determined by reverse transcriptase PCR (RT-PCR). Twelve ORFs were expressed, whereas no transcription of the 7 other ORFs of pCLP was detected. Five of the 12 transcribed ORFs detected by RT-PCR are of unknown function. Sequence analysis revealed similar loci in both M. celatum pCLP and the Mycobacterium tuberculosis chromosome, including transposase-related sequences. This result suggests horizontal transfer between these two organisms. pCLP also contains ORFs that are similar to genes of bacterial circular plasmids involved in partition (par operon) and postsegregational (pem operon) mechanisms. Functional analysis of these ORFs suggests that they probably carry out similar maintenance roles in pCLP. PMID:11244052

  3. Identification and functional analysis of the transfer region of plasmid pMEA300 of the methylotrophic actinomycete Amycolatopsis methanolica.

    PubMed Central

    Vrijbloed, J W; van der Put, N M; Dijkhuizen, L

    1995-01-01

    Amycolatopsis methanolica contains a 13.3-kb plasmid (pMEA300) that is present either as an integrated element or as an autonomously replicating plasmid. Conjugational transfer of pMEA300 results in pock formation, zones of growth inhibition that become apparent when plasmid-carrying donor cells develop in a confluent lawn of plasmid-lacking recipient cells. A 6.2-kb pMEA300 DNA region specifying the functions of conjugation and pock formation was sequenced, revealing 10 open reading frames. This is the first sequence of the transfer region of a plasmid from a nonstreptomycete actinomycete. No clear similarities were found between the deduced sequences of the 10 putative Tra proteins of pMEA300 and those of Streptomyces plasmids. All Tra proteins of pMEA300 thus may represent unfamiliar types. A detailed mutational analysis showed that at least four individual proteins, TraG (9,488 Da), TraH (12,586 Da), TraI (40,468 Da), and TraJ (81,109 Da), are required for efficient transfer of pMEA300. Their disruption resulted in a clear reduction in the conjugational transfer frequencies, ranging from (5.2 x 10(1))-fold (TraG) to (2.3 x 10(6))-fold (TraJ), and in reduced pock sizes. At least two putative proteins, TraA (10,698 Da) and TraB (31,442 Da), were shown to be responsible for pock formation specifically. Specific binding of the pMEA300-encoded KorA protein to the traA-korA intragenic region was observed. PMID:7592426

  4. Transfer of the virulence-associated protein A-bearing plasmid between field strains of virulent and avirulent Rhodococcus equi

    USDA-ARS?s Scientific Manuscript database

    Virulent and avirulent isolates coexist in equine feces and the environment and serve as a source of infection for foals. The extent to which conjugative plasmid transfer occurs between these strains is unknown and is important for understanding the epidemiology of Rhodococcus equi infections of fo...

  5. Plasmid Biopharmaceuticals.

    PubMed

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  6. Expression Characteristics of the Transfer-Related kilB Gene Product of Streptomyces Plasmid pIJ101: Implications for the Plasmid Spread Function

    PubMed Central

    Pettis, Gregg S.; Ward, Naomi; Schully, Kevin L.

    2001-01-01

    Intermycelial transfer of Streptomyces plasmid pIJ101 occurs prior to cellular differentiation and is mediated by plasmid functions that are also required for production of zones of growth-inhibited recipient cells (i.e., pocks) that develop around individual donors during mating on agar medium. Several other pIJ101 functions, including that of the kilB gene, whose unregulated expression on pIJ101 is lethal, are required for normal pock size and so have been postulated to mediate intramycelial spread of the plasmid throughout recipient cells. Using antibodies raised against a KilB fusion protein expressed in Escherichia coli, native KilB protein was detected throughout development of pIJ101-containing Streptomyces lividans cells, with the concentration of KilB increasing dramatically and reaching a maximum during the final stages (i.e., sporulation and secondary metabolism) of cellular differentiation. Insertion of the kilB gene of pIJ101 into the S. lividans chromosome in cells lacking the pIJ101 KorB protein, which normally represses kilB gene transcription, resulted in elevated but still temporally increasing amounts of KilB. The increased expression or accumulation of the KilB spread protein throughout cellular differentiation of S. lividans, which leads to maximum KilB concentrations during developmental stages that occur far later than when intermycelial transfer of pIJ101 is mediated, supports the existence of a subsequent intramycelial component to the pIJ101 spread function. The results also suggest that intramycelial spread of pIJ101 molecules within the recipient extends beyond intercompartmental movements within the substrate mycelia and includes undetermined steps within the spore-yielding aerial hyphae as well. PMID:11157947

  7. Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

    PubMed Central

    Merryweather, A; Rees, C E; Smith, N M; Wilkins, B M

    1986-01-01

    The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell. Images Fig. 2. Fig. 3. Fig. 4. PMID:3024972

  8. Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation.

    PubMed

    Lee, Min-Jae; Cho, Soon-Shin; Jang, Hyung-Suk; Lim, Young Shin; You, Ji-Ran; Park, Jangwon; Suh, Hearan; Kim, Jeong-a; Park, Jong-Sang; Kim, Duk-Kyung

    2002-09-30

    In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.

  9. Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host

    PubMed Central

    Gaimster, Hannah; Summers, David

    2015-01-01

    Regulation by non-coding RNAs was found to be widespread among plasmids and other mobile elements of bacteria well before its ubiquity in the eukaryotic world was suspected. As an increasing number of examples was characterised, a common mechanism began to emerge. Non-coding RNAs, such as CopA and Sok from plasmid R1, or RNAI from ColE1, exerted regulation by refolding the secondary structures of their target RNAs or modifying their translation. One regulatory RNA that seemed to swim against the tide was Rcd, encoded within the multimer resolution site of ColE1. Required for high fidelity maintenance of the plasmid in recombination-proficient hosts, Rcd was found to have a protein target, elevating indole production by stimulating tryptophanase. Rcd production is up-regulated in dimer-containing cells and the consequent increase in indole is part of the response to the rapid accumulation of dimers by over-replication (known as the dimer catastrophe). It is proposed that indole simultaneously inhibits cell division and plasmid replication, stopping the catastrophe and allowing time for the resolution of dimers to monomers. The idea of a plasmid-mediated cell division checkpoint, proposed but then discarded in the 1980s, appears to be enjoying a revival. PMID:25446541

  10. Plasmids in the driving seat: The regulatory RNA Rcd gives plasmid ColE1 control over division and growth of its E. coli host.

    PubMed

    Gaimster, Hannah; Summers, David

    2015-03-01

    Regulation by non-coding RNAs was found to be widespread among plasmids and other mobile elements of bacteria well before its ubiquity in the eukaryotic world was suspected. As an increasing number of examples was characterised, a common mechanism began to emerge. Non-coding RNAs, such as CopA and Sok from plasmid R1, or RNAI from ColE1, exerted regulation by refolding the secondary structures of their target RNAs or modifying their translation. One regulatory RNA that seemed to swim against the tide was Rcd, encoded within the multimer resolution site of ColE1. Required for high fidelity maintenance of the plasmid in recombination-proficient hosts, Rcd was found to have a protein target, elevating indole production by stimulating tryptophanase. Rcd production is up-regulated in dimer-containing cells and the consequent increase in indole is part of the response to the rapid accumulation of dimers by over-replication (known as the dimer catastrophe). It is proposed that indole simultaneously inhibits cell division and plasmid replication, stopping the catastrophe and allowing time for the resolution of dimers to monomers. The idea of a plasmid-mediated cell division checkpoint, proposed but then discarded in the 1980s, appears to be enjoying a revival.

  11. Horizontal Gene Transfer of the Secretome Drives the Evolution of Bacterial Cooperation and Virulence

    PubMed Central

    Nogueira, Teresa; Rankin, Daniel J.; Touchon, Marie; Taddei, François; Brown, Sam P.; Rocha, Eduardo P.C.

    2009-01-01

    Summary Background Microbes engage in a remarkable array of cooperative behaviors, secreting shared proteins that are essential for foraging, shelter, microbial warfare, and virulence. These proteins are costly, rendering populations of cooperators vulnerable to exploitation by nonproducing cheaters arising by gene loss or migration. In such conditions, how can cooperation persist? Results Our model predicts that differential gene mobility drives intragenomic variation in investment in cooperative traits. More mobile loci generate stronger among-individual genetic correlations at these loci (higher relatedness) and thereby allow the maintenance of more cooperative traits via kin selection. By analyzing 21 Escherichia genomes, we confirm that genes coding for secreted proteins—the secretome—are very frequently lost and gained and are associated with mobile elements. We show that homologs of the secretome are overrepresented among human gut metagenomics samples, consistent with increased relatedness at secretome loci across multiple species. The biosynthetic cost of secreted proteins is shown to be under intense selective pressure, even more than for highly expressed proteins, consistent with a cost of cooperation driving social dilemmas. Finally, we demonstrate that mobile elements are in conflict with their chromosomal hosts over the chimeric ensemble's social strategy, with mobile elements enforcing cooperation on their otherwise selfish hosts via the cotransfer of secretome genes with “mafia strategy” addictive systems (toxin-antitoxin and restriction-modification). Conclusion Our analysis matches the predictions of our model suggesting that horizontal transfer promotes cooperation, as transmission increases local genetic relatedness at mobile loci and enforces cooperation on the resident genes. As a consequence, horizontal transfer promoted by agents such as plasmids, phages, or integrons drives microbial cooperation. PMID:19800234

  12. Horizontal gene transfer of the secretome drives the evolution of bacterial cooperation and virulence.

    PubMed

    Nogueira, Teresa; Rankin, Daniel J; Touchon, Marie; Taddei, François; Brown, Sam P; Rocha, Eduardo P C

    2009-11-03

    Microbes engage in a remarkable array of cooperative behaviors, secreting shared proteins that are essential for foraging, shelter, microbial warfare, and virulence. These proteins are costly, rendering populations of cooperators vulnerable to exploitation by nonproducing cheaters arising by gene loss or migration. In such conditions, how can cooperation persist? Our model predicts that differential gene mobility drives intragenomic variation in investment in cooperative traits. More mobile loci generate stronger among-individual genetic correlations at these loci (higher relatedness) and thereby allow the maintenance of more cooperative traits via kin selection. By analyzing 21 Escherichia genomes, we confirm that genes coding for secreted proteins-the secretome-are very frequently lost and gained and are associated with mobile elements. We show that homologs of the secretome are overrepresented among human gut metagenomics samples, consistent with increased relatedness at secretome loci across multiple species. The biosynthetic cost of secreted proteins is shown to be under intense selective pressure, even more than for highly expressed proteins, consistent with a cost of cooperation driving social dilemmas. Finally, we demonstrate that mobile elements are in conflict with their chromosomal hosts over the chimeric ensemble's social strategy, with mobile elements enforcing cooperation on their otherwise selfish hosts via the cotransfer of secretome genes with "mafia strategy" addictive systems (toxin-antitoxin and restriction-modification). Our analysis matches the predictions of our model suggesting that horizontal transfer promotes cooperation, as transmission increases local genetic relatedness at mobile loci and enforces cooperation on the resident genes. As a consequence, horizontal transfer promoted by agents such as plasmids, phages, or integrons drives microbial cooperation.

  13. Plasmid-mediated gene transfer in neurons using the biolistics technique.

    PubMed

    Biewenga, J E; Destrée, O H; Schrama, L H

    1997-01-01

    Biolistics has been developed as a system for gene delivery into plant cells, but has recently been introduced for transfection into mammalian tissue, including few attempts in neural cells. Basically, in this system the plasmid DNA of interest is coated onto small particles, that are accelerated by a particular driving force. The combination of several so-called 'ballistic' parameters and tissue parameters determine the transfection efficiency. The main advantage of the system is that it is, unlike other available transfection methods, a mechanical way to cross the plasma membrane and therefore less dependent on target cell characteristics. In terms of transfection efficiency, biolistics seems favorable above conventional techniques, like calcium phosphate precipitation and lipofection. Compared to viral techniques biolistics may be less efficient, but is quicker and easier to handle and seems to produce fewer complications for in vivo gene delivery. Therefore, although the technique is only in a developmental stage, preliminary results seem promising, and optimalization of the method may prove useful in scientific research and/or clinical use.

  14. Power transfer device for four-wheel drive vehicles

    SciTech Connect

    Ida, S.

    1988-10-04

    This patent describes a power transfer device for four-wheel drive vehicles, comprising: a housing an input shaft rotatably mounted within the housing for drive connection to an output shaft of a power transmission; a first output shaft rotatably mounted within the housing in a coaxial relationship with the input shaft; a second output shaft rotatably within the housing in a concentric relationship with the input shaft; and a planetary gear unit including a sun gear, a carrier, a ring gear, and planet gears journaled on the carrier and in mesh with the sun and ring gears and being axially slidably mounted on the input and output shafts, wherein the planetary gear unit is shiftable between first, second and third positions to establish a high speed drive mode in the first position wherein the carrier is engaged with the input shaft, and the sun and ring gears are each engaged with the output shafts to permit differential action between the output shafts. The planetary gear unit being arranged to establish a high speed drive mode in the second position wherein the carrier is engaged with the input shaft and the first output shaft, and the sun gear is engaged with the input shaft and the second output shaft to restrict the differential action between the output shafts and being further arranged to establish a low speed drive mode in the third position wherein the sun gear is engaged with the input shaft, the carrier is engaged with the output shafts, and the ring gear is locked by engagement with the housing to restrict the differential action between the output shafts.

  15. Novel R-plasmid conjugal transfer inhibitory and antibacterial activities of phenolic compounds from Mallotus philippensis (Lam.) Mull. Arg.

    PubMed

    Oyedemi, Blessing O M; Shinde, Vaibhav; Shinde, Kamlesh; Kakalou, Dionysia; Stapleton, Paul D; Gibbons, Simon

    2016-06-01

    Antimicrobial resistance severely limits the therapeutic options for many clinically important bacteria. In Gram-negative bacteria, multidrug resistance is commonly facilitated by plasmids that have the ability to accumulate and transfer refractory genes amongst bacterial populations. The aim of this study was to isolate and identify bioactive compounds from the medicinal plant Mallotus philippensis (Lam.) Mull. Arg. with both direct antibacterial properties and the capacity to inhibit plasmid conjugal transfer. A chloroform-soluble extract of M. philippensis was subjected to bioassay-guided fractionation using chromatographic and spectrometric techniques that led to the isolation of the known compounds rottlerin [5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-cinnamoyl-1,2-chromene] and the red compound (8-cinnamoyl-5,7-dihydroxy-2,2,6-trimethylchromene). Both compounds were characterised and elucidated using one-dimensional and two-dimensional nuclear magnetic resonance (NMR). Rottlerin and the red compound showed potent activities against a panel of clinically relevant Gram-positive bacteria, including meticillin-resistant Staphylococcus aureus (MRSA). No significant direct activities were observed against Gram-negative bacteria. However, both rottlerin and the red compound strongly inhibited conjugal transfer of the plasmids pKM101, TP114, pUB307 and R6K amongst Escherichia coli at a subinhibitory concentration of 100mg/L. Interestingly, despite the planar nature of the compounds, binding to plasmid DNA could not be demonstrated by a DNA electrophoretic mobility shift assay. These results show that rottlerin and the red compound are potential candidates for antibacterial drug lead development. Further studies are needed to elucidate the mode of inhibition of the conjugal transfer of plasmids.

  16. Identifying changes in EEG information transfer during drowsy driving by transfer entropy

    PubMed Central

    Huang, Chih-Sheng; Pal, Nikhil R.; Chuang, Chun-Hsiang; Lin, Chin-Teng

    2015-01-01

    Drowsy driving is a major cause of automobile accidents. Previous studies used neuroimaging based approaches such as analysis of electroencephalogram (EEG) activities to understand the brain dynamics of different cortical regions during drowsy driving. However, the coupling between brain regions responding to this vigilance change is still unclear. To have a comprehensive understanding of neural mechanisms underlying drowsy driving, in this study we use transfer entropy, a model-free measure of effective connectivity based on information theory. We investigate the pattern of information transfer between brain regions when the vigilance level, which is derived from the driving performance, changes from alertness to drowsiness. Results show that the couplings between pairs of frontal, central, and parietal areas increased at the intermediate level of vigilance, which suggests that an enhancement of the cortico-cortical interaction is necessary to maintain the task performance and prevent behavioral lapses. Additionally, the occipital-related connectivity magnitudes monotonically decreases as the vigilance level declines, which further supports the cortical gating of sensory stimuli during drowsiness. Neurophysiological evidence of mutual relationships between brain regions measured by transfer entropy might enhance the understanding of cortico-cortical communication during drowsy driving. PMID:26557069

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

    PubMed

    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.

  18. An In Vitro Chicken Gut Model Demonstrates Transfer of a Multidrug Resistance Plasmid from Salmonella to Commensal Escherichia coli

    PubMed Central

    Card, Roderick M.; Cawthraw, Shaun A.; Nunez-Garcia, Javier; Ellis, Richard J.; Kay, Gemma; Pallen, Mark J.; Woodward, Martin J.

    2017-01-01

    ABSTRACT The chicken gastrointestinal tract is richly populated by commensal bacteria that fulfill various beneficial roles for the host, including helping to resist colonization by pathogens. It can also facilitate the conjugative transfer of multidrug resistance (MDR) plasmids between commensal and pathogenic bacteria which is a significant public and animal health concern as it may affect our ability to treat bacterial infections. We used an in vitro chemostat system to approximate the chicken cecal microbiota, simulate colonization by an MDR Salmonella pathogen, and examine the dynamics of transfer of its MDR plasmid harboring several genes, including the extended-spectrum beta-lactamase blaCTX-M1. We also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity. Bacterial community profiles obtained by culture-independent methods showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha diversity and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered. MDR plasmid transfer from Salmonella to commensal Escherichia coli was demonstrated by PCR and whole-genome sequencing of isolates purified from agar plates containing cefotaxime. Transfer occurred to seven E. coli sequence types at high rates, even in the absence of cefotaxime, with resistant strains isolated within 3 days. Our chemostat system provides a good representation of bacterial interactions, including antibiotic resistance transfer in vivo. It can be used as an ethical and relatively inexpensive approach to model dissemination of antibiotic resistance within the gut of any animal or human and refine interventions that mitigate its spread before employing in vivo studies. PMID:28720731

  19. Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region.

    PubMed Central

    Furuya, N; Komano, T

    1996-01-01

    A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized. Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance. The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation. The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation. The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein. The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance. The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene. It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree. Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency. DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization. PMID:8626273

  20. Transfer of plasmid DNA to clinical coagulase-negative staphylococcal pathogens by using a unique bacteriophage.

    PubMed

    Winstel, Volker; Kühner, Petra; Krismer, Bernhard; Peschel, Andreas; Rohde, Holger

    2015-04-01

    Genetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a unique Staphylococcus aureus strain via a specific S. aureus bacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinical Staphylococcus epidermidis isolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

  1. Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

    PubMed Central

    Kühner, Petra; Krismer, Bernhard; Peschel, Andreas; Rohde, Holger

    2015-01-01

    Genetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a unique Staphylococcus aureus strain via a specific S. aureus bacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinical Staphylococcus epidermidis isolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion. PMID:25616805

  2. Plasmid transferability of KPC into a virulent K2 serotype Klebsiella pneumoniae

    PubMed Central

    2014-01-01

    Background KPC-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are associated with high mortality; however, their virulence determinants are not well defined. Methods We investigated the virulence and plasmid transferability among KPC-containing K. pneumoniae isolates. Results KPC-2 and -3 were successfully conjugated and retained by a virulent K2 K. pneumoniae recipient isolate. Antimicrobial susceptibility testing showed KPC-2 and -3 donor strains were resistant to more than four classes of antibiotics while the K2 isolate was only initially resistant to ampicillin. After conjugation of KPC-2 and -3, the K2 K. pneumoniae transconjugants became resistant to all beta-lactams. Additionally, the KPC K2 K. pneumoniae transconjugants continued to retain its high serum resistance and murine lethality. Conclusions Conjugation and retainment of KPC by virulent K2 K. pneumoniae and the ability of the tranconjugants to maintain its high serum resistance and murine lethality after conjugation was demonstrated in this study. These findings are concerning for the potential of KPC-like genes to disseminate among virulent K. pneumoniae isolates. PMID:24678611

  3. Plasmid transferability of KPC into a virulent K2 serotype Klebsiella pneumoniae.

    PubMed

    Siu, Leung-Kei Kristopher; Huang, David B; Chiang, Tom

    2014-03-31

    KPC-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are associated with high mortality; however, their virulence determinants are not well defined. We investigated the virulence and plasmid transferability among KPC-containing K. pneumoniae isolates. KPC-2 and -3 were successfully conjugated and retained by a virulent K2 K. pneumoniae recipient isolate. Antimicrobial susceptibility testing showed KPC-2 and -3 donor strains were resistant to more than four classes of antibiotics while the K2 isolate was only initially resistant to ampicillin. After conjugation of KPC-2 and -3, the K2 K. pneumoniae transconjugants became resistant to all beta-lactams. Additionally, the KPC K2 K. pneumoniae transconjugants continued to retain its high serum resistance and murine lethality. Conjugation and retainment of KPC by virulent K2 K. pneumoniae and the ability of the tranconjugants to maintain its high serum resistance and murine lethality after conjugation was demonstrated in this study. These findings are concerning for the potential of KPC-like genes to disseminate among virulent K. pneumoniae isolates.

  4. Transferable cefoxitin resistance in enterobacteria from Greek hospitals and characterization of a plasmid-mediated group 1 beta-lactamase (LAT-2).

    PubMed Central

    Gazouli, M; Tzouvelekis, L S; Prinarakis, E; Miriagou, V; Tzelepi, E

    1996-01-01

    Cefoxitin resistance in Klebsiella pneumoniae from Escherichia coli strains isolated in Greek hospitals was found to be due to the acquisition of similar plasmids coding for group 1 beta-lactamases. The plasmids were not self-transferable but were mobilized by conjugative plasmids. These elements have also been spread to Enterobacter aerogenes. The most common enzyme was a Citrobacter freundii-derived cephalosporinase (LAT-2) which differed from LAT-1 by three amino acids. PMID:8807075

  5. Effect of chromosomal homology on plasmid transfer and transformation in Streptococcus pneumoniae

    SciTech Connect

    Lacks, S.A.; Stassi, D.L.; Lopez, P.; Espinosa, M.; Greenberg, B.

    1982-01-01

    The obvious rarity of simultanaeous uptake of two recombinant plasmids bearing the same chromosomal segment militated against the likelihood of cloning such segments by the conventional approach. However, it was possible to clone chromosomal genes in S. pneumoniae, and clones of two well-studied pneumococcal genes, malM, which codes for the amylomaltase essential for maltose utilization, and sul-d, which confers sulfonamide resistance, were obtained. The availability of recombinant plasmids carrying DNA segments homologous to the chromosome allowed investigation of the effects of such homology on plasmid establishment. In addition to facilitation of plasmid establishment, evidence was obtained for a subsequent process, in which alleles were equilibrated between the chromosome and the plasmid pool.

  6. Plasmid transfer in Streptomyces lividans: identification of a kil-kor system associated with the transfer region of pIJ101.

    PubMed Central

    Kendall, K J; Cohen, S N

    1987-01-01

    The 8.9-kilobase Streptomyces plasmid pIJ101 is self-transmissible at high frequency into recipient strains. By genetic analysis of the transfer region of the plasmid, we identified six plasmid-encoded loci involved in gene transfer and the associated pocking phenomenon. Two loci, kilA and kilB, could not be cloned into Streptomyces lividans on a minimal pIJ101-based replicon unless suitable kil-override (kor) genes were present, either in cis or in trans. korA could control the lethal effects of both kilA and kilB, whereas korB could control only the effects of kilB. KilB mutants were defective in their pocking reaction; kilA mutants produced no visible pocks whatsoever. Mutations in two other loci, tra and spd, produced no pocks and defective pocks, respectively. These results suggest that kilA, kilB, tra, and spd are intimately involved in plasmid transfer and that the actions of kilA and kilB are regulated by the products of the korA and korB genes. PMID:3040681

  7. The animal food supplement sepiolite promotes a direct horizontal transfer of antibiotic resistance plasmids between bacterial species.

    PubMed

    Rodríguez-Beltrán, Jerónimo; Rodríguez-Rojas, Alexandro; Yubero, Elva; Blázquez, Jesús

    2013-06-01

    Animal fodder is routinely complemented with antibiotics together with other food supplements to improve growth. For instance, sepiolite is currently used as a dietary coadjuvant in animal feed, as it increases animal growth parameters and improves meat and derived final product quality. This type of food additive has so far been considered innocuous for the development and spread of antibiotic resistance. In this study, we demonstrate that sepiolite promotes the direct horizontal transfer of antibiotic resistance plasmids between bacterial species. The conditions needed for plasmid transfer (sepiolite and friction forces) occur in the digestive tracts of farm animals, which routinely receive sepiolite as a food additive. Furthermore, this effect may be aggravated by the use of antibiotics supplied as growth promoters.

  8. Plasmid-based transient human stromal cell-derived factor-1 gene transfer improves cardiac function in chronic heart failure

    PubMed Central

    Sundararaman, S; Miller, T J; Pastore, J M; Kiedrowski, M; Aras, R; Penn, M S

    2011-01-01

    We previously demonstrated that transient stromal cell-derived factor-1 alpha (SDF-1) improved cardiac function when delivered via cell therapy in ischemic cardiomyopathy at a time remote from acute myocardial infarction (MI) rats. We hypothesized that non-viral gene transfer of naked plasmid DNA-expressing hSDF-1 could similarly improve cardiac function. To optimize plasmid delivery, we tested SDF-1 and luciferase plasmids driven by the cytomegalovirus (CMV) promoter with (pCMVe) or without (pCMV) translational enhancers or α myosin heavy chain (pMHC) promoter in a rodent model of heart failure. In vivo expression of pCMVe was 10-fold greater than pCMV and pMHC expression and continued over 30 days. We directly injected rat hearts with SDF-1 plasmid 1 month after MI and assessed heart function. At 4 weeks after plasmid injection, we observed a 35.97 and 32.65% decline in fractional shortening (FS) in control (saline) animals and pMHC-hSDF1 animals, respectively, which was sustained to 8 weeks. In contrast, we observed a significant 24.97% increase in animals injected with the pCMVe-hSDF1 vector. Immunohistochemistry of cardiac tissue revealed a significant increase in vessel density in the hSDF-1-treated animals compared with control animals. Increasing SDF-1 expression promoted angiogenesis and improved cardiac function in rats with ischemic heart failure along with evidence of scar remodeling with a trend toward decreased myocardial fibrosis. These data demonstrate that stand-alone non-viral hSDF-1 gene transfer is a strategy for improving cardiac function in ischemic cardiomyopathy. PMID:21472007

  9. Crossed-beam energy transfer in direct-drive implosions

    SciTech Connect

    Seka, W; Edgell, D H; Michel, D T; Froula, D H; Goncharov, V N; Craxton, R S; Divol, L; Epstein, R; Follett, R; Kelly, J H; Kosc, T Z; Maximov, A V; McCrory, R L; Meyerhofer, D D; Michel, P; Myatt, J F; Sangster, T C; Shvydky, A; Skupsky, S; Stoeckl, C

    2012-05-22

    Direct-drive-implosion experiments on the OMEGA laser [T. R. Boehly et al., Opt. Commun. 133, 495 (1997)] have showed discrepancies between simulations of the scattered (non-absorbed) light levels and measured ones that indicate the presence of a mechanism that reduces laser coupling efficiency by 10%-20%. This appears to be due to crossed-beam energy transfer (CBET) that involves electromagnetic-seeded, low-gain stimulated Brillouin scattering. CBET scatters energy from the central portion of the incoming light beam to outgoing light, reducing the laser absorption and hydrodynamic efficiency of implosions. One-dimensional hydrodynamic simulations including CBET show good agreement with all observables in implosion experiments on OMEGA. Three strategies to mitigate CBET and improve laser coupling are considered: the use of narrow beams, multicolor lasers, and higher-Z ablators. Experiments on OMEGA using narrow beams have demonstrated improvements in implosion performance.

  10. Mapping of transfer and H pilus coding regions of the IncHII plasmid pHH1508a.

    PubMed

    Yan, W; Taylor, D E

    1989-02-01

    The IncHII plasmid pHH1508a (208 kilobases) encodes resistance to potassium tellurite, trimethoprim, and streptomycin. Conjugative pili encoded by pHH1508a were isolated, purified, and used for preparation of anti-H pilus antiserum. Immuno-gold labelling experiments using H pilus specific antiserum showed that antigenic determinants were located along the entire length of the H pilus. Immuno-gold labelling and lysis studies using pilH alpha, a bacteriophage specific for H pili, were used to investigate transfer-deficient mutants of pHH1508a obtained by Tn5 mutagenesis and an in vitro constructed derivative of 96 kilobases, pDT1178, which also conferred resistance to potassium tellurite, trimethoprim, and streptomycin. The transfer-deficient mutants did not specify H pili, whereas pDT1178, which transferred at low frequency (1 x 10(-4) transconjugants per recipient), specified a small number of H pili. A naturally occurring plasmid, pMG110, was found to encode the production of H pili, but was completely transfer deficient (less than 1 x 10(-7) transconjugants per recipient). This study suggests that genes required for H pilus production and assembly as well as low level transfer are located separately within the 96-kilobase fragment of pDT1178 and that other genes, located outside this region, are essential for the regulation and full expression of conjugative transfer.

  11. Lubrication mechanism in power transfer device for four wheel drive

    SciTech Connect

    Hamano, H.; Funato, Y.; Ida, S.

    1989-06-27

    A power transfer device for automotive vehicles is described, comprising: a housing, an input shaft rotatably mounted within the housing; an output shaft rotatably mounted with the housing and arranged coaxially with the input shaft for relative rotation thereto; an ancillary change-speed mechanism arranged within the housing and mounted on the input shaft, the change-speed mechanism having an input element mounted on the input shaft for rotation therewith and an output element connectable to the output shaft; a clutch sleeve axially slidably mounted on the output shaft and shiftable between a first position in which it is retained to effect a drive connection between the input and output shafts and a second position in which it is retained to effect a drive connection between the output element of the change-speed mechanism and the output shaft; and an oil pump assembly arranged within the housing to pump lubricating oil stored in the housing and supply it into an axial bore in the input shaft for lubrication of components of the change-speed mechanism.

  12. Plasmids from Euryarchaeota.

    PubMed

    Forterre, Patrick; Krupovic, Mart; Raymann, Kasie; Soler, Nicolas

    2014-12-01

    Many plasmids have been described in Euryarchaeota, one of the three major archaeal phyla, most of them in salt-loving haloarchaea and hyperthermophilic Thermococcales. These plasmids resemble bacterial plasmids in terms of size (from small plasmids encoding only one gene up to large megaplasmids) and replication mechanisms (rolling circle or theta). Some of them are related to viral genomes and form a more or less continuous sequence space including many integrated elements. Plasmids from Euryarchaeota have been useful for designing efficient genetic tools for these microorganisms. In addition, they have also been used to probe the topological state of plasmids in species with or without DNA gyrase and/or reverse gyrase. Plasmids from Euryarchaeota encode both DNA replication proteins recruited from their hosts and novel families of DNA replication proteins. Euryarchaeota form an interesting playground to test evolutionary hypotheses on the origin and evolution of viruses and plasmids, since a robust phylogeny is available for this phylum. Preliminary studies have shown that for different plasmid families, plasmids share a common gene pool and coevolve with their hosts. They are involved in gene transfer, mostly between plasmids and viruses present in closely related species, but rarely between cells from distantly related archaeal lineages. With few exceptions (e.g., plasmids carrying gas vesicle genes), most archaeal plasmids seem to be cryptic. Interestingly, plasmids and viral genomes have been detected in extracellular membrane vesicles produced by Thermococcales, suggesting that these vesicles could be involved in the transfer of viruses and plasmids between cells.

  13. Mercury resistance is encoded by transferable giant linear plasmids in two chesapeake bay Streptomyces strains.

    PubMed

    Ravel, J; Schrempf, H; Hill, R T

    1998-09-01

    The Streptomyces strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two and one, respectively, giant linear plasmids which carry terminally bound proteins. The plasmids pRJ3L (322 kb), from CHR3, and pRJ28 (330 kb), from CHR28, carry genes homologous to the previously characterized chromosomal Streptomyces lividans 66 operon encoding resistance against mercuric compounds. Both plasmids are transmissible (without any detectable rearrangement) to the chloramphenicol-resistant S. lividans TK24 strain lacking plasmids and carrying a chromosomal deletion of the mer operon. S. lividans TK24 conjugants harboring pRJ3L or pRJ28 exhibited profiles of mercury resistance to mercuric compounds similar to those of Streptomyces strains CHR3 and CHR28.

  14. Mercury Resistance Is Encoded by Transferable Giant Linear Plasmids in Two Chesapeake Bay Streptomyces Strains†

    PubMed Central

    Ravel, Jacques; Schrempf, Hildgund; Hill, Russell T.

    1998-01-01

    The Streptomyces strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two and one, respectively, giant linear plasmids which carry terminally bound proteins. The plasmids pRJ3L (322 kb), from CHR3, and pRJ28 (330 kb), from CHR28, carry genes homologous to the previously characterized chromosomal Streptomyces lividans 66 operon encoding resistance against mercuric compounds. Both plasmids are transmissible (without any detectable rearrangement) to the chloramphenicol-resistant S. lividans TK24 strain lacking plasmids and carrying a chromosomal deletion of the mer operon. S. lividans TK24 conjugants harboring pRJ3L or pRJ28 exhibited profiles of mercury resistance to mercuric compounds similar to those of Streptomyces strains CHR3 and CHR28. PMID:9726886

  15. Wavelength Detuning Cross-Beam Energy Transfer Mitigation for Polar Direct Drive and Symmetric Direct Drive

    NASA Astrophysics Data System (ADS)

    Marozas, J. A.; Collins, T. J. B.; McKenty, P. W.; Radha, P. B.; Hohenberger, M.; Rosenberg, M. J.

    2016-10-01

    Cross-beam energy transfer (CBET) results from two-beam energy exchange via stimulated Brillouin scattering, which reduces absorbed light and implosion velocity, alters time-resolved scattered-light spectra, and redistributes absorbed and scattered light. These effects reduce target performance in polar direct drive (PDD) and symmetric direct drive (SDD) at the National Ignition Facility (NIF) and on the OMEGA Laser System. The CBET package (Adaawam) incorporated into the 2-D hydrodynamics code DRACO is an integral part of the 3-D ray-trace package (Mazinisin). Detuning the initial laser wavelength (dλ0) reduces the CBET interaction volume, which can be combined with other mitigation domains (e.g., spatial and temporal). Recent PDD experiments on the NIF explored this option using a cone-swapping technique with dλ0 = +/- 2.34 Å UV, which are compared with DRACO simulations. DRACO simulations of wavelength detuning in SDD on OMEGA predict the expected mitigation using OMEGA's three main amplifier chains in both near-term dλ0 = { - 3 , 0 , + 3 } -Å and long-term dλ0 = { - 6 , 0 , + 6 } -Å UV configurations. The detuning simulations predict improved performance and changes in 2-D and 3-D morphology in both PDD and SDD. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DE-NA0001944.

  16. Transferable Multiresistance Plasmids Carrying cfr in Enterococcus spp. from Swine and Farm Environment

    PubMed Central

    Liu, Yang; Wang, Yang; Schwarz, Stefan; Li, Yun; Shen, Zhangqi; Zhang, Qijing; Wu, Congming

    2013-01-01

    Seventy-seven porcine Enterococcus isolates with florfenicol MICs of ≥16 μg of were/ml screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. Three isolates—Enterococcus thailandicus 3-38 (from a porcine rectal swab collected at a pig farm), Enterococcus thailandicus W3, and Enterococcus faecalis W9-2 (the latter two from sewage at a different farm), carried the cfr gene. The SmaI pulsed-field gel electrophoresis patterns of the three isolates differed distinctly. In addition, E. faecalis W9-2 was assigned to a new multilocus sequence type ST469. Mating experiments and Southern blot analysis indicated that cfr is located on conjugative plasmids pW3 (∼75 kb) from E. thailandicus W3, p3-38 (∼72 kb) from E. thailandicus 3-38, and pW9-2 (∼55 kb) from E. faecalis W9-2; these plasmids differed in their sizes, additional resistance genes, and the analysis of the segments encompassing the cfr gene. Sequence analysis revealed that all plasmids harbored a 4,447-bp central region, in which cfr was bracketed by two copies of the novel insertion sequence ISEnfa4 located in the same orientation. The sequences flanking the central regions of these plasmids, including the partial tra gene regions and a ω-ε-ζ toxin-antitoxin module, exhibited >95% nucleotide sequence identity to the conjugative plasmid pAMβ1 from E. faecalis. Conjugative plasmids carrying cfr appear to play an important role in the dissemination and maintenance of the multiresistance gene cfr among enterococcal isolates and possibly other species of Gram-positive bacteria. PMID:23070165

  17. Transferable multiresistance plasmids carrying cfr in Enterococcus spp. from swine and farm environment.

    PubMed

    Liu, Yang; Wang, Yang; Schwarz, Stefan; Li, Yun; Shen, Zhangqi; Zhang, Qijing; Wu, Congming; Shen, Jianzhong

    2013-01-01

    Seventy-seven porcine Enterococcus isolates with florfenicol MICs of ≥16 μg of were/ml screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. Three isolates-Enterococcus thailandicus 3-38 (from a porcine rectal swab collected at a pig farm), Enterococcus thailandicus W3, and Enterococcus faecalis W9-2 (the latter two from sewage at a different farm), carried the cfr gene. The SmaI pulsed-field gel electrophoresis patterns of the three isolates differed distinctly. In addition, E. faecalis W9-2 was assigned to a new multilocus sequence type ST469. Mating experiments and Southern blot analysis indicated that cfr is located on conjugative plasmids pW3 (∼75 kb) from E. thailandicus W3, p3-38 (∼72 kb) from E. thailandicus 3-38, and pW9-2 (∼55 kb) from E. faecalis W9-2; these plasmids differed in their sizes, additional resistance genes, and the analysis of the segments encompassing the cfr gene. Sequence analysis revealed that all plasmids harbored a 4,447-bp central region, in which cfr was bracketed by two copies of the novel insertion sequence ISEnfa4 located in the same orientation. The sequences flanking the central regions of these plasmids, including the partial tra gene regions and a ω-ε-ζ toxin-antitoxin module, exhibited >95% nucleotide sequence identity to the conjugative plasmid pAMβ1 from E. faecalis. Conjugative plasmids carrying cfr appear to play an important role in the dissemination and maintenance of the multiresistance gene cfr among enterococcal isolates and possibly other species of Gram-positive bacteria.

  18. Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4.

    PubMed Central

    Balzer, D.; Pansegrau, W.; Lanka, E.

    1994-01-01

    Two essential transfer genes of the conjugative plasmid RP4 were altered by site-directed mutagenesis: traG of the primase operon and traI of the relaxase operon. To evaluate effects on the transfer phenotype of the point mutations, we have reconstituted the RP4 transfer system by fusion of the transfer regions Tra1 and Tra2 to the small multicopy replicon ColD. Deletions in traG or traI served to determine the Tra phenotype of mutant plasmids by trans complementation. Two motifs of TraG which are highly conserved among TraG-like proteins in several other conjugative DNA transfer systems were found to be essential for TraG function. One of the motifs resembles that of a nucleotide binding fold of type B. The relaxase (TraI) catalyzes the specific cleaving-joining reaction at the transfer origin needed to initiate and terminate conjugative DNA transfer (W. Pansegrau, W. Schröder, and E. Lanka, Proc. Natl. Acad. Sci. USA 90:2925-2929, 1993). Phenotypes of mutations in three motifs that belong to the active center of the relaxase confirmed previously obtained biochemical evidence for the contributions of the motifs to the catalytic activity of TraI. Expression of the relaxase operon is greatly increased in the absence of an intact TraI protein. This finding suggests that the relaxosome which assembles only in the presence of the TraI in addition to its enzymatic activity plays a role in gene regulation. Images PMID:8021214

  19. Osa Protein Constitutes a Strong Oncogenic Suppression System That Can Block vir-Dependent Transfer of IncQ Plasmids between Agrobacterium Cells and the Establishment of IncQ Plasmids in Plant Cells

    PubMed Central

    Lee, Lan-Ying; Gelvin, Stanton B.

    2004-01-01

    The osa (oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by Agrobacterium tumefaciens. osa confers oncogenic suppression by inhibiting VirE2 protein export. This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010. We conducted a series of experiments to compare oncogenic suppression by these two systems. Agrobacterium strains harboring plasmids containing osa are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives. When osa is present within a donor Agrobacterium strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked. Oncogenic suppression is still effected when the osa gene is integrated into the Agrobacterium chromosome, suggesting that it is the osa gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids. Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself. Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms. PMID:15489437

  20. A novel peptide delivers plasmids across blood-brain barrier into neuronal cells as a single-component transfer vector.

    PubMed

    Fu, Ailing; Zhang, Miaomiao; Gao, Feiyan; Xu, Xingran; Chen, Zhangbao

    2013-01-01

    There is no data up to now to show that peptide can deliver plasmid into brain as a single-component transfer vector. Here we show that a novel peptide, RDP (consisted of 39 amino acids), can be exploited as an efficient plasmid vector for brain-targeting delivery. The plasmids containing Lac Z reporter gene (pVAX-Lac Z) and BDNF gene (pVAX-BDNF) are complexed with RDP and intravenously injected into mice. The results of gel retardation assay show that RDP enables to bind DNA in a dose-dependent manner, and the X-Gal staining identity that Lac Z is specifically expressed in the brain. Also, the results of Western blot and immunofluorescence staining of BDNF indicate that pVAX-BDNF complexed with RDP can be delivered into brain, and show neuroprotective properties in experimental Parkinson's disease (PD) model. The results demonstrate that RDP enables to bind and deliver DNA into the brain, resulting in specific gene expression in the neuronal cells. This strategy provides a novel, simple and effective approach for non-viral gene therapy of brain diseases.

  1. The repABC Plasmids with Quorum-Regulated Transfer Systems in Members of the Rhizobiales Divide into Two Structurally and Separately Evolving Groups

    PubMed Central

    Wetzel, Margaret E.; Olsen, Gary J.; Chakravartty, Vandana; Farrand, Stephen K.

    2015-01-01

    The large repABC plasmids of the order Rhizobiales with Class I quorum-regulated conjugative transfer systems often define the nature of the bacterium that harbors them. These otherwise diverse plasmids contain a core of highly conserved genes for replication and conjugation raising the question of their evolutionary relationships. In an analysis of 18 such plasmids these elements fall into two organizational classes, Group I and Group II, based on the sites at which cargo DNA is located. Cladograms constructed from proteins of the transfer and quorum-sensing components indicated that those of the Group I plasmids, while coevolving, have diverged from those coevolving proteins of the Group II plasmids. Moreover, within these groups the phylogenies of the proteins usually occupy similar, if not identical, tree topologies. Remarkably, such relationships were not seen among proteins of the replication system; although RepA and RepB coevolve, RepC does not. Nor do the replication proteins coevolve with the proteins of the transfer and quorum-sensing systems. Functional analysis was mostly consistent with phylogenies. TraR activated promoters from plasmids within its group, but not between groups and dimerized with TraR proteins from within but not between groups. However, oriT sequences, which are highly conserved, were processed by the transfer system of plasmids regardless of group. We conclude that these plasmids diverged into two classes based on the locations at which cargo DNA is inserted, that the quorum-sensing and transfer functions are coevolving within but not between the two groups, and that this divergent evolution extends to function. PMID:26590210

  2. The repABC plasmids with quorum-regulated transfer systems in members of the Rhizobiales divide into two structurally and separately evolving groups

    DOE PAGES

    Wetzel, Margaret E.; Olsen, Gary J.; Chakravartty, Vandana; ...

    2015-11-19

    The large repABC plasmids of the order Rhizobiales with Class I quorum-regulated conjugative transfer systems often define the nature of the bacterium that harbors them. These otherwise diverse plasmids contain a core of highly conserved genes for replication and conjugation raising the question of their evolutionary relationships. In an analysis of 18 such plasmids these elements fall into two organizational classes, Group I and Group II, based on the sites at which cargo DNA is located. Cladograms constructed from proteins of the transfer and quorum-sensing components indicated that those of the Group I plasmids, while coevolving, have diverged from thosemore » coevolving proteins of the Group II plasmids. Moreover, within these groups the phylogenies of the proteins usually occupy similar, if not identical, tree topologies. Remarkably, such relationships were not seen among proteins of the replication system; although RepA and RepB coevolve, RepC does not. Nor do the replication proteins coevolve with the proteins of the transfer and quorum-sensing systems. Functional analysis was mostly consistent with phylogenies. TraR activated promoters from plasmids within its group, but not between groups and dimerized with TraR proteins from within but not between groups. However, oriT sequences, which are highly conserved, were processed by the transfer system of plasmids regardless of group. Here, we conclude that these plasmids diverged into two classes based on the locations at which cargo DNA is inserted, that the quorum-sensing and transfer functions are coevolving within but not between the two groups, and that this divergent evolution extends to function.« less

  3. The repABC plasmids with quorum-regulated transfer systems in members of the Rhizobiales divide into two structurally and separately evolving groups

    SciTech Connect

    Wetzel, Margaret E.; Olsen, Gary J.; Chakravartty, Vandana; Farrand, Stephen K.

    2015-11-19

    The large repABC plasmids of the order Rhizobiales with Class I quorum-regulated conjugative transfer systems often define the nature of the bacterium that harbors them. These otherwise diverse plasmids contain a core of highly conserved genes for replication and conjugation raising the question of their evolutionary relationships. In an analysis of 18 such plasmids these elements fall into two organizational classes, Group I and Group II, based on the sites at which cargo DNA is located. Cladograms constructed from proteins of the transfer and quorum-sensing components indicated that those of the Group I plasmids, while coevolving, have diverged from those coevolving proteins of the Group II plasmids. Moreover, within these groups the phylogenies of the proteins usually occupy similar, if not identical, tree topologies. Remarkably, such relationships were not seen among proteins of the replication system; although RepA and RepB coevolve, RepC does not. Nor do the replication proteins coevolve with the proteins of the transfer and quorum-sensing systems. Functional analysis was mostly consistent with phylogenies. TraR activated promoters from plasmids within its group, but not between groups and dimerized with TraR proteins from within but not between groups. However, oriT sequences, which are highly conserved, were processed by the transfer system of plasmids regardless of group. Here, we conclude that these plasmids diverged into two classes based on the locations at which cargo DNA is inserted, that the quorum-sensing and transfer functions are coevolving within but not between the two groups, and that this divergent evolution extends to function.

  4. Differences in the carriage and the ability to utilize the serotype associated virulence plasmid in strains of Salmonella enterica serotype Typhimurium investigated by use of a self-transferable virulence plasmid, pOG669.

    PubMed

    Olsen, John E; Brown, Derek J; Thomsen, Line E; Platt, David J; Chadfield, Mark S

    2004-06-01

    Most strains of Salmonella enterica subspecies enterica serotype typhimurium (S. typhimurium) naturally harbour a virulence plasmid which carries the salmonella plasmid virulence (spv) genes. However, isolates belonging to certain phage types are generally found without the plasmid. We have utilized a self-transferable virulence plasmid, pOG669 to investigate the effect of introduction of spv genes into strains of such phage types. The use of the co-integrate plasmid, pOG669, was validated on a diverse collection of strains. pOG669 was transferred into strains of serotypes that are normally associated with the possession of virulence plasmids. All strains maintained the wild type level of virulence in a mouse model, except that introduction of pOG669 restored normal virulence levels in an avirulent, plasmid free strain of S. dublin and resulted in a decrease in virulence in a strain of S. dublin from clonal line Du3. S. gallinarum did not become virulent in mice, but pOG669 was functionally interchangeable with the wild type plasmid when strains were tested in a chicken model. Strains of serotypes not normally associated with the carriage of a virulence plasmid did not increase in virulence upon the introduction of pOG669. An IncX plasmid pOG670 that was included as control was incompatible with the virulence plasmid in a strain of S. dublin, demonstrating that the common virulence plasmid of this serotype is of a different incompatibility group than other virulence plasmids. Strains of S. typhimurium from phage types that do not normally carry a virulence plasmid responded differently to attempts to introduce pOG669. No transconjugants were observed with the strains of DT5 and DT21. The introduction of pOG669 did not alter the virulence of JEO3942(DT10), DT35 and JEO3949(DT66) significantly, while DT1 and DT27 became more virulent. DT27 became as virulent as wild type C5, while logVC(10) of DT1 only increased from 4.1 to 5.7. The ability to express spv-genes was

  5. Plasmid diversity in neisseriae.

    PubMed

    van Passel, Mark W J; van der Ende, Arie; Bart, Aldert

    2006-08-01

    Horizontal gene transfer constitutes an important force in prokaryotic genome evolution, and it is well-known that plasmids are vehicles for DNA transfer. Chromosomal DNA is frequently exchanged between pathogenic and commensal neisseriae, but relatively little is known about plasmid diversity and prevalence among these nasopharyngeal inhabitants. We investigated the plasmid contents of 18 Neisseria lactamica isolates and 20 nasopharyngeal Neisseria meningitidis isolates. Of 18 N. lactamica strains, 9 harbored one or more plasmids, whereas only one N. meningitidis isolate contained a plasmid. Twelve plasmids were completely sequenced, while five plasmid sequences from the public databases were also included in the analyses. On the basis of nucleic acid sequences, mobilization, and replicase protein alignments, we distinguish six different plasmid groups (I to VI). Three plasmids from N. lactamica appeared to be highly similar on the nucleotide level to the meningococcal plasmids pJS-A (>99%) and pJS-B (>75%). The genetic organizations of two plasmids show a striking resemblance with that of the recently identified meningococcal disease-associated (MDA) phage, while four putative proteins encoded by these plasmids show 25% to 39% protein identity to those encoded by the MDA phage. The putative promoter of the gene encoding the replicase on these plasmids contains a polycytidine tract, suggesting that replication is subjected to phase variation. In conclusion, extensive plasmid diversity is encountered among commensal neisseriae. Members of three plasmid groups are found in both pathogenic and commensal neisseriae, indicating plasmid exchange between these species. Resemblance between plasmids and MDA phage may be indicative of dissemination of phage-related sequences among pathogenic and commensal neisseriae.

  6. Transferable, multiple antibiotic and mercury resistance in Atlantic Canadian isolates of Aeromonas salmonicida subsp. salmonicida is associated with carriage of an IncA/C plasmid similar to the Salmonella enterica plasmid pSN254

    PubMed Central

    McIntosh, Douglas; Cunningham, Michelle; Ji, Baijing; Fekete, Frank A.; Parry, Erin M.; Clark, Sarah E.; Zalinger, Zachary B.; Gilg, Ilana C.; Danner, G. Russell; Johnson, Keith A.; Beattie, Mike; Ritchie, Rachael

    2008-01-01

    Objectives The aim of this study was to examine the molecular basis for multiple antibiotic and mercury resistance in Canadian isolates of Aeromonas salmonicida subsp. salmonicida. Methods Phenotypic and genotypic methods were employed to identify plasmid-associated antibiotic and mercury resistance genes and to determine the organization of those genes in multidrug-resistant (MDR) A. salmonicida isolates. Results The MDR phenotype was transferable via conjugation using Escherichia coli, Aeromonas hydrophila and Edwardseilla tarda as recipients. Antibiotic and mercury resistance genes were carried by a conjugative IncA/C plasmid. Three distinct antibiotic resistance cassettes were characterized; first a class I integron containing an aadA7 gene encoding for an aminoglycoside-3′-adenyltransferase, the second cassette showed 99.9% nucleotide sequence homology to a cassette previously identified in the Salmonella enterica IncA/C plasmid pSN254, containing floR, tetA, sulII and strA/strB sequences. The third cassette showed 100% nucleotide sequence similarity to a transposon-like element, containing a blaCMY-2 β-lactamase in association with sugE and blc sequences. This element is known to be widely distributed among clinical and food-borne Salmonella and other Enterobacteriaceae throughout Asia and the United States. Mercury resistance was linked to the presence of a mer operon that showed 100% nucleotide sequence homology to the mer operon carried by plasmid pSN254. Conclusions Each MDR A. salmonicida isolate carried the same plasmid, which was related to plasmid pSN254. This is the first report of plasmid-mediated florfenicol-resistant A. salmonicida in North America. In addition, it is the first report of a plasmid-associated AmpC β-lactamase sequence in a member of the Aeromonadaceae. PMID:18375380

  7. Evidence for Transfer of CMY-2 AmpC β-Lactamase Plasmids between Escherichia coli and Salmonella Isolates from Food Animals and Humans

    PubMed Central

    Winokur, P. L.; Vonstein, D. L.; Hoffman, L. J.; Uhlenhopp, E. K.; Doern, G. V.

    2001-01-01

    Escherichia coli is an important pathogen that shows increasing antimicrobial resistance in isolates from both animals and humans. Our laboratory recently described Salmonella isolates from food animals and humans that expressed an identical plasmid-mediated, AmpC-like β-lactamase, CMY-2. In the present study, 59 of 377 E. coli isolates from cattle and swine (15.6%) and 6 of 1,017 (0.6%) isolates of human E. coli from the same geographic region were resistant to both cephamycins and extended-spectrum cephalosporins. An ampC gene could be amplified with CMY-2 primers in 94.8% of animal and 33% of human isolates. Molecular epidemiological studies of chromosomal DNA revealed little clonal relatedness among the animal and human E. coli isolates harboring the CMY-2 gene. The ampC genes from 10 animal and human E. coli isolates were sequenced, and all carried an identical CMY-2 gene. Additionally, all were able to transfer a plasmid containing the CMY-2 gene to a laboratory strain of E. coli. CMY-2 plasmids demonstrated two different plasmid patterns that each showed strong similarities to previously described Salmonella CMY-2 plasmids. Additionally, Southern blot analyses using a CMY-2 probe demonstrated conserved fragments among many of the CMY-2 plasmids identified in Salmonella and E. coli isolates from food animals and humans. These data demonstrate that common plasmids have been transferred between animal-associated Salmonella and E. coli, and identical CMY-2 genes carried by similar plasmids have been identified in humans, suggesting that the CMY-2 plasmid has undergone transfer between different bacterial species and may have been transmitted between food animals and humans. PMID:11557460

  8. Novel Conjugative Transferable Multiple Drug Resistance Plasmid pAQU1 from Photobacterium damselae subsp. damselae Isolated from Marine Aquaculture Environment

    PubMed Central

    Nonaka, Lisa; Maruyama, Fumito; Miyamoto, Manabu; Miyakoshi, Masatoshi; Kurokawa, Ken; Masuda, Michiaki

    2012-01-01

    The emergence of drug-resistant bacteria is a severe problem in aquaculture. The ability of drug resistance genes to transfer from a bacterial cell to another is thought to be responsible for the wide dissemination of these genes in the aquaculture environment; however, little is known about the gene transfer mechanisms in marine bacteria. In this study, we show that a tetracycline-resistant strain of Photobacterium damselae subsp. damselae, isolated from seawater at a coastal aquaculture site in Japan, harbors a novel multiple drug resistance plasmid. This plasmid named pAQU1 can be transferred to Escherichia coli by conjugation. Nucleotide sequencing showed that the plasmid was 204,052 base pairs and contained 235 predicted coding sequences. Annotation showed that pAQU1 did not have known repA, suggesting a new replicon, and contained seven drug resistance genes: blaCARB-9-like, floR, mph(A)-like, mef(A)-like, sul2, tet(M) and tet(B). The plasmid has a complete set of genes encoding the apparatus for the type IV secretion system with a unique duplication of traA. Phylogenetic analysis of the deduced amino acid sequence of relaxase encoded by traI in pAQU1 demonstrated that the conjugative transfer system of the plasmid belongs to MOBH12, a sub-group of the MOBH plasmid family, closely related to the IncA/C type of plasmids and SXT/R391 widely distributed among species of Enterobacteriaceae and Vibrionaceae. Our data suggest that conjugative transfer is involved in horizontal gene transfer among marine bacteria and provide useful insights into the molecular basis for the dissemination of drug resistance genes among bacteria in the aquaculture environment. PMID:22446310

  9. Energy transfer in strained graphene assisted by discrete breathers excited by external ac driving

    NASA Astrophysics Data System (ADS)

    Evazzade, Iman; Lobzenko, Ivan P.; Korznikova, Elena A.; Ovid'ko, Ilya A.; Roknabadi, Mahmood Rezaee; Dmitriev, Sergey V.

    2017-01-01

    In the present molecular-dynamics study, external ac driving is used at frequencies outside the phonon spectrum to excite gap DBs in uniformly strained graphene nanoribbon. Harmonic displacement or harmonic force is applied to a zigzag atomic chain of graphene. In the former case, nonpropagating DBs are excited on the atoms next to the driven atoms, while in the latter case the excited DBs propagate along the nanoribbon. The energy transfer along the nanoribbon assisted by the DBs is investigated in detail, and the differences between harmonic displacement driving and harmonic force driving are discussed. It is concluded that the amplitude of external driving at out-of-phonon spectrum frequencies should not necessarily be large to obtain a noticeable energy transfer to the system. Overall, our results suggest that external harmonic driving even at relatively small driving amplitudes can be used to control excitation of DBs and consequently the energy transfer to the system.

  10. Successful gene transfer into dendritic cells with cationized gelatin and plasmid DNA complexes via a phagocytosis-dependent mechanism.

    PubMed

    Inada, Satoshi; Fujiwara, Hitoshi; Atsuji, Kiyoto; Takashima, Kazuhiro; Araki, Yasunobu; Kubota, Takeshi; Tabata, Yasuhiko; Yamagishi, Hisakazu

    2006-01-01

    The use of gene-modified dendritic cells (DC) is a powerful tool to enhance antitumor immune responses stimulated by these cells in cancer immunotherapy. Cationized gelatin is preferably incorporated via phagocytosis and is gradually degraded by proteolysis while buffering lysosomal activity. This may be appropriate for gene transfer into phagocytic cells, such as immature DC. In the present study, successful transfection into monocyte-derived immature DC was demonstrated using cationized gelatin and plasmid DNA complexes. A high transfection efficiency, approaching 16%, was obtained upon transfection of the enhanced green fluorescent protein (EGFP) gene as evaluated by flow cytometry. Transgene expression of EGFP and murine interleukin 12 were also detected by RT-PCR. The antigen-presenting capacity of the transfected DC was equal to that of untransfected DC as evaluated by the allogeneic mixed lymphocyte reaction. Cationized gelatin has the potential to be a unique non-viral vector for gene transfer into DC.

  11. The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

    PubMed

    Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

    2014-01-31

    The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

  12. Crossed-Beam Energy Transfer in Direct-Drive Implosions

    NASA Astrophysics Data System (ADS)

    Igumenshchev, I. V.

    2011-10-01

    Direct-drive-implosion experiments on OMEGA have revealed the importance of crossed-beam energy transfer (CBET), which is caused by stimulated Brillouin scattering. The CBET reduces the laser absorption in a target corona by ~10% to 20% and, therefore, decreases the implosion performance. The signature of CBET is observed in time-resolved, reflected-light spectra as a suppression of red-shifted light during the main laser pulse. Simulations without CBET typically predict an earlier bang time and overestimate the laser absorption in high-compression, low-adiabat implosions. Simulations using a CBET model and a nonlocal heat-transport model explain well the scattered-light and bang-timing measurements. This talk will summarize the possible mitigation strategies for CBET required for robust ignition designs. CBET most effectively scatters incoming light that interacts with outgoing light originated from laser beam edges. This makes it possible to mitigate CBET by reducing the beam diameter with respect to the target diameter. Implosion experiments using large 1400- μm-diam plastic shells and in-focus and defocus laser beams have demonstrated the reduction of CBET in implosions with a smaller ratio of the beam-to-target diameters. Simulations predict the optimum range of this ratio to be 0.7 to 0.8. Another mitigation strategy involves splitting the incident light into two or more colors. This reduces CBET by shifting and suppressing the coupling resonances. The reduction in scattered light caused by CBET is predicted to be up to a factor of 2 when incident light colors are separated by δλ > 6 Ã. This work was supported by the U.S. Department of Energy Office of Inertial Confinement Fusion under Cooperative Agreement No. DE-FC52-08NA28302. In collaboration with W. Seka, D. H. Edgell, D. H. Froula, V. N. Goncharov, R. S. Craxton, R. L. McCrory, A. V. Maximov, D. D. Meyerhofer, J. F. Myatt, T. C. Sangster, A. Shvydky, S. Skupsky, and C. Stoeckl. I. V. Igumenshchevet

  13. Characterization of a transferable bcrABC and cadAC genes-harboring plasmid in Listeria monocytogenes strain isolated from food products of animal origin.

    PubMed

    Xu, Dongyang; Nie, Qing; Wang, Wenyan; Shi, Lei; Yan, He

    2016-01-18

    In this study, we characterized a bcrABC cassette and its genetic environment harbored by a plasmid in Listeria monocytogenes (L. monocytogenes) 11GZL18, a strain isolated from raw meat in 2011. The bcrABC cassette and its genetic environment were characterized, with a total of 33,727 nt nucleotide sequence obtained. The nucleotide sequences of the bcrABC cassette in strain 11GZL18 exhibited 100% identity to that on plasmid pLM80, which is harbored by L. monocytogenes strain H7550 and H7858, and the neighboring 21,678 nt nucleotide sequence of bcrABC cassette showed 99% identity with plasmid pLM80. The plasmid curing experiment demonstrated the role of the plasmid in conferring benzalkonium chloride (BC) and cadmium (Cd) tolerance in this strain. The bcrABC cassette and cadAC genes from the L. monocytogenes 11GZL18 were harbored by plasmid, functional and transmissible, and led to the acquired tolerance in Gram-negative Escherichia coli (E. coli) DH5α by chemical and natural transformation. Besides, the efflux pump activity that is conferring tolerance to BC and Cd was observed in strain 11GZL18, while not in a plasmid-cured strain 11GZL18-C, confirming that efflux pumps play a role in plasmid-mediated tolerance to BC and Cd in L. monocytogenes 11GZL18. In this study we characterized the genetic organization of a novel BC and Cd tolerance determinants-harboring plasmid in a L. monocytogenes strain isolated from raw meat of animal origin, and demonstrated the potential horizontal transferability of this bcrABC cassette-harboring plasmid to E. coli. The findings will further improve our understanding of the adaptations of this organism to disinfectants such as BC and may contribute to elucidating possible dissemination of BC tolerance in foodborne L. monocytogenes.

  14. Evidence for the Role of Horizontal Transfer in Generating pVT1, a Large Mosaic Conjugative Plasmid from the Clam Pathogen, Vibrio tapetis

    PubMed Central

    Bidault-Toffin, Adeline; Le Chevalier, Patrick; Bouloc, Philippe; Paillard, Christine; Jacq, Annick

    2011-01-01

    The marine bacterium Vibrio tapetis is the causative agent of the brown ring disease, which affects the clam Ruditapes philippinarum and causes heavy economic losses in North of Europe and in Eastern Asia. Further characterization of V. tapetis isolates showed that all the investigated strains harbored at least one large plasmid. We determined the sequence of the 82,266 bp plasmid pVT1 from the CECT4600T reference strain and analyzed its genetic content. pVT1 is a mosaic plasmid closely related to several conjugative plasmids isolated from Vibrio vulnificus strains and was shown to be itself conjugative in Vibrios. In addition, it contains DNA regions that have similarity with several other plasmids from marine bacteria (Vibrio sp., Shewanella sp., Listonella anguillarum and Photobacterium profundum). pVT1 contains a number of mobile elements, including twelve Insertion Sequences or inactivated IS genes and an RS1 phage element related to the CTXphi phage of V. cholerae. The genetic organization of pVT1 underscores an important role of horizontal gene transfer through conjugative plasmid shuffling and transposition events in the acquisition of new genetic resources and in generating the pVT1 modular organization. In addition, pVT1 presents a copy number of 9, relatively high for a conjugative plasmid, and appears to belong to a new type of replicon, which may be specific to Vibrionaceae and Shewanelleacae. PMID:21326607

  15. Horizontal Gene Transfer of a ColV Plasmid Has Resulted in a Dominant Avian Clonal Type of Salmonella enterica Serovar Kentucky

    PubMed Central

    Johnson, Timothy J.; Thorsness, Jessica L.; Anderson, Cole P.; Lynne, Aaron M.; Foley, Steven L.; Han, Jing; Fricke, W. Florian; McDermott, Patrick F.; White, David G.; Khatri, Mahesh; Stell, Adam L.; Flores, Cristian; Singer, Randall S.

    2010-01-01

    Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard. PMID:21203520

  16. A Silenced vanA Gene Cluster on a Transferable Plasmid Caused an Outbreak of Vancomycin-Variable Enterococci

    PubMed Central

    Sivertsen, Audun; Pedersen, Torunn; Larssen, Kjersti Wik; Bergh, Kåre; Rønning, Torunn Gresdal; Radtke, Andreas

    2016-01-01

    We report an outbreak of vancomycin-variable vanA+ enterococci (VVE) able to escape phenotypic detection by current guidelines and demonstrate the molecular mechanisms for in vivo switching into vancomycin resistance and horizontal spread of the vanA cluster. Forty-eight vanA+ Enterococcus faecium isolates and one Enterococcus faecalis isolate were analyzed for clonality with pulsed-field gel electrophoresis (PFGE), and their vanA gene cluster compositions were assessed by PCR and whole-genome sequencing of six isolates. The susceptible VVE strains were cultivated in brain heart infusion broth containing vancomycin at 8 μg/ml for in vitro development of resistant VVE. The transcription profiles of susceptible VVE and their resistant revertants were assessed using quantitative reverse transcription-PCR. Plasmid content was analyzed with S1 nuclease PFGE and hybridizations. Conjugative transfer of vanA was assessed by filter mating. The only genetic difference between the vanA clusters of susceptible and resistant VVE was an ISL3-family element upstream of vanHAX, which silenced vanHAX gene transcription in susceptible VVE. Furthermore, the VVE had an insertion of IS1542 between orf2 and vanR that attenuated the expression of vanHAX. Growth of susceptible VVE occurred after 24 to 72 h of exposure to vancomycin due to excision of the ISL3-family element. The vanA gene cluster was located on a transferable broad-host-range plasmid also detected in outbreak isolates with different pulsotypes, including one E. faecalis isolate. Horizontally transferable silenced vanA able to escape detection and revert into resistance during vancomycin therapy represents a new challenge in the clinic. Genotypic testing of invasive vancomycin-susceptible enterococci by vanA-PCR is advised. PMID:27139479

  17. Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.

    PubMed

    Pontes, Daniela; Innocentin, Silvia; Del Carmen, Silvina; Almeida, Juliana Franco; Leblanc, Jean-Guy; de Moreno de Leblanc, Alejandra; Blugeon, Sébastien; Cherbuy, Claire; Lefèvre, François; Azevedo, Vasco; Miyoshi, Anderson; Langella, Philippe; Chatel, Jean-Marc

    2012-01-01

    Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.

  18. A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions.

    PubMed Central

    Suwanto, A; Kaplan, S

    1992-01-01

    Rhodobacter sphaeroides 2.4.1 naturally harbors five cryptic endogenous plasmids (C. S. Fornari, M. Watkins, and S. Kaplan, Plasmid 11:39-47, 1984). The smallest plasmid (pRS241e), with a molecular size of 42 kb, was observed to be a self-transmissible plasmid which can transfer only to certain strains of R. sphaeroides. Transfer frequencies can be as high as 10(-2) to 10(-3) per donor under optimal mating conditions in liquid media in the absence of oxygen. pRS241e, designated the S factor, was also shown to possess a narrow host range, failing either to replicate or to be maintained in Escherichia coli, Agrobacterium tumefaciens, and Rhizobium meliloti. It was further revealed that one of the remaining four endogenous plasmids, pRS241d, was also transmissible at a frequency similar to that of the S. factor. As a cointegrate with pSUP203, S was maintained in E. coli, providing sufficient DNA from which a physical map of S could be constructed. Progressive subcloning of S-factor DNA, in conjunction with assays of plasmid transfer, led to the localization and identification of oriV (IncA), IncB, and the putative oriT locus. The DNA sequence of the 427 bp containing oriTs revealed topological similarity to other described oriT sequences, consisting of an A-T-rich DNA region, several direct and inverted repeats, and putative integration host factor (IHF)-binding sites, and was shown to be functional in promoting plasmid transfer. Images PMID:1735707

  19. Conjugative transfer of p42a from rhizobium etli CFN42, which is required for mobilization of the symbiotic plasmid, is regulated by quorum sensing.

    PubMed

    Tun-Garrido, Cristina; Bustos, Patricia; González, Víctor; Brom, Susana

    2003-03-01

    Rhizobium etli CFN42 contains six plasmids. Only one of them, p42a, is self-conjugative at high frequency. This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym). To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated. Its sequence showed that most of the tra genes are highly similar to genes of Agrobacterium tumefaciens pTiC58 and other related plasmids. Four putative regulatory genes were identified; three of these (traI, traR, and cinR) belong to the LuxR-LuxI family. Mutagenesis of these genes confirmed their requirement for p42a transfer. We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer. Although R. etli has been shown to produce different N-acyl-homoserine lactones, only one of them, a 3-oxo-C(8)-homoserine lactone encoded by the traI gene described here, was involved in transfer. Mutagenesis of the fourth regulatory gene, traM, had no effect on transfer. Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer. Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C(8)-homoserine lactone synthesized by TraI. The traI gene was autoregulated by these elements and positively regulated by CinR, while cinR expression required traI. Finally, we did not detect expression of traM, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a.

  20. Genetic and physical characterization of recombinant plasmids associated with cell aggregation and high-frequency conjugal transfer in Streptococcus lactis ML3.

    PubMed

    Anderson, D G; McKay, L L

    1984-06-01

    Restriction mapping was employed to characterize the 104-kilobase (kb) cointegrate lactose plasmids from 15 independent transconjugants derived from Streptococcus lactis ML3 as well as the 55-kb lactose plasmid ( pSK08 ) and a previously uncharacterized 48.4-kb plasmid ( pRS01 ) from S. lactis ML3. The data revealed that the 104-kb plasmids were cointegrates of pSK08 and pRS01 and were structurally distinct. The replicon fusion event occurred within adjacent 13.8- or 7.3-kb PvuII fragments of pSK08 and interrupted apparently random regions of pRS01 . Correlation of the transconjugants' clumping and conjugal transfer capabilities with the interrupted region of pRS01 identified pRS01 regions coding for these properties. In the 104-kb plasmids, the pRS01 region was present in both orientations with respect to the pSK08 region. The replicon fusion occurred in recombination-deficient (Rec-) strains and appeared to introduce a 0.8 to 1.0-kb segment of DNA within the junction fragments. The degeneration of the cointegrate plasmids was monitored by examining the lactose plasmids from nonclumping derivatives of clumping transconjugants. These plasmids displayed either precise or imprecise excision of pRS01 sequences or had dramatically reduced copy numbers. Both alterations occurred by rec-independent mechanisms. Alterations of a transconjugant 's clumping phenotype also occurred by rec-independent inversion of a 4.3-kb KpnI-PvuII fragment within the pRS01 sequences of the cointegrate plasmid.

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

    PubMed

    Licht, Tine Rask; Laugesen, Dorthe; Jensen, Lars Bogø; Jacobsen, Bodil Lund

    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 OG1 was investigated in the model. The plasmid encodes resistance to tetracycline. Numbers of recipient, donor, and transconjugant bacteria were monitored by selective plating of fecal samples, and transconjugants were subsequently verified by PCR. After being ingested by the mini-pigs, the recipient strain persisted in the intestine at levels between 10(6) and 10(7) CFU per g of feces throughout the experiment. The donor strain, which carried different resistance markers but was otherwise chromosomally isogenic to the recipient strain, was given to the pigs 3 weeks after the recipient strain. The donor cells were initially present in high numbers (10(6) CFU per g) in feces, but they did not persist in the intestine at detectable levels. Immediately after introduction of the donor bacteria, transconjugant cells appeared and persisted in fecal samples at levels between 10(3) and 10(4) CFU per g until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted in the intestine.

  2. Non-invasive determination of conjugative transfer of plasmids bearing antibiotic-resistance genes in biofilm-bound bacteria: effects of substrate loading and antibiotic selection

    PubMed Central

    Ma, Hongyan; Bryers, James D.

    2012-01-01

    Biofilms cause much of all human microbial infections. Attempts to eradicate biofilm-based infections rely on disinfectants and antibiotics. Unfortunately, biofilm bacteria are significantly less responsive to antibiotic stressors than their planktonic counterparts. Sublethal doses of antibiotics can actually enhance biofilm formation. Here, we have developed a non-invasive microscopic image analyses to quantify plasmid conjugation within a developing biofilm. Corroborating destructive samples were analyzed by a cultivation-independent flow cytometry analysis and a selective plate count method to cultivate transconjugants. Increases in substrate loading altered biofilm 3-D architecture and subsequently affected the frequency of plasmid conjugation (decreases at least two times) in the absence of any antibiotic selective pressure. More importantly, donor populations in biofilms exposed to a sublethal dose of kanamycin exhibited enhanced transfer efficiency of plasmids containing the kanamycin resistance gene, up to tenfold. However, when stressed with a different antibiotic, imipenem, transfer of plasmids containing the kanR+ gene was not enhanced. These preliminary results suggest biofilm bacteria “sense” antibiotics to which they are resistant, which enhances the spread of that resistance. Confocal scanning microscopy coupled with our non-invasive image analysis was able to estimate plasmid conjugative transfer efficiency either averaged over the entire biofilm landscape or locally with individual biofilm clusters. PMID:22669634

  3. Interspecies Dissemination of a Mobilizable Plasmid Harboring blaIMP-19 and the Possibility of Horizontal Gene Transfer in a Single Patient

    PubMed Central

    Gomi, Ryota; Matsuda, Tomonari; Tanaka, Michio; Nagao, Miki; Takakura, Shunji; Uemoto, Shinji; Ichiyama, Satoshi

    2016-01-01

    Carbapenemase-producing Gram-negative bacilli have been a global concern over the past 2 decades because these organisms can cause severe infections with high mortality rates. Carbapenemase genes are often carried by mobile genetic elements, and resistance plasmids can be transferred through conjugation. We conducted whole-genome sequencing (WGS) to demonstrate that the same plasmid harboring a metallo-β-lactamase gene was detected in two different species isolated from a single patient. Metallo-β-lactamase-producing Achromobacter xylosoxidans (KUN4507), non-metallo-β-lactamase-producing Klebsiella pneumoniae (KUN4843), and metallo-β-lactamase-producing K. pneumoniae (KUN5033) were sequentially isolated from a single patient and then analyzed in this study. Antimicrobial susceptibility testing, molecular typing (pulsed-field gel electrophoresis and multilocus sequence typing), and conjugation analyses were performed by conventional methods. Phylogenetic and molecular clock analysis of K. pneumoniae isolates were performed with WGS, and the nucleotide sequences of plasmids detected from these isolates were determined using WGS. Conventional molecular typing revealed that KUN4843 and KUN5033 were identical, whereas the phylogenetic tree analysis revealed a slight difference. These two isolates were separated from the most recent common ancestor 0.74 years before they were isolated. The same resistance plasmid harboring blaIMP-19 was detected in metallo-β-lactamase-producing A. xylosoxidans and K. pneumoniae. Although this plasmid was not self-transferable, the conjugation of this plasmid from A. xylosoxidans to non-metallo-β-lactamase-producing K. pneumoniae was successfully performed. The susceptibility patterns for metallo-β-lactamase-producing K. pneumoniae and the transconjugant were similar. These findings supported the possibility of the horizontal transfer of plasmid-borne blaIMP-19 from A. xylosoxidans to K. pneumoniae in a single patient. PMID

  4. MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer.

    PubMed

    Godziszewska, Jolanta; Kulińska, Anna; Jagura-Burdzy, Grażyna

    2014-09-04

    The IncU conjugative transfer module represents highly efficient promiscuous system widespread among conjugative plasmids of different incompatibility groups. Despite its frequent occurrence the mechanisms of relaxosome formation/action are far from understood. Here we analyzed the putative transfer auxiliary protein MobC of the conjugative plasmid RA3 from the IncU incompatibility group. MobC is a protein of 176 amino acids encoded in the bicistronic operon mobC-nic adjacent to oriT. MobC is homologous to prokaryotic transcription factors of the ribbon-helix-helix (RHH) superfamily. Conserved LxxugxNlNQiaxxLn motif clusters MobC with the clade of conjugative transfer auxilliary proteins of MobP relaxases. MobC forms dimers in solution and autoregulates the expression of mobCp by binding to an imperfect palindromic sequence (OM) located between putative -35 and -10 motifs of the promoter. Medium-copy number test plasmid containing the oriT-mobCp region is mobilized with a high frequency by the RA3 conjugative system. The mutations introduced into OM that abolished MobC binding in vitro decreased 2-3 fold the frequency of mobilization of the test plasmids. The deletion of OM within the RA3 conjugative module had no effect on transfer if the mobC-nic operon was expressed from the heterologous promoter. If only nic was expressed from the heterologous promoter (no mobC) the conjugative transfer frequency of such plasmid was 1000-fold lower. The MobC is an auxiliary transfer protein of dual function. It autoregulates the expression of mobC-nic operon while its presence significantly stimulates transfer efficiency.

  5. Transfer Protein TraY of Plasmid R1 Stimulates TraI-Catalyzed oriT Cleavage In Vivo

    PubMed Central

    Karl, Wolfgang; Bamberger, Martina; Zechner, Ellen L.

    2001-01-01

    The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (oriT) in vivo was investigated. As expected, the cleavage reaction was not detected in Escherichia coli cells expressing tral and the integration host factor (IHF) in the absence of other transfer proteins. The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY. Thus, the TraY and TraM proteins could each enhance cleaving activity at oriT in the absence of the other. In contrast, no detectable intracellular cleaving activity was exhibited by TraI in an IHF mutant strain despite the additional presence of both TraM and TraY. An essential role for IHF in this reaction in vivo is, therefore, implied. Mobilization experiments employing recombinant R1 oriT constructions and a heterologous conjugative helper plasmid were used to investigate the independent contributions of TraY and TraM to the R1 relaxosome during bacterial conjugation. In accordance with earlier observations, traY was dispensable for mobilization in the presence of traM, but mobilization did not occur in the absence of both traM and traY. Interestingly, although the cleavage assays demonstrate that TraM and TraY independently promote strand scission in vivo, TraM remained essential for mobilization of the R1 origin even in the presence of TraY. These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally performs a second function that is essential for successful conjugative transmission of plasmid DNA. PMID:11208788

  6. Efficient in vivo gene transfer by intraperitoneal injection of plasmid DNA and calcium carbonate microflowers in mice.

    PubMed

    Fumoto, Shintaro; Nakajima, Sayuri; Mine, Toyoharu; Yoshikawa, Naoki; Kitahara, Takashi; Sasaki, Hitoshi; Miyamoto, Hirotaka; Nishida, Koyo

    2012-07-02

    Gene transfer to intraperitoneal organs is thought to be a promising approach to treat such conditions as peritoneal fibrosis and peritoneal dissemination of cancers. We previously discovered that simple instillation of naked plasmid DNA (pDNA) onto intraperitoneal organs such as the liver and stomach could effectively transfer foreign genes in mice. In this study, we developed a novel nonviral method to enhance transfection efficiency of naked pDNA to intraperitoneal organs using a calcium carbonate suspension containing pDNA. Using commercially available calcium carbonate, we successfully transfected pDNA to the stomach. Handling of commercially available calcium carbonate, however, was troublesome owing to rapid precipitation and caking. To obtain slowly settling particles of calcium carbonate, we tried to synthesize novel versions of such particles and succeeded in creating flower-shaped particles, named calcium carbonate microflowers. Sedimentation of calcium carbonate microflowers was sufficiently slow for in vivo experiments. Moreover, the transfection efficiency of the suspension of calcium carbonate microflowers to the stomach was more effective than that of commercially available calcium carbonate, especially at low concentrations. Intraperitoneal injection of the suspension of calcium carbonate microflowers containing pDNA greatly enhanced naked pDNA transfer to whole intraperitoneal organs in mice. Furthermore, lactate dehydrogenase activities in intraperitoneal fluid and plasma were not raised by the suspension of calcium carbonate microflowers.

  7. R-plasmid transfer in Salmonella spp. isolated from wastewater and sewage-contaminated surface waters.

    PubMed Central

    Alcaide, E; Garay, E

    1984-01-01

    A total of 865 Salmonella isolates from wastewaters and sewage-contaminated natural waters were tested for antimicrobial resistance by using NR10 medium and incubation at 43 degrees C. Of the strains, 12.7% were resistant to one or more of the compounds tested, and 30% transferred resistance to an Escherichia coli recipient. The highest minimal inhibitory concentrations were ca. 1,000 micrograms/ml. Transfer frequencies ranged from 10(-3) to 10(-7). PMID:6486785

  8. Horizontal Transfer of a Multi-Drug Resistance Plasmid between Coliform Bacteria of Human and Bovine Origin in a Farm Environment

    PubMed Central

    Oppegaard, Hanne; Steinum, Terje M.; Wasteson, Yngvild

    2001-01-01

    Multi-drug-resistant coliform bacteria were isolated from feces of cattle exposed to antimicrobial agents and humans associated with the animals. Isolates from both cattle and humans harbored an R plasmid of 65 kb (pTMS1) that may have been transferred between them due to selective antibiotic pressure in the farm environment. PMID:11472956

  9. Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors.

    PubMed

    Newby, D T; Gentry, T J; Pepper, I L

    2000-08-01

    A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.

  10. Comparison of 2,4-Dichlorophenoxyacetic Acid Degradation and Plasmid Transfer in Soil Resulting from Bioaugmentation with Two Different pJP4 Donors

    PubMed Central

    Newby, D. T.; Gentry, T. J.; Pepper, I. L.

    2000-01-01

    A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful. PMID:10919798

  11. Conjugative plasmid transfer in Xylella fastidiosa is dependent on tra and trb operon functions

    USDA-ARS?s Scientific Manuscript database

    The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer and recombination, leading to diversity between strains and the categorization of X. fastidiosa into multiple subspecies. Although natural transformation is shown to occur at high rates in X. fa...

  12. Plasmid-mediated transfer of CTX-M-55 extended-spectrum beta-lactamase among different strains of Salmonella and Shigella spp. in the Republic of Korea.

    PubMed

    Kim, Jin Seok; Kim, Soojin; Park, Jungsun; Shin, Eunkyung; Yun, Young-Sun; Lee, Deog-Yong; Kwak, Hyo-Sun; Seong, Won Keun; Chung, Gyung Tae; Kim, Junyoung

    2017-09-01

    We screened 10 CTX-M-55-producing Shigella and Salmonella isolates from a national surveillance in Korea. The blaCTX-M-55 was located on the IncI1 (n=5), IncA/C (n=4) and IncZ (n=1) plasmids, downstream of ISEcp1, IS26-ISEcp1 and ISEcp1-IS5 sequences, respectively. These results indicate that CTX-M-55 has disseminated to other bacteria by lateral plasmid transfer. Copyright © 2017. Published by Elsevier Inc.

  13. Plasmid transfer by conjugation as a possible route of horizontal gene transfer and recombination in Xylella fastidiosa

    USDA-ARS?s Scientific Manuscript database

    Horizontal gene transfer is an important component of evolution and adaptation of bacterial species. Xylella fastidiosa has the ability to incorporate exogenous DNA into its genome by homologous recombination at relatively high rates. This genetic recombination is believed to play a role in adaptati...

  14. The Transfer of Cognitive Speed of Processing Training to Older Adults' Driving Mobility Across 5 Years.

    PubMed

    Ross, Lesley A; Edwards, Jerri D; O'Connor, Melissa L; Ball, Karlene K; Wadley, Virginia G; Vance, David E

    2016-01-01

    Multilevel models assessed the effects of cognitive speed of processing training (SPT) on older adults' self-reported driving using intention-to-treat (ITT, randomization to training or control conditions) and dosage (treatment-received via number of training sessions) analyses across 5 years. Participants randomized to SPT (n = 598) were compared with those randomized to either the no-contact control (n = 598) or memory training, which served as an active control (n = 610). Driving mobility (frequency, exposure, and space) was assessed over time. No significant effects were found within the ITT analyses. However, number of SPT sessions did affect driving mobility outcomes. In the full sample (N = 1,806), higher SPT doses were associated with maintained driving frequency as compared with both control groups, but no effects were found for driving exposure or space. Subsample analyses (n = 315) revealed that persons at-risk for mobility declines (i.e., poor initial processing speed) who received additional booster SPT sessions reported greater maintenance of both driving frequency and exposure over time as compared with the no-contact and active control groups. These results and prior research indicate that cognitive SPT transfers to prolonged driving mobility among older adults. Future research should investigate the mechanisms behind transfer effects to real-world activities, such as driving. © The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. The Transfer of Cognitive Speed of Processing Training to Older Adults’ Driving Mobility Across 5 Years

    PubMed Central

    Edwards, Jerri D.; O’Connor, Melissa L.; Ball, Karlene K.; Wadley, Virginia G.; Vance, David E.

    2016-01-01

    Objectives. Multilevel models assessed the effects of cognitive speed of processing training (SPT) on older adults’ self-reported driving using intention-to-treat (ITT, randomization to training or control conditions) and dosage (treatment-received via number of training sessions) analyses across 5 years. Method. Participants randomized to SPT (n = 598) were compared with those randomized to either the no-contact control (n = 598) or memory training, which served as an active control (n = 610). Driving mobility (frequency, exposure, and space) was assessed over time. Results. No significant effects were found within the ITT analyses. However, number of SPT sessions did affect driving mobility outcomes. In the full sample (N = 1,806), higher SPT doses were associated with maintained driving frequency as compared with both control groups, but no effects were found for driving exposure or space. Subsample analyses (n = 315) revealed that persons at-risk for mobility declines (i.e., poor initial processing speed) who received additional booster SPT sessions reported greater maintenance of both driving frequency and exposure over time as compared with the no-contact and active control groups. Discussion. These results and prior research indicate that cognitive SPT transfers to prolonged driving mobility among older adults. Future research should investigate the mechanisms behind transfer effects to real-world activities, such as driving. PMID:25878053

  16. Plasmid partitioning systems of conjugative plasmids from Clostridium perfringens.

    PubMed

    Adams, Vicki; Watts, Thomas D; Bulach, Dieter M; Lyras, Dena; Rood, Julian I

    2015-07-01

    Many pathogenic strains of Clostridium perfringens carry several highly similar toxin or antibiotic resistance plasmids that have 35 to 40 kb of very closely related syntenous sequences, including regions that carry the genes encoding conjugative transfer, plasmid replication and plasmid maintenance functions. Key questions are how are these closely related plasmids stably maintained in the same cell and what is the basis for plasmid incompatibility in C. perfringens. Comparative analysis of the Rep proteins encoded by these plasmids suggested that this protein was not the basis for plasmid incompatibility since plasmids carried in a single strain often encoded an almost identical Rep protein. These plasmids all carried a similar, but not identical, parMRC plasmid partitioning locus. Phylogenetic analysis of the deduced ParM proteins revealed that these proteins could be divided into ten separate groups. Importantly, in every strain that carried more than one of these plasmids, the respective ParM proteins were from different phylogenetic groups. Similar observations were made from the analysis of phylogenetic trees of the ParR proteins and the parC loci. These findings provide evidence that the basis for plasmid incompatibility in the conjugative toxin and resistance plasmid family from C. perfringens resides in subtle differences in the parMRC plasmid partitioning loci carried by these plasmids.

  17. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids.

    PubMed

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  18. Degradative plasmids from sphingomonads.

    PubMed

    Stolz, Andreas

    2014-01-01

    Large plasmids ('megaplasmids') are commonly found in members of the Alphaproteobacterial family Sphingomonadaceae ('sphingomonads'). These plasmids contribute to the extraordinary catabolic flexibility of this group of organisms, which degrade a broad range of recalcitrant xenobiotic compounds. The genomes of several sphingomonads have been sequenced during the last years. In the course of these studies, also the sequences of several plasmids have been determined. The analysis of the published information and the sequences deposited in the public databases allowed a first classification of these plasmids into a restricted number of groups according to the proteins involved in the initiation of replication, plasmid partition and conjugation. The sequence comparisons demonstrated that the plasmids from sphingomonads encode for four main groups of replication initiation (Rep) proteins. These Rep proteins belong to the protein superfamilies RepA_C (Pfam 04796), Rep_3 (Pfam 01051), RPA (Pfam 10134) and HTH-36 (Pfam 13730). The 'degradative megaplasmids' pNL2, pCAR3, pSWIT02, pCHQ1, pISP0, and pISP1, which code for genes involved in the degradation of aromatic hydrocarbons, carbazole, dibenzo-p-dioxin and γ-hexachlorocyclohexane, carry Rep proteins which either belong to the RepA_C- (plasmids pNL2, pCAR3, pSWIT02), Rep-3- (plasmids pCHQ1, pISP0) or RPA-superfamily (pISP1). The classification of these 'degradative megaplasmids' into three groups is also supported by sequence comparisons of the proteins involved in plasmid partition (ParAB) and the organization of the three genes on the respective plasmids. All analysed 'degradative megaplasmids' carry genes, which might allow a conjugative transfer of the plasmids. Sequence comparisons of these genes suggest the presence of at least two types of transfer functions, which either are closer related to the tra- or vir-genes previously described for plasmids from other sources.

  19. The qacC Gene Has Recently Spread between Rolling Circle Plasmids of Staphylococcus, Indicative of a Novel Gene Transfer Mechanism

    PubMed Central

    Wassenaar, Trudy M.; Ussery, David W.; Ingmer, Hanne

    2016-01-01

    Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfectants and biocides, can be attributed to qac genes. Most qac gene products belong to the Small Multidrug Resistant (SMR) protein family, and are often encoded by rolling-circle (RC) replicating plasmids. Four classes of SMR-type qac gene families have been described in Staphylococcus species: qacC, qacG, qacJ, and qacH. Within their class, these genes are highly conserved, but qacC genes are extremely conserved, although they are found in variable plasmid backgrounds. The lower degree of sequence identity of these plasmids compared to the strict nucleotide conservation of their qacC means that this gene has recently spread. In the absence of insertion sequences or other genetic elements explaining the mobility, we sought for an explanation of mobilization by sequence comparison. Publically available sequences of qac genes, their flanking genes and the replication gene that is invariably present in RC-plasmids were compared to reconstruct the evolutionary history of these plasmids and to explain the recent spread of qacC. Here we propose a new model that explains how qacC is mobilized and transferred to acceptor RC-plasmids without assistance of other genes, by means of its location in between the Double Strand replication Origin (DSO) and the Single-Strand replication Origin (SSO). The proposed mobilization model of this DSO-qacC-SSO element represents a novel mechanism of gene mobilization in RC-plasmids, which has also been employed by other genes, such as lnuA (conferring lincomycin resistance). The proposed gene mobility has aided to the wide spread of clinically relevant resistance genes in Staphylococcus populations. PMID:27729906

  20. Sequence-based analysis of pQBR103; a representative of a unique, transfer-proficient mega plasmid resident in the microbial community of sugar beet.

    PubMed

    Tett, Adrian; Spiers, Andrew J; Crossman, Lisa C; Ager, Duane; Ciric, Lena; Dow, J Maxwell; Fry, John C; Harris, David; Lilley, Andrew; Oliver, Anna; Parkhill, Julian; Quail, Michael A; Rainey, Paul B; Saunders, Nigel J; Seeger, Kathy; Snyder, Lori A S; Squares, Rob; Thomas, Christopher M; Turner, Sarah L; Zhang, Xue-Xian; Field, Dawn; Bailey, Mark J

    2007-08-01

    The plasmid pQBR103 was found within Pseudomonas populations colonizing the leaf and root surfaces of sugar beet plants growing at Wytham, Oxfordshire, UK. At 425 kb it is the largest self-transmissible plasmid yet sequenced from the phytosphere. It is known to enhance the competitive fitness of its host, and parts of the plasmid are known to be actively transcribed in the plant environment. Analysis of the complete sequence of this plasmid predicts a coding sequence (CDS)-rich genome containing 478 CDSs and an exceptional degree of genetic novelty; 80% of predicted coding sequences cannot be ascribed a function and 60% are orphans. Of those to which function could be assigned, 40% bore greatest similarity to sequences from Pseudomonas spp, and the majority of the remainder showed similarity to other gamma-proteobacterial genera and plasmids. pQBR103 has identifiable regions presumed responsible for replication and partitioning, but despite being tra+ lacks the full complement of any previously described conjugal transfer functions. The DNA sequence provided few insights into the functional significance of plant-induced transcriptional regions, but suggests that 14% of CDSs may be expressed (11 CDSs with functional annotation and 54 without), further highlighting the ecological importance of these novel CDSs. Comparative analysis indicates that pQBR103 shares significant regions of sequence with other plasmids isolated from sugar beet plants grown at the same geographic location. These plasmid sequences indicate there is more novelty in the mobile DNA pool accessible to phytosphere pseudomonas than is currently appreciated or understood.

  1. The qacC Gene Has Recently Spread between Rolling Circle Plasmids of Staphylococcus, Indicative of a Novel Gene Transfer Mechanism.

    PubMed

    Wassenaar, Trudy M; Ussery, David W; Ingmer, Hanne

    2016-01-01

    Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfectants and biocides, can be attributed to qac genes. Most qac gene products belong to the Small Multidrug Resistant (SMR) protein family, and are often encoded by rolling-circle (RC) replicating plasmids. Four classes of SMR-type qac gene families have been described in Staphylococcus species: qacC, qacG, qacJ, and qacH. Within their class, these genes are highly conserved, but qacC genes are extremely conserved, although they are found in variable plasmid backgrounds. The lower degree of sequence identity of these plasmids compared to the strict nucleotide conservation of their qacC means that this gene has recently spread. In the absence of insertion sequences or other genetic elements explaining the mobility, we sought for an explanation of mobilization by sequence comparison. Publically available sequences of qac genes, their flanking genes and the replication gene that is invariably present in RC-plasmids were compared to reconstruct the evolutionary history of these plasmids and to explain the recent spread of qacC. Here we propose a new model that explains how qacC is mobilized and transferred to acceptor RC-plasmids without assistance of other genes, by means of its location in between the Double Strand replication Origin (DSO) and the Single-Strand replication Origin (SSO). The proposed mobilization model of this DSO-qacC-SSO element represents a novel mechanism of gene mobilization in RC-plasmids, which has also been employed by other genes, such as lnuA (conferring lincomycin resistance). The proposed gene mobility has aided to the wide spread of clinically relevant resistance genes in Staphylococcus populations.

  2. Iteron Plasmids.

    PubMed

    Konieczny, Igor; Bury, Katarzyna; Wawrzycka, Aleksandra; Wegrzyn, Katarzyna

    2014-12-01

    Iteron-containing plasmids are model systems for studying the metabolism of extrachromosomal genetic elements in bacterial cells. Here we describe the current knowledge and understanding of the structure of iteron-containing replicons, the structure of the iteron plasmid encoded replication initiation proteins, and the molecular mechanisms for iteron plasmid DNA replication initiation. We also discuss the current understanding of control mechanisms affecting the plasmid copy number and how host chaperone proteins and proteases can affect plasmid maintenance in bacterial cells.

  3. Design of a 7kW power transfer solar array drive mechanism

    NASA Technical Reports Server (NTRS)

    Sheppard, J. G.

    1982-01-01

    With the availability of the Shuttle and the European launcher, Ariane, there will be a continuing trend towards large payload satellite missions requiring high-power, high-inertia, flexible solar arrays. The need arises for a solar array drive with a large power transfer capability which can rotate these solar arrays without disturbing the satellite body pointing. The modular design of such a Solar Array Drive Mechanism (SADM) which is capable of transferring 7kW of power or more is described. Total design flexibility has been achieved, enabling different spacecraft power requirements to be accommodated within the SADM design.

  4. Methanosarcina spp. Drive Vinyl Chloride Dechlorination via Interspecies Hydrogen Transfer

    PubMed Central

    Heimann, Axel C.; Batstone, Damien J.; Jakobsen, Rasmus

    2006-01-01

    Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-14C]acetate to 14CO2 when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H2) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H2 levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H2 levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H2 as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO2 plus H2, driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides. PMID:16598001

  5. Methanosarcina spp. drive vinyl chloride dechlorination via interspecies hydrogen transfer.

    PubMed

    Heimann, Axel C; Batstone, Damien J; Jakobsen, Rasmus

    2006-04-01

    Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-(14)C]acetate to (14)CO(2) when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H(2)) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H(2) levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H(2) levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H(2) as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO(2) plus H(2), driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.

  6. Phenotypic plasticity in bacterial plasmids.

    PubMed Central

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  7. Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

    PubMed Central

    Kishida, Kouhei; Inoue, Kei; Ohtsubo, Yoshiyuki; Nagata, Yuji

    2016-01-01

    ABSTRACT NAH7 and pWW0 from gammaproteobacterial Pseudomonas putida strains are IncP-9 conjugative plasmids that carry the genes for degradation of naphthalene and toluene, respectively. Although such genes on these plasmids are well-characterized, experimental investigation of their conjugation systems remains at a primitive level. To clarify these conjugation systems, in this study, we investigated the NAH7-encoded conjugation system by (i) analyzing the origin of its conjugative transfer (oriT)-containing region and its relaxase, which specifically nicks within the oriT region for initiation of transfer, and (ii) comparing the conjugation systems between NAH7 and pWW0. The NAH7 oriT (oriTN) region was located within a 430-bp fragment, and the strand-specific nicking (nic) site and its upstream sequences that were important for efficient conjugation in the oriTN region were identified. Unlike many other relaxases, the NAH7 relaxase exhibited unique features in its ability to catalyze, in a conjugation-independent manner, the site-specific intramolecular recombination between two copies of the oriTN region, between two copies of the pWW0 oriT (oriTW) region (which is clearly different from the oriTN region), and between the oriTN and oriTW regions. The pWW0 relaxase, which is also clearly different from the NAH7 relaxase, was strongly suggested to have the ability to conjugatively and efficiently mobilize the oriTN-containing plasmid. Such a plasmid was, in the presence of the NAH7Δnic derivative, conjugatively transferable to alphaproteobacterial and betaproteobacterial strains in which the NAH7 replication machinery is nonfunctional, indicating that the NAH7 conjugation system has a broader host range than its replication system. IMPORTANCE Various studies have strongly suggested an important contribution of conjugative transfer of catabolic plasmids to the rapid and wide dissemination of the plasmid-loaded degradation genes to microbial populations

  8. Plasmid Partition Mechanisms.

    PubMed

    Baxter, Jamie C; Funnell, Barbara E

    2014-12-01

    The stable maintenance of low-copy-number plasmids in bacteria is actively driven by partition mechanisms that are responsible for the positioning of plasmids inside the cell. Partition systems are ubiquitous in the microbial world and are encoded by many bacterial chromosomes as well as plasmids. These systems, although different in sequence and mechanism, typically consist of two proteins and a DNA partition site, or prokaryotic centromere, on the plasmid or chromosome. One protein binds site-specifically to the centromere to form a partition complex, and the other protein uses the energy of nucleotide binding and hydrolysis to transport the plasmid, via interactions with this partition complex inside the cell. For plasmids, this minimal cassette is sufficient to direct proper segregation in bacterial cells. There has been significant progress in the last several years in our understanding of partition mechanisms. Two general areas that have developed are (i) the structural biology of partition proteins and their interactions with DNA and (ii) the action and dynamics of the partition ATPases that drive the process. In addition, systems that use tubulin-like GTPases to partition plasmids have recently been identified. In this chapter, we concentrate on these recent developments and the molecular details of plasmid partition mechanisms.

  9. CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

    PubMed Central

    Price, Valerie J.; Huo, Wenwen; Sharifi, Ardalan

    2016-01-01

    ABSTRACT Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E

  10. In vivo Acquisition of Carbapenemase Gene blaKPC-2 in Multiple Species of Enterobacteriaceae through Horizontal Transfer of Insertion Sequence or Plasmid.

    PubMed

    Ding, Baixing; Shen, Zhen; Hu, Fupin; Ye, Meiping; Xu, Xiaogang; Guo, Qinglan; Wang, Minggui

    2016-01-01

    Objectives: Current worldwide spread of carbapenem resistance in Enterobacteriaceae constitutes a critical public health threat. This study aims to investigate how carbapenem resistance is acquired in Enterobacteriaceae in patients during antimicrobial therapy. Methods: Clinical strains from the same anatomical site of the same patients that converted from carbapenem-susceptible to resistant during antimicrobial therapy and showed identical or similar PFGE patterns were identified. The similarly sized plasmids carried by the susceptible and resistant strains, the latter containing the carbapenemase genes, were sequenced and analyzed. Results: Paired strains were identified from four patients: three had neurosurgical conditions while the other had acute exacerbation of COPD. Two pairs of Klebsiella pneumoniae (KP1-S/R and KP2-S/R, S and R indicating susceptible and resistant strains, respectively), one pair of Morganella morganii (MM-S/R) and one pair of Enterobacter aerogenes (EA-S/R) were collected. All four carbapenem-resistant strains carried plasmids harboring blaKPC-2. Compared with the similarly sized plasmids in KP1-S and KP2-S, an insertion sequence that includes ISKpn6-like, blaKPC-2 and ISKpn8 was noted in pKP1-R and pKP2-R. Strains MM-R and EA-R had blaKPC-2-carrying plasmids not resembling plasmids in strains MM-S and EA-S suggesting their new acquisition while on therapy. Conclusions: Enterobacteriaceae can acquire carbapenem resistance during antimicrobial therapy through horizontal transfer of an insertion sequence or plasmid.

  11. In vivo Acquisition of Carbapenemase Gene blaKPC-2 in Multiple Species of Enterobacteriaceae through Horizontal Transfer of Insertion Sequence or Plasmid

    PubMed Central

    Ding, Baixing; Shen, Zhen; Hu, Fupin; Ye, Meiping; Xu, Xiaogang; Guo, Qinglan; Wang, Minggui

    2016-01-01

    Objectives: Current worldwide spread of carbapenem resistance in Enterobacteriaceae constitutes a critical public health threat. This study aims to investigate how carbapenem resistance is acquired in Enterobacteriaceae in patients during antimicrobial therapy. Methods: Clinical strains from the same anatomical site of the same patients that converted from carbapenem-susceptible to resistant during antimicrobial therapy and showed identical or similar PFGE patterns were identified. The similarly sized plasmids carried by the susceptible and resistant strains, the latter containing the carbapenemase genes, were sequenced and analyzed. Results: Paired strains were identified from four patients: three had neurosurgical conditions while the other had acute exacerbation of COPD. Two pairs of Klebsiella pneumoniae (KP1-S/R and KP2-S/R, S and R indicating susceptible and resistant strains, respectively), one pair of Morganella morganii (MM-S/R) and one pair of Enterobacter aerogenes (EA-S/R) were collected. All four carbapenem-resistant strains carried plasmids harboring blaKPC−2. Compared with the similarly sized plasmids in KP1-S and KP2-S, an insertion sequence that includes ISKpn6-like, blaKPC−2 and ISKpn8 was noted in pKP1-R and pKP2-R. Strains MM-R and EA-R had blaKPC−2-carrying plasmids not resembling plasmids in strains MM-S and EA-S suggesting their new acquisition while on therapy. Conclusions: Enterobacteriaceae can acquire carbapenem resistance during antimicrobial therapy through horizontal transfer of an insertion sequence or plasmid. PMID:27818649

  12. Identification and Characterization of the Conjugal Transfer Region of the pCg1 plasmid from Naphthalene-Degrading Pseudomonas putida Cg1

    PubMed Central

    Park, Woojun; Jeon, Che Ok; Hohnstock-Ashe, Amy M.; Winans, Stephen C.; Zylstra, Gerben J.; Madsen, Eugene L.

    2003-01-01

    Hybridization and restriction fragment length polymorphism data (K. G. Stuart-Keil, A. M. Hohnstock, K. P. Drees, J. B. Herrick, and E. L. Madsen, Appl. Environ. Microbiol. 64:3633-3640, 1998) have shown that pCg1, a naphthalene catabolic plasmid carried by Pseudomonas putida Cg1, is homologous to the archetypal naphthalene catabolic plasmid, pDTG1, in P. putida NCIB 9816-4. Sequencing of the latter plasmid allowed PCR primers to be designed for amplifying and sequencing the conjugal transfer region in pCg1. The mating pair formation (mpf) gene, mpfA encoding the putative precursor of the conjugative pilin subunit from pCg1, was identified along with other trb-like mpf genes. Sequence comparison revealed that the 10 mpf genes in pCg1 and pDTG1 are closely related (61 to 84% identity) in sequence and operon structure to the putative mpf genes of catabolic plasmid pWW0 (TOL plasmid of P. putida) and pM3 (antibiotic resistance plasmid of Pseudomonas. spp). A polar mutation caused by insertional inactivation in mpfA of pCg1 and reverse transcriptase PCR analysis of mRNA showed that this mpf region was involved in conjugation and was transcribed from a promoter located upstream of an open reading frame adjacent to mpfA. lacZ transcriptional fusions revealed that mpf genes of pCg1 were expressed constitutively both in liquid and on solid media. This expression did not respond to host exposure to naphthalene. Conjugation frequency on semisolid media was consistently 10- to 100-fold higher than that in liquid media. Thus, conjugation of pCg1 in P. putida Cg1 was enhanced by expression of genes in the mpf region and by surfaces where conditions fostering stable, high-density cell-to-cell contact are manifest. PMID:12788725

  13. In vivo transfer of plasmid-encoded ACC-1 AmpC from Klebsiella pneumoniae to Escherichia coli in an infant and selection of impermeability to imipenem in K. pneumoniae.

    PubMed

    Bidet, Philippe; Burghoffer, Béatrice; Gautier, Valérie; Brahimi, Naïma; Mariani-Kurkdjian, Patricia; El-Ghoneimi, Alaa; Bingen, Edouard; Arlet, Guillaume

    2005-08-01

    We describe in vivo selection of a Klebsiella pneumoniae strain with diminished imipenem susceptibility attributable to plasmid-encoded ACC-1 beta-lactamase production and loss of a 36-kDa major outer membrane protein, together with transfer of this plasmid from K. pneumoniae to Escherichia coli in a Tunisian infant.

  14. Electronic resonance with anticorrelated pigment vibrations drives photosynthetic energy transfer outside the adiabatic framework

    PubMed Central

    Tiwari, Vivek; Peters, William K.; Jonas, David M.

    2013-01-01

    The delocalized, anticorrelated component of pigment vibrations can drive nonadiabatic electronic energy transfer in photosynthetic light-harvesting antennas. In femtosecond experiments, this energy transfer mechanism leads to excitation of delocalized, anticorrelated vibrational wavepackets on the ground electronic state that exhibit not only 2D spectroscopic signatures attributed to electronic coherence and oscillatory quantum energy transport but also a cross-peak asymmetry not previously explained by theory. A number of antennas have electronic energy gaps matching a pigment vibrational frequency with a small vibrational coordinate change on electronic excitation. Such photosynthetic energy transfer steps resemble molecular internal conversion through a nested intermolecular funnel. PMID:23267114

  15. The master regulator of IncA/C plasmids is recognized by the Salmonella Genomic island SGI1 as a signal for excision and conjugal transfer.

    PubMed

    Kiss, János; Papp, Péter Pál; Szabó, Mónika; Farkas, Tibor; Murányi, Gábor; Szakállas, Erik; Olasz, Ferenc

    2015-10-15

    The genomic island SGI1 and its variants, the important vehicles of multi-resistance in Salmonella strains, are integrative elements mobilized exclusively by the conjugative IncA/C plasmids. Integration and excision of the island are carried out by the SGI1-encoded site-specific recombinase Int and the recombination directionality factor Xis. Chromosomal integration ensures the stable maintenance and vertical transmission of SGI1, while excision is the initial step of horizontal transfer, followed by conjugation and integration into the recipient. We report here that SGI1 not only exploits the conjugal apparatus of the IncA/C plasmids but also utilizes the regulatory mechanisms of the conjugation system for the exact timing and activation of excision to ensure efficient horizontal transfer. This study demonstrates that the FlhDC-family activator AcaCD, which regulates the conjugation machinery of the IncA/C plasmids, serves as a signal of helper entry through binding to SGI1 xis promoter and activating SGI1 excision. Promoters of int and xis genes have been identified and the binding site of the activator has been located by footprinting and deletion analyses. We prove that expression of xis is activator-dependent while int is constitutively expressed, and this regulatory mechanism is presumably responsible for the efficient transfer and stable maintenance of SGI1.

  16. The master regulator of IncA/C plasmids is recognized by the Salmonella Genomic island SGI1 as a signal for excision and conjugal transfer

    PubMed Central

    Kiss, János; Papp, Péter Pál; Szabó, Mónika; Farkas, Tibor; Murányi, Gábor; Szakállas, Erik; Olasz, Ferenc

    2015-01-01

    The genomic island SGI1 and its variants, the important vehicles of multi-resistance in Salmonella strains, are integrative elements mobilized exclusively by the conjugative IncA/C plasmids. Integration and excision of the island are carried out by the SGI1-encoded site-specific recombinase Int and the recombination directionality factor Xis. Chromosomal integration ensures the stable maintenance and vertical transmission of SGI1, while excision is the initial step of horizontal transfer, followed by conjugation and integration into the recipient. We report here that SGI1 not only exploits the conjugal apparatus of the IncA/C plasmids but also utilizes the regulatory mechanisms of the conjugation system for the exact timing and activation of excision to ensure efficient horizontal transfer. This study demonstrates that the FlhDC-family activator AcaCD, which regulates the conjugation machinery of the IncA/C plasmids, serves as a signal of helper entry through binding to SGI1 xis promoter and activating SGI1 excision. Promoters of int and xis genes have been identified and the binding site of the activator has been located by footprinting and deletion analyses. We prove that expression of xis is activator-dependent while int is constitutively expressed, and this regulatory mechanism is presumably responsible for the efficient transfer and stable maintenance of SGI1. PMID:26209134

  17. Transfer of the symbiotic plasmid of Rhizobium etli CFN42 requires cointegration with p42a, which may be mediated by site-specific recombination.

    PubMed

    Brom, Susana; Girard, Lourdes; Tun-Garrido, Cristina; García-de los Santos, Alejandro; Bustos, Patricia; González, Víctor; Romero, David

    2004-11-01

    Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.

  18. Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42 Requires Cointegration with p42a, Which May Be Mediated by Site-Specific Recombination

    PubMed Central

    Brom, Susana; Girard, Lourdes; Tun-Garrido, Cristina; García-de los Santos, Alejandro; Bustos, Patricia; González, Víctor; Romero, David

    2004-01-01

    Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer. PMID:15516565

  19. Conjugative Transfer of Insecticidal Plasmid pHT73 from Bacillus thuringiensis to B. anthracis and Compatibility of This Plasmid with pXO1 and pXO2 ▿

    PubMed Central

    Yuan, Yongming; Zheng, Dasheng; Hu, Xiaomin; Cai, Quanxin; Yuan, Zhiming

    2010-01-01

    Bacillus anthracis, the etiologic agent of anthrax, is genetically close to and commonly shares a giant gene pool with B. cereus and B. thuringiensis. In view of the human pathogenicity and the long persistence in the environment of B. anthracis, there is growing concern about the effects of genetic exchange with B. anthracis on public health. In this work, we demonstrate that an insecticidal plasmid, pHT73, from B. thuringiensis strain KT0 could be efficiently transferred into two attenuated B. anthracis strains, Ba63002R (pXO1+ pXO2−) and Ba63605R (pXO1− pXO2+), by conjugation in liquid medium in the laboratory, with transfer rates of 2.3 × 10−4 and 1.6 × 10−4 CFU/donor, respectively. The B. anthracis transconjugants containing both pHT73 and pXO1 or pXO2 could produce crystal protein Cry1Ac encoded by plasmid pHT73 and had high toxicity to Helicoverpa armigera larvae. Furthermore, the compatibility and stability of pHT73 with pXO1/pXO2 were demonstrated. The data are informative for further investigation of the safety of B. thuringiensis and closely related strains in food and in the environment. PMID:19948871

  20. Parallel compensatory evolution stabilizes plasmids across the parasitism-mutualism continuum.

    PubMed

    Harrison, Ellie; Guymer, David; Spiers, Andrew J; Paterson, Steve; Brockhurst, Michael A

    2015-08-03

    Plasmids drive genomic diversity in bacteria via horizontal gene transfer [1, 2]; nevertheless, explaining their survival in bacterial populations is challenging [3]. Theory predicts that irrespective of their net fitness effects, plasmids should be lost: when parasitic (costs outweigh benefits), plasmids should decline due to purifying selection [4-6], yet under mutualism (benefits outweigh costs), selection favors the capture of beneficial accessory genes by the chromosome and loss of the costly plasmid backbone [4]. While compensatory evolution can enhance plasmid stability within populations [7-15], the propensity for this to occur across the parasitism-mutualism continuum is unknown. We experimentally evolved Pseudomonas fluorescens and its mercury resistance mega-plasmid, pQBR103 [16], across an environment-mediated parasitism-mutualism continuum. Compensatory evolution stabilized plasmids by rapidly ameliorating the cost of plasmid carriage in all environments. Genomic analysis revealed that, in both parasitic and mutualistic treatments, evolution repeatedly targeted the gacA/gacS bacterial two-component global regulatory system while leaving the plasmid sequence intact. Deletion of either gacA or gacS was sufficient to completely ameliorate the cost of plasmid carriage. Mutation of gacA/gacS downregulated the expression of ∼17% of chromosomal and plasmid genes and appears to have relieved the translational demand imposed by the plasmid. Chromosomal capture of mercury resistance accompanied by plasmid loss occurred throughout the experiment but very rarely invaded to high frequency, suggesting that rapid compensatory evolution can limit this process. Compensatory evolution can explain the widespread occurrence of plasmids and allows bacteria to retain horizontally acquired plasmids even in environments where their accessory genes are not immediately useful.

  1. Plasmid Detection, Characterization, and Ecology.

    PubMed

    Smalla, Kornelia; Jechalke, Sven; Top, Eva M

    2015-02-01

    Plasmids are important vehicles for rapid adaptation of bacterial populations to changing environmental conditions. It is thought that to reduce the cost of plasmid carriage, only a fraction of a local population carries plasmids or is permissive to plasmid uptake. Plasmids provide various accessory traits which might be beneficial under particular conditions. The genetic variation generated by plasmid carriage within populations ensures the robustness toward environmental changes. Plasmid-mediated gene transfer plays an important role not only in the mobilization and dissemination of antibiotic resistance genes but also in the spread of degradative pathways and pathogenicity determinants of pathogens. Here we summarize the state-of-the-art methods to study the occurrence, abundance, and diversity of plasmids in environmental bacteria. Increasingly, cultivation-independent total-community DNA-based methods are being used to characterize and quantify the diversity and abundance of plasmids in relation to various biotic and abiotic factors. An improved understanding of the ecology of plasmids and their hosts is crucial in the development of intervention strategies for antibiotic-resistance-gene spread. We discuss the potentials and limitations of methods used to determine the host range of plasmids, as the ecology of plasmids is tightly linked to their hosts. The recent advances in sequencing technologies provide an enormous potential for plasmid classification, diversity, and evolution studies, but numerous challenges still exist.

  2. Co-resident plasmids travel together.

    PubMed

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation.

    PubMed

    Genco, C A; Clark, V L

    1988-12-01

    We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.

  4. Characterization of IntA, a Bidirectional Site-Specific Recombinase Required for Conjugative Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42

    PubMed Central

    Hernández-Tamayo, Rogelio; Sohlenkamp, Christian; Puente, José Luis; Brom, Susana

    2013-01-01

    Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination. PMID:23935046

  5. Characterization of IntA, a bidirectional site-specific recombinase required for conjugative transfer of the symbiotic plasmid of Rhizobium etli CFN42.

    PubMed

    Hernández-Tamayo, Rogelio; Sohlenkamp, Christian; Puente, José Luis; Brom, Susana; Romero, David

    2013-10-01

    Site-specific recombination occurs at short specific sequences, mediated by the cognate recombinases. IntA is a recombinase from Rhizobium etli CFN42 and belongs to the tyrosine recombinase family. It allows cointegration of plasmid p42a and the symbiotic plasmid via site-specific recombination between attachment regions (attA and attD) located in each replicon. Cointegration is needed for conjugative transfer of the symbiotic plasmid. To characterize this system, two plasmids harboring the corresponding attachment sites and intA were constructed. Introduction of these plasmids into R. etli revealed IntA-dependent recombination events occurring at high frequency. Interestingly, IntA promotes not only integration, but also excision events, albeit at a lower frequency. Thus, R. etli IntA appears to be a bidirectional recombinase. IntA was purified and used to set up electrophoretic mobility shift assays with linear fragments containing attA and attD. IntA-dependent retarded complexes were observed only with fragments containing either attA or attD. Specific retarded complexes, as well as normal in vivo recombination abilities, were seen even in derivatives harboring only a minimal attachment region (comprising the 5-bp central region flanked by 9- to 11-bp inverted repeats). DNase I-footprinting assays with IntA revealed specific protection of these zones. Mutations that disrupt the integrity of the 9- to 11-bp inverted repeats abolish both specific binding and recombination ability, while mutations in the 5-bp central region severely reduce both binding and recombination. These results show that IntA is a bidirectional recombinase that binds to att regions without requiring neighboring sequences as enhancers of recombination.

  6. Mobility of plasmids.

    PubMed

    Smillie, Chris; Garcillán-Barcia, M Pilar; Francia, M Victoria; Rocha, Eduardo P C; de la Cruz, Fernando

    2010-09-01

    Plasmids are key vectors of horizontal gene transfer and essential genetic engineering tools. They code for genes involved in many aspects of microbial biology, including detoxication, virulence, ecological interactions, and antibiotic resistance. While many studies have decorticated the mechanisms of mobility in model plasmids, the identification and characterization of plasmid mobility from genome data are unexplored. By reviewing the available data and literature, we established a computational protocol to identify and classify conjugation and mobilization genetic modules in 1,730 plasmids. This allowed the accurate classification of proteobacterial conjugative or mobilizable systems in a combination of four mating pair formation and six relaxase families. The available evidence suggests that half of the plasmids are nonmobilizable and that half of the remaining plasmids are conjugative. Some conjugative systems are much more abundant than others and preferably associated with some clades or plasmid sizes. Most very large plasmids are nonmobilizable, with evidence of ongoing domestication into secondary chromosomes. The evolution of conjugation elements shows ancient divergence between mobility systems, with relaxases and type IV coupling proteins (T4CPs) often following separate paths from type IV secretion systems. Phylogenetic patterns of mobility proteins are consistent with the phylogeny of the host prokaryotes, suggesting that plasmid mobility is in general circumscribed within large clades. Our survey suggests the existence of unsuspected new relaxases in archaea and new conjugation systems in cyanobacteria and actinobacteria. Few genes, e.g., T4CPs, relaxases, and VirB4, are at the core of plasmid conjugation, and together with accessory genes, they have evolved into specific systems adapted to specific physiological and ecological contexts.

  7. Large IncHI2-plasmids encode extended-spectrum β-lactamases (ESBLs) in Enterobacter spp. bloodstream isolates, and support ESBL-transfer to Escherichia coli.

    PubMed

    Nilsen, E; Haldorsen, B C; Sundsfjord, A; Simonsen, G S; Ingebretsen, A; Naseer, U; Samuelsen, O

    2013-11-01

    We investigated the prevalence of extended-spectrum β-lactamases (ESBLs) in Enterobacter spp. bloodstream isolates from 19 hospital laboratories in Norway during 2011. A total of 62/230 (27%) isolates were resistant to third-generation cephalosporins and four (1.7%) were ESBL-positive; blaCTX -M-15 (n = 3) and blaSHV -12 (n = 1). This is comparable to the prevalence of ESBLs in clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway during the same period. All ESBL-positive isolates were multidrug resistant (MDR) and harboured plasmid-mediated quinolone resistance. Three isolates supported transfer of large IncHI2-plasmids harbouring ESBL- and MDR-encoding genes to E. coli recipients by in vitro conjugation. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

  8. [Characterization of the transfer-related tra region of the conjugative plasmid p19 from a Bacillus subtilis soil strain].

    PubMed

    Poluéktova, E U; Gagarina, E Iu; Nezametdinova, V Z; Shilovskiĭ, I P; Rodionova, S A; Prozorov, A A

    2010-01-01

    The nucleotide sequences of three DNA fragments (total size 30574 bp) of the plasmid p19 from the Bacillus subtilis 19 soil strain have been determined. Thirty open reading frames (ORFs) have been identified in these fragments. oriT of the plasmid has also been identified. As shown by the search for homologs of hypothetical protein products of these ORFs in databases, such homology exists for 18 ORFs. The protein products of nine ORFs can be assumed to have specific functions. Several ORFs were inactivated via insertional mutagenesis, and the conjugation capacity of the mutant plasmids was estimated. According to the data on homology of protein products and the results of ORF inactivation, regions of a total size of about 20 kb from the DNA fragments sequenced by us were inferred to belong to the tra region of p19. As follows from the analysis of the identified ORFs of the p19 tra region, it differs from the earlier described tra regions of other plasmids, irrespective of a certain similarity with the corresponding regions of plasmids of gram-positive bacteria from the genera Bacillus, Clostridium, and Listeria.

  9. PrgK, a Multidomain Peptidoglycan Hydrolase, Is Essential for Conjugative Transfer of the Pheromone-Responsive Plasmid pCF10

    PubMed Central

    Laverde Gomez, Jenny A.; Bhatty, Minny

    2014-01-01

    Peptidoglycan (PG) hydrolases associated with bacterial type IV secretion systems (T4SSs) are thought to generate localized lesions in the PG layer to facilitate assembly of the translocation channel. The pheromone-responsive plasmid pCF10 of Enterococcus faecalis encodes a putative cell wall hydrolase, PrgK, and here we report that a prgK deletion abolished functionality of the pCF10-encoded T4SS as monitored by pCF10 conjugative transfer. Expression in trans of wild-type prgK fully complemented this mutation. PrgK has three potential hydrolase motifs resembling staphylococcal LytM, soluble lytic transglycosylase (SLT), and cysteine-, histidine-dependent amidohydrolase/peptidase (CHAP) domains. Complementation analyses with mutant alleles established that PrgK bearing two hydrolase domains in any combination supported near-wild-type plasmid transfer, and PrgK bearing a single hydrolase domain supported at least a low level of transfer in filter matings. When exported to the Escherichia coli periplasm, each domain disrupted cell growth, and combinations of domains additionally induced cell rounding and blebbing and conferred enhanced sensitivity to osmotic shock. Each domain bound PG in vitro, but only the SLT domain exhibited detectable hydrolase activity, as shown by zymographic analyses and release of fluorescent PG fragments. Genes encoding three T4SS-associated, putative hydrolases, Lactococcus lactis CsiA, Tn925 Orf14, and pIP501 TraG, partially complemented the ΔprgK mutation. Our findings establish that PrgK is an essential component of the pCF10-encoded Prg/Pcf T4SS and that its hydrolase domains coordinate their activities for full PrgK function. PrgK is indispensable for plasmid transfer in liquid matings, suggestive of a role in formation or stabilization of mating junctions. PMID:24244005

  10. Genes encoding conserved hypothetical proteins localized in the conjugative transfer region of plasmid pRet42a from Rhizobium etli CFN42 participate in modulating transfer and affect conjugation from different donors

    PubMed Central

    López-Fuentes, Eunice; Torres-Tejerizo, Gonzalo; Cervantes, Laura; Brom, Susana

    2015-01-01

    Among sequenced genomes, it is common to find a high proportion of genes encoding proteins that cannot be assigned a known function. In bacterial genomes, genes related to a similar function are often located in contiguous regions. The presence of genes encoding conserved hypothetical proteins (chp) in such a region may suggest that they are related to that particular function. Plasmid pRet42a from Rhizobium etli CFN42 is a conjugative plasmid containing a segment of approximately 30 Kb encoding genes involved in conjugative transfer. In addition to genes responsible for Dtr (DNA transfer and replication), Mpf (Mating pair formation) and regulation, it has two chp-encoding genes (RHE_PA00163 and RHE_PA00164) and a transcriptional regulator (RHE_PA00165). RHE_PA00163 encodes an uncharacterized protein conserved in bacteria that presents a COG4634 conserved domain, and RHE_PA00164 encodes an uncharacterized conserved protein with a DUF433 domain of unknown function. RHE_PA00165 presents a HTH_XRE domain, characteristic of DNA-binding proteins belonging to the xenobiotic response element family of transcriptional regulators. Interestingly, genes similar to these are also present in transfer regions of plasmids from other bacteria. To determine if these genes participate in conjugative transfer, we mutagenized them and analyzed their conjugative phenotype. A mutant in RHE_PA00163 showed a slight (10 times) but reproducible increase in transfer frequency from Rhizobium donors, while mutants in RHE_PA00164 and RHE_PA00165 lost their ability to transfer the plasmid from some Agrobacterium donors. Our results indicate that the chp-encoding genes located among conjugation genes are indeed related to this function. However, the participation of RHE_PA00164 and RHE_PA00165 is only revealed under very specific circumstances, and is not perceived when the plasmid is transferred from the original host. RHE_PA00163 seems to be a fine-tuning modulator for conjugative transfer

  11. Escherichia coli DNA helicase I catalyzes a sequence-specific cleavage/ligation reaction at the F plasmid origin of transfer.

    PubMed

    Sherman, J A; Matson, S W

    1994-10-21

    Recent studies have shown that the Escherichia coli F plasmid-encoded traI gene product (TraIp), also known as DNA helicase I, catalyzes the formation of the site- and strand-specific nick that initiates F plasmid DNA transfer. Scission of the phosphodiester bond at the nic site within the origin of transfer (oriT) is accompanied by the covalent attachment of TraIp to the 5'-phosphate of the nicked DNA strand. This mechanism suggests that TraIp may also be capable of catalyzing a DNA ligation reaction using the energy stored in the protein-DNA intermediate. To test this possibility, an in vitro assay was designed that utilized short single-stranded DNA oligonucleotides of different lengths derived from the region within oriT that spanned the nic site. Purified TraIp was capable of efficiently cleaving single-stranded DNA that contained a nic site, and upon cleavage, the protein became covalently linked to the 5'-end of the nic site. When TraIp was incubated with two oligonucleotides of different length that contained the nic site, there was formation of novel recombinant products resulting from a TraIp-catalyzed cleavage/ligation reaction. Furthermore, the cleavage and ligation reactions were both sequence-specific. These data suggest that TraIp plays an important role in the initiation and termination of conjugative DNA transfer.

  12. Toxin Plasmids of Clostridium perfringens

    PubMed Central

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  13. Regulation of the transfer genes of Streptomyces plasmid pSN22: in vivo and in vitro study of the interaction of TraR with promoter regions.

    PubMed Central

    Kataoka, M; Kosono, S; Seki, T; Yoshida, T

    1994-01-01

    Expression of the tra operon, essential for conjugative transfer of the 11-kb Streptomyces nigrifaciens plasmid pSN22, is negatively regulated by traR, which is located upstream of the tra operon and transcribed in the opposite orientation. The transcriptional start points for the tra and traR mRNAs were determined by primer extension; they are 72 bp apart and have identical -10 promoter sequences. The TraR protein was overexpressed in Escherichia coli and used for gel retardation and DNase I protection experiments. It bound specifically to the bidirectional tra-traR promoter region and protected four DNA regions, each of which contains a similar 12-bp sequence. The binding was strongest to the region downstream of the tra promoter, probably ensuring that expression of the potentially lethal traB gene is turned off before traR. The efficiency of intramycelial plasmid transfer was decreased by the mutation at the downstream region. Images PMID:7961501

  14. Conjugative transfer of a derivative of the IncP-1α plasmid RP4 and establishment of transconjugants in the indigenous bacterial community of poplar plants

    PubMed Central

    Ulrich, Andreas; Becker, Regina; Ulrich, Kristina; Ewald, Dietrich

    2015-01-01

    The persistence of traits introduced into the indigenous bacterial community of poplar plants was investigated using bioluminescence mediated by the luc gene. Three endophytic bacterial strains provided with the IncP-1α plasmid RP4-Tn-luc were used to inoculate poplar cuttings at different phenological stages. Screening of isolates by bioluminescence and real-time PCR detection of the luc gene revealed stable persistence for at least 10 weeks. Although the inoculated strains became established with a high population density after inoculation at leaf development (April) and senescence (October), the strains were suppressed by the indigenous bacteria at stem elongation (June). Transconjugants could be detected only at this phenological stage. Indigenous bacteria harbouring RP4-Tn-luc became established with densities ranging from 2 × 105 to 9 × 106 CFU g−1 fresh weight 3 and 10 weeks after inoculation. The increased colonization of the cuttings by indigenous bacteria at stem elongation seemed to strongly compete with the introduced strains. Otherwise, the phenological stage of the plants as well as the density of the indigenous recipients could serve as the driver for a more frequent conjugative plasmid transfer. A phylogenetic assignment of transconjugants indicated the transfer of RP4-Tn-luc into six genera of Proteobacteria, mainly Sphingomonas, Stenotrophomonas and Xanthomonas. PMID:26490946

  15. Conjugal transfer of the Sinorhizobium meliloti 1021 symbiotic plasmid is governed through the concerted action of one- and two-component signal transduction regulators.

    PubMed

    Nogales, Joaquina; Blanca-Ordóñez, Helena; Olivares, José; Sanjuán, Juan

    2013-03-01

    Conjugal transfer of Sinorhizobium meliloti and Rhizobium etli symbiotic plasmids are repressed by the transcriptional regulator RctA. Here we report on new key players in the signal transduction cascade towards S. meliloti pSym conjugation. We have identified S. meliloti pSymA gene SMa0974 as an orthologue of the R. etli rctB gene which is required to antagonize repression by RctA. In S. meliloti two additional genes, rctR and rctC participate in control of rctB expression. rctR (SMa0955) encodes a protein of the GntR family of transcriptional regulators involved in repression of rctB. A rctR mutant promotes pSymA conjugal transfer and displays increased transcription of tra, virB and rctB genes even in presence of wild-type rctA gene. Among genes repressed by RctR, rctC (SMa0961) encodes a response regulator required to activate rctB transcription and therefore for derepression of plasmid conjugative functions. We conclude that in both R. etli and S. meliloti pSym conjugal transfer is derepressed via rctB, however the regulatory cascades to achieve activation of rctB are probably different. Upstream of rctB, the S. meliloti pSym conjugal transfer is regulated through the concerted action of genes representing one- (rctR) and two-component (rctC) signal transduction systems in response to yet unidentified signals. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  16. 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

    PubMed Central

    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.

    2015-01-01

    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 < 0.001) and increased movement of the 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. PMID:25769824

  17. 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.

    PubMed

    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 < 0.001) and increased movement of the 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.

  18. Production of Plasmid DNA as Pharmaceutical.

    PubMed

    Schmeer, Marco; Schleef, Martin

    2015-01-01

    Pharmaceutical applications of plasmid DNA require certain quality standards, depending on the intended use of the plasmids. That is, for direct gene transfer into human, GMP Grade is mandatory, however, for GMP production of for example viral vectors (AAV or mRNA etc.), the plasmid DNA used has not to be produced under GMP necessarily. Here we summarize important features of producing plasmid DNA, ensuring the required quality for the intended (pharmaceutical) application.

  19. Expression and plasmid transfer of genes coding for the fimbrial antigen F107 in porcine Escherichia coli strains.

    PubMed

    Wittig, W; Prager, R; Stamm, M; Streckel, W; Tschäpe, H

    1994-08-01

    Expression of the fimbrial antigen F107 of porcine E. coli strains on agar plates was achieved by microaerobic cultivation. For part of the strains of some types, addition of alizarin yellow and eosin to the agar medium proved to be necessary. Some of these strains reacted distinctly positive only when the small colonies growing between the larger ones on alizarin-yellow agar were tested. The fimbrial antigen of the Swiss strain 107/86 was provisionally designated the F107ab variant and that of the Hungarian strain 2134P and the Czech strain 8813, the F107ac variant. The F107 genetic determinants were found to be often linked with those encoding haemolysin production and are frequently carried by plasmids.

  20. Multiple plasmid interference - Pledging allegiance to my enemy's enemy.

    PubMed

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Transfer of herpes simplex virus thymidine kinase synthesized in bacteria by a high-expression plasmid to tissue culture cells by protoplast fusion

    SciTech Connect

    Waldman, A.S.; Milman, G.

    1984-08-01

    The introduction of a protein into living tissue culture cells may permit the in vivo study of functions of the protein. The authors have previously described a high-efficiency-expression plasmid, pHETK2, containing the herpes simplex virus type 1 thymidine kinase (TK) gene which, upon temperature induction, causes TK to be synthesized as greater than 4% of the bacterial protein. In this report it is shown that enzymatically active TK was transferred to mouse Ltk- cells by polyethylene glycol-mediated fusion with protoplasts prepared from bacteria containing induced levels of TK. The presence of TK in the Ltk- cells was detected by the incorporation of (/sup 3/H)thymidine into cell nuclei as measured by autoradiography.

  2. MESSENGER observations of dayside flux transfer events: Do they drive Mercury's substorm cycle? (Invited)

    NASA Astrophysics Data System (ADS)

    Imber, S. M.; Slavin, J. A.; Boardsen, S. A.; Anderson, B. J.; Korth, H.; Baker, D. N.; McNutt, R. L.; Solomon, S. C.

    2013-12-01

    The large-scale dynamics of Mercury's magnetosphere appear to be predominantly driven by magnetic reconnection, which promotes the transfer of energy and momentum from the solar wind to the magnetosphere. Reconnection thus also drives Mercury's substorm cycle, which circulates magnetic flux through the system. The rate of reconnection taking place at any given time can be determined by observations of reconnection-related signatures such as plasma jets, large magnetic field components normal to the magnetopause, and flux transfer events (FTEs). FTEs are flux ropes formed by reconnection at Mercury's magnetopause which transport magnetic flux from the dayside to the lobes of the tail. Ninety orbits of magnetic field data were surveyed, taken in February and May 2012 when the MESSENGER orbit was generally in a noon-midnight configuration. Fifty-eight FTEs were identified in the magnetosheath, with a core field larger than the planetary magnetic field just inside the magnetopause. The orientation of the events was determined by minimum variance analysis of the magnetic field data, and the results suggest that the FTEs were formed by multiple X-line reconnection at low latitudes on the dayside magnetopause. We show that these large-amplitude FTEs can carry substantial amounts of magnetic flux, driving a cross-polar-cap potential of tens of kV. Further, we show that the substorm cycle at Mercury is both shorter in duration and larger in relative amplitude than that observed at the Earth, and that these large-amplitude FTEs play a major role in magnetic flux transport throughout the magnetosphere.

  3. Genetic Analysis of the Role of the Transfer Gene, traN, of the F and R100-1 Plasmids in Mating Pair Stabilization during Conjugation

    PubMed Central

    Klimke, William A.; Frost, Laura S.

    1998-01-01

    Mating pair stabilization occurs during conjugative DNA transfer whereby the donor and recipient cells form a tight junction which requires pili as well as TraN and TraG in the donor cell. The role of the outer membrane protein, TraN, during conjugative transfer was examined by introduction of a chloramphenicol resistance cassette into the traN gene on an F plasmid derivative, pOX38, to produce pOX38N1::CAT. pOX38N1::CAT was greatly reduced in its ability to transfer DNA, indicating that TraN plays a greater role in conjugation than previously thought. F and R100-1 traN were capable of complementing pOX38N1::CAT transfer equally well when wild-type recipients were used. F traN, but not R100-1 traN, supported a much lower level of transfer when there was an ompA mutation or lipopolysaccharide (LPS) deficiency in the recipient cell, suggesting receptor specificity. The R100-1 traN gene was sequenced, and the gene product was found to exhibit 82.3% overall similarity with F TraN. The differences were mainly located within a central region of the proteins (amino acids 162 to 333 of F and 162 to 348 of R100-1). Deletion analysis of F traN suggested that this central portion might be responsible for the receptor specificity displayed by TraN. TraN was not responsible for TraT-dependent surface exclusion. Thus, TraN, and not the F pilus, appears to interact with OmpA and LPS moieties during conjugation, resulting in mating pair stabilization, the first step in efficient mobilization of DNA. PMID:9696748

  4. Drive-Response Analysis of Global Ice Volume, CO2, and Insolation using Information Transfer

    NASA Astrophysics Data System (ADS)

    Brendryen, J.; Hannisdal, B.

    2014-12-01

    The processes and interactions that drive global ice volume variability and deglaciations are a topic of considerable debate. Here we analyze the drive-response relationships between data sets representing global ice volume, CO2 and insolation over the past 800 000 years using an information theoretic approach. Specifically, we use a non-parametric measure of directional information transfer (IT) based on the construct of transfer entropy to detect the relative strength and directionality of interactions in the potentially chaotic and non-linear glacial-interglacial climate system. Analyses of unfiltered data suggest a tight coupling between CO2 and ice volume, detected as strong, symmetric information flow consistent with a two-way interaction. In contrast, IT from Northern Hemisphere (NH) summer insolation to CO2 is highly asymmetric, suggesting that insolation is an important driver of CO2. Conditional analysis further suggests that CO2 is a dominant influence on ice volume, with the effect of insolation also being significant but limited to smaller-scale variability. However, the strong correlation between CO2 and ice volume renders them information redundant with respect to insolation, confounding further drive-response attribution. We expect this information redundancy to be partly explained by the shared glacial-interglacial "sawtooth" pattern and its overwhelming influence on the transition probability distributions over the target interval. To test this, we filtered out the abrupt glacial terminations from the ice volume and CO2 records to focus on the residual variability. Preliminary results from this analysis confirm insolation as a driver of CO2 and two-way interactions between CO2 and ice volume. However, insolation is reduced to a weak influence on ice volume. Conditional analyses support CO2 as a dominant driver of ice volume, while ice volume and insolation both have a strong influence on CO2. These findings suggest that the effect of orbital

  5. Clostridium perfringens type A-E toxin plasmids.

    PubMed

    Freedman, John C; Theoret, James R; Wisniewski, Jessica A; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2015-05-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell.

  6. Clostridium perfringens type A–E toxin plasmids

    PubMed Central

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  7. Origin and Evolution of Rickettsial Plasmids

    PubMed Central

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    Background 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. Results 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. Conclusion Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via

  8. Origin and Evolution of Rickettsial Plasmids.

    PubMed

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    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 transfer as well as

  9. Conjugative Plasmids of Neisseria gonorrhoeae

    PubMed Central

    Pachulec, Emilia; van der Does, Chris

    2010-01-01

    Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM) determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones) and with and without different tetM determinants (Dutch and American type tetM determinants) have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233) or containing Dutch (pEP5289) or American (pEP5050) type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1α, β, γ, δ and ε subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids conjugated efficiently between

  10. Feline neural progenitor cells II: use of novel plasmid vector and hybrid promoter to drive expression of glial cell line-derived neurotrophic factor transgene.

    PubMed

    You, X Joann; Yang, Jing; Gu, Ping; Liew, Chee Gee; Klassen, Henry J

    2012-01-01

    Sustained transgene expression is required for the success of cell transplant-based gene therapy. Most widely used are lentiviral-based vectors which integrate into the host genome and thereby maintain sustained transgene expression. This requires integration into the nuclear genome, and potential risks include activation of oncogenes and inactivation of tumor suppressor genes. Plasmids have been used; however lack of sustained expression presents an additional challenge. Here we used the pCAG-PyF101-eGFP plasmid to deliver the human GDNF gene to cat neural progenitor cells (cNPCs). This vector consists of a CAGG composite promoter linked to the polyoma virus mutant enhancer PyF101. Expression of an episomal eGFP reporter and GDNF transgene were stably maintained by the cells, even following induction of differentiation. These genetically modified cells appear suitable for use in allogeneic models of cell-based delivery of GDNF in the cat and may find veterinary applications should such strategies prove clinically beneficial.

  11. mcr-1.2, a New mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain of Sequence Type 512

    PubMed Central

    Di Pilato, Vincenzo; Arena, Fabio; Tascini, Carlo; Cannatelli, Antonio; Henrici De Angelis, Lucia; Fortunato, Simona; Giani, Tommaso; Menichetti, Francesco

    2016-01-01

    A novel mcr variant, named mcr-1.2, encoding a Gln3-to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniae isolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2 gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1-bearing plasmids previously found in Escherichia coli and K. pneumoniae strains from geographically distant sites (Estonia, China, and South Africa). PMID:27401575

  12. mcr-1.2, a New mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain of Sequence Type 512.

    PubMed

    Di Pilato, Vincenzo; Arena, Fabio; Tascini, Carlo; Cannatelli, Antonio; Henrici De Angelis, Lucia; Fortunato, Simona; Giani, Tommaso; Menichetti, Francesco; Rossolini, Gian Maria

    2016-09-01

    A novel mcr variant, named mcr-1.2, encoding a Gln3-to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniae isolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2 gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1-bearing plasmids previously found in Escherichia coli and K. pneumoniae strains from geographically distant sites (Estonia, China, and South Africa).

  13. Evolutionary Thrift: Mycobacteria Repurpose Plasmid Diversity during Adaptation of Type VII Secretion Systems

    PubMed Central

    Mortimer, Tatum D.; Weber, Alexandra M.

    2017-01-01

    Mycobacteria have a distinct secretion system, termed type VII (T7SS), which is encoded by paralogous chromosomal loci (ESX) and associated with pathogenesis, conjugation, and metal homeostasis. Evolution of paralogous gene families is of interest because duplication is an important mechanism by which novel genes evolve, but there are potential conflicts between adaptive forces that stabilize duplications and those that enable evolution of new functions. Our objective was to delineate the adaptive forces underlying diversification of T7SS. Plasmid-borne ESX were described recently, and we found evidence that the initial duplication and divergence of ESX systems occurred on plasmids and was driven by selection for advantageous mutations. Plasmid conjugation has been linked to T7SS and type IV secretion systems (T4SS) in mycobacteria, and we discovered that T7SS and T4SS genes evolved in concert on the plasmids. We hypothesize that differentiation of plasmid ESX helps to prevent conjugation among cells harboring incompatible plasmids. Plasmid ESX appear to have been repurposed following migration to the chromosome, and there is evidence of positive selection driving further differentiation of chromosomal ESX. We hypothesize that ESX loci were initially stabilized on the chromosome by mediating their own transfer. These results emphasize the diverse adaptive paths underlying evolution of novelty, which in this case involved plasmid duplications, selection for advantageous mutations in the mobile and core genomes, migration of the loci between plasmids and chromosomes, and lateral transfer among chromosomes. We discuss further implications for the choice of model organism to study ESX functions in Mycobacterium tuberculosis. PMID:28391322

  14. Enterococcus faecalis pCF10-encoded Surface Proteins PrgA, PrgB (Aggregation Substance), and PrgC Contribute to Plasmid Transfer, Biofilm Formation, and Virulence

    PubMed Central

    Bhatty, Minny; Cruz, Melissa R.; Frank, Kristi L.; Gomez, Jenny A. Laverde; Andrade, Fernando; Garsin, Danielle A.; Dunny, Gary M.; Kaplan, Heidi B.; Christie, Peter J.

    2014-01-01

    SUMMARY Enterococcus faecalis pCF10 transfers at high frequencies upon pheromone induction of the prgQ transfer operon. This operon codes for three cell-wall-anchored proteins - PrgA, PrgB (aggregation substance), and PrgC - and a type IV secretion system through which the plasmid is delivered to recipient cells. Here, we defined the contributions of the Prg surface proteins to plasmid transfer, biofilm formation, and virulence using the Caenorhabditis elegans infection model. We report that a combination of PrgB and extracellular DNA (eDNA), but not PrgA or PrgC, was required for extensive cellular aggregation and pCF10 transfer at wild-type frequencies. In addition to PrgB and eDNA, production of PrgA was necessary for extensive binding of enterococci to abiotic surfaces and development of robust biofilms. However, although PrgB is a known virulence factor in mammalian infection models, we determined that PrgA and PrgC, but not PrgB, were required for efficient killing in the worm infection model. We propose that the pheromone-responsive, conjugative plasmids of E. faecalis have retained Prg-like surface functions over evolutionary time for attachment, colonization and robust biofilm development. In natural settings, these biofilms are polymicrobial in composition and constitute optimal environments for signal exchange, mating pair formation, and widespread lateral gene transfer. PMID:25431047

  15. Horizontal gene transfer drives adaptive colonization of apple trees by the fungal pathogen Valsa mali

    PubMed Central

    Yin, Zhiyuan; Zhu, Baitao; Feng, Hao; Huang, Lili

    2016-01-01

    Horizontal gene transfer (HGT) often has strong benefits for fungi. In a study of samples from apple canker in Shaanxi Province, China, diverse microbes, along with the necrotrophic pathogen Valsa mali, were found to colonize the apple bark, thus providing ample opportunity for HGT to occur. In the present study, we identified 32 HGT events in V. mali by combining phyletic distribution-based methods with phylogenetic analyses. Most of these HGTs were from bacteria, whereas several others were from eukaryotes. Three HGTs putatively functioned in competition with actinomycetes, some of which showed a significant inhibitory effect on V. mali. Three HGTs that were probably involved in nitrogen uptake were also identified. Ten HGTs were thought to be involved in pathogenicity because they were related to known virulence factors, including cell wall-degrading enzymes and candidate effector proteins. HGT14, together with HGT32, was shown to contribute to bleomycin resistance of V. mali.These results suggest that HGT drives the adaptive evolution of V. mali. The HGTs identified here provide new clues for unveiling the adaptation mechanisms and virulence determinants of V. mali. PMID:27634406

  16. Building mosaics of therapeutic plasmid gene vectors.

    PubMed

    Tolmachov, Oleg E

    2011-12-01

    Plasmids are circular or linear DNA molecules propagated extra-chromosomally in bacteria. Evolution shaped plasmids are inherently mosaic structures with individual functional units represented by distinct segments in the plasmid genome. The patchwork of plasmid genetic modules is a convenient template and a model for the generation of artificial plasmids used as vehicles for gene delivery into human cells. Plasmid gene vectors are an important tool in gene therapy and in basic biomedical research, where these vectors offer efficient transgene expression in many settings in vitro and in vivo. Plasmid vectors can be attached to nuclear directing ligands or transferred by electroporation as naked DNA to deliver the payload genes to the nuclei of the target cells. Transgene expression silencing by plasmid sequences of bacterial origin and immune stimulation by bacterial unmethylated CpG motifs can be avoided by the generation of plasmid-based minimized DNA vectors, such as minicircles. Systems of efficient site-specific integration into human chromosomes and stable episomal maintenance in human cells are being developed for further reduction of the chances for transgene silencing. The successful generation of plasmid vectors is governed by a number of vector design rules, some of which are common to all gene vectors, while others are specific to plasmid vectors. This review is focused both on the guiding principles and on the technical know-how of plasmid gene vector design.

  17. The mechanism of plasmid curing in bacteria.

    PubMed

    Spengler, Gabriella; Molnár, Annamária; Schelz, Zsuzsanna; Amaral, Leonard; Sharples, Derek; Molnár, Joseph

    2006-07-01

    plasmids. The inhibition of conjugational transfer of antibiotic resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of plasmid replication at various stages, as shown in the "rolling circle" model (replication, partition, conjugal transfer) may also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and antibiotic resistance. The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing mutations.

  18. Plasmids in diatom species.

    PubMed Central

    Hildebrand, M; Corey, D K; Ludwig, J R; Kukel, A; Feng, T Y; Volcani, B E

    1991-01-01

    We have discovered plasmids in 5 of 18 diatom species surveyed. In several species, more than one type of plasmid is present. Several of the plasmids show similarity by hybridization previously characterized plasmids in Cylindrotheca fusiformis (J. D. Jacobs et al., unpublished data). Additionally, there is similarity between the plasmids found in C. fusiformis and chloroplast DNA in three diatom species. These results add to the evidence that the plasmids have features of mobile genetic elements. Images PMID:1885558

  19. Autotransmissible resident plasmid of Rhizobium meliloti.

    PubMed

    Bedmar, E J; Olivares, J

    1980-01-01

    A resident plasmid of wild-type strains of Rhizobium meliloti of 59.6 megadaltons has been shown to be transferred at a high frequency to "cured" strains of this bacterial species. This plasmid, named pEZ1, that confers phage-sensitivity to cells carrying it is also transmissible to Escherichia coli and from it to "cured" R. meliloti strains.

  20. Plasmid-encoded trimethoprim resistance in staphylococci.

    PubMed Central

    Archer, G L; Coughter, J P; Johnston, J L

    1986-01-01

    High-level (greater than 1,000 micrograms/ml) resistance to the antimicrobial agent trimethoprim was found in 17 of 101 (17%) coagulase-negative staphylococci and 5 of 51 (10%) Staphylococcus aureus from a number of different hospitals in the United States. Resistance was plasmid encoded and could be transferred by conjugation in 4 of the 17 (24%) Tpr coagulase-negative staphylococci and 3 of the 5 (60%) Tpr S. aureus. A 1.2-kilobase segment of plasmid DNA from one of the plasmids (pG01) was cloned on a high-copy-number vector in Escherichia coli and expressed high-level Tpr (MIC, 1,025 micrograms/ml) in the gram-negative host. In situ filter hybridization demonstrated homology between the cloned Tpr gene probe and plasmid DNA from each conjugative Tpr plasmid, a single nonconjugative plasmid from a United States Staphylococcus epidermidis isolate, a nonconjugative plasmid from an Australian methicillin-resistant S. aureus isolate, and chromosomal DNA from three Tpr S. epidermidis isolates that did not contain any plasmid DNA that was homologous with the probe. No homology was seen between the probe and staphylococcal plasmids not mediating Tpr, plasmid DNA from 12 Tpr S. epidermidis isolates not transferring Tpr by conjugation, or plasmid-encoded Tpr genes derived from gram-negative bacteria. Plasmid-encoded Tpr appears to be a relatively new gene in staphylococci and, because it can be transferred by conjugation, could become more prevalent in nonsocomial isolates. Images PMID:3729338

  1. Genetic and functional characterization of a yet-unclassified rhizobial Dtr (DNA-transfer-and-replication) region from a ubiquitous plasmid conjugal system present in Sinorhizobium meliloti, in Sinorhizobium medicae, and in other nonrhizobial Gram-negative bacteria.

    PubMed

    Giusti, María de los Ángeles; Pistorio, Mariano; Lozano, Mauricio J; Tejerizo, Gonzalo A Torres; Salas, María Eugenia; Martini, María Carla; López, José Luis; Draghi, Walter O; Del Papa, María Florencia; Pérez-Mendoza, Daniel; Sanjuán, Juan; Lagares, Antonio

    2012-05-01

    Rhizobia are Gram-negative bacteria that live in soils and associate with leguminous plants to establish nitrogen-fixing symbioses. The ability of these bacteria to undergo horizontal gene transfer (HGT) is thought to be one of the main features to explain both the origin of their symbiotic life-style and the plasticity and dynamics of their genomes. In our laboratory we have previously characterized at the species level the non-pSym plasmid mobilome in Sinorhizobium meliloti, the symbiont of Medicago spp., and have found a high incidence of conjugal activity in many plasmids (Pistorio et al., 2008). In this work we characterized the Dtr (DNA-transfer-and-replication) region of one of those plasmids, pSmeLPU88b. This mobilization region was found to represent a previously unclassified Dtr type in rhizobia (hereafter type-IV), highly ubiquitous in S. meliloti and found in other genera of Gram-negative bacteria as well; including Agrobacterium, Ochrobactrum, and Chelativorans. The oriT of the type-IV Dtr described here could be located by function within a DNA fragment of 278 bp, between the divergent genes parA and mobC. The phylogenetic analysis of the cognate relaxase MobZ indicated that this protein groups close to the previously defined MOB(P3) and MOB(P4) type of enzymes, but is located in a separate and novel cluster that we have designated MOB(P0). Noteworthy, MOB(P0) and MOB(P4) relaxases were frequently associated with plasmids present in rhizospheric soil bacteria. A comparison of the nod-gene locations with the phylogenetic topology of the rhizobial relaxases revealed that the symbiotic genes are found on diverse plasmids bearing any of the four Dtr types, thus indicating that pSym plasmids are not specifically associated with any particular mobilization system. Finally, we demonstrated that the type-IV Dtr promoted the mobilization of plasmids from S. meliloti to Sinorhizobium medicae as well as from these rhizobia to other bacteria by means of their own

  2. Goals, Family, and Community: What Drives Tribal College Transfer Student Success

    ERIC Educational Resources Information Center

    Makomenaw, Matthew

    2014-01-01

    This article examines success factors for American Indian tribal college students who transfer to 4-year predominantly White institutions. The study examined the experiences of 8 tribal college transfer students to Midwest universities. Using an indigenous methodology, 3 themes were found to help American Indian tribal college transfer students…

  3. Goals, Family, and Community: What Drives Tribal College Transfer Student Success

    ERIC Educational Resources Information Center

    Makomenaw, Matthew

    2014-01-01

    This article examines success factors for American Indian tribal college students who transfer to 4-year predominantly White institutions. The study examined the experiences of 8 tribal college transfer students to Midwest universities. Using an indigenous methodology, 3 themes were found to help American Indian tribal college transfer students…

  4. Structural basis of cooperative DNA recognition by the plasmid conjugation factor, TraM

    PubMed Central

    Wong, Joyce J. W.; Lu, Jun; Edwards, Ross A.; Frost, Laura S.; Glover, J. N. Mark

    2011-01-01

    The conjugative transfer of F-like plasmids such as F, R1, R100 and pED208, between bacterial cells requires TraM, a plasmid-encoded DNA-binding protein. TraM tetramers bridge the origin of transfer (oriT) to a key component of the conjugative pore, the coupling protein TraD. Here we show that TraM recognizes a high-affinity DNA-binding site, sbmA, as a cooperative dimer of tetramers. The crystal structure of the TraM–sbmA complex from the plasmid pED208 shows that binding cooperativity is mediated by DNA kinking and unwinding, without any direct contact between tetramers. Sequence-specific DNA recognition is carried out by TraM’s N-terminal ribbon–helix–helix (RHH) domains, which bind DNA in a staggered arrangement. We demonstrate that both DNA-binding specificity, as well as selective interactions between TraM and the C-terminal tail of its cognate TraD mediate conjugation specificity within the F-like family of plasmids. The ability of TraM to cooperatively bind DNA without interaction between tetramers leaves the C-terminal TraM tetramerization domains free to make multiple interactions with TraD, driving recruitment of the plasmid to the conjugative pore. PMID:21565799

  5. Transformation of Bacillus polymyxa with plasmid DNA.

    PubMed Central

    Mallonee, D H; Speckman, R A

    1989-01-01

    A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media. Images PMID:2604393

  6. F33: A-: B-, IncHI2/ST3, and IncI1/ST71 plasmids drive the dissemination of fosA3 and blaCTX−M−55/−14/−65 in Escherichia coli from chickens in China

    PubMed Central

    Yang, Xiaoyun; Liu, Wuling; Liu, Yiyun; Wang, Jing; Lv, Luchao; Chen, Xiaojie; He, Dandan; Yang, Tong; Hou, Jianxia; Tan, Yinjuan; Xing, Li; Zeng, Zhenling; Liu, Jian-Hua

    2014-01-01

    The purpose of this study was to examine the occurrence of fosfomycin-resistant Escherichia coli from chickens and to characterize the plasmids carrying fosA3. A total of 661 E. coli isolates of chicken origin collected from 2009 to 2011 were screened for plasmid-mediated fosfomycin resistance determinants by PCR. Plasmids were characterized using PCR-based replicon typing, plasmid multilocus sequence typing, and restriction fragment length polymorphisms. Associated addiction systems and resistance genes were identified by PCR. PCR-mapping was used for analysis of the genetic context of fosA3. Fosfomycin resistance was detected in 58 isolates that also carried the fosA3 gene. Fifty-seven, 17, and 52 FosA3-producers also harbored blaCTX−M, rmtB, and floR genes, respectively. Most of the 58 fosA3-carrying isolates were clonally unrelated, and all fosA3 genes were located on plasmids belonged to F33:A-:B- (n = 18), IncN-F33:A-:B- (n = 7), IncHI2/ST3 (n = 10), IncI1/ST71 (n = 3), IncI1/ST108 (n = 3), and others. The genetic structures, IS26-ISEcp1-blaCTX−M−55-orf477-blaTEM-1-IS26-fosA3-1758bp-IS26 and ISEcp1-blaCTX−M−65-IS903-iroN-IS26-fosA3-536bp-IS26 were located on highly similar F33:A-:B- plasmids. In addition, blaCTX−M−14-fosA3-IS26 was frequently present on similar IncHI2/ST3 plasmids. IncFII plasmids had a significantly higher frequency of addiction systems (mean 3.5) than other plasmids. Our results showed a surprisingly high prevalence of fosA3 gene in E. coli isolates recovered from chicken in China. The spread of fosA3 can be attributed to horizontal dissemination of several epidemic plasmids, especially F33:A-:B- plasmids. Since coselection by other antimicrobials is the major driving force for the diffusion of the fosA3 gene, a strict antibiotic use policy is urgently needed in China. PMID:25566207

  7. Solvent fluctuations drive the hole transfer in DNA: a mixed quantum-classical study.

    PubMed

    Kubar, Tomás; Kleinekathöfer, Ulrich; Elstner, Marcus

    2009-10-01

    We studied the hole transfer across adenine bridges in double-stranded DNA by means of a multiscale approach, propagating the hole in the framework of time-dependent DFT coupled to classical molecular dynamics simulation using a QM/MM scheme. The hole transfer in DNA is codetermined by the large fluctuations of site energies on the order of 0.4 eV, induced by the solvent degrees of freedom. These fluctuations lead to charge-transfer active conformations with large transfer efficiency, which are characterized by a favorable alignment of site energies along the DNA strand. This reduces the barrier for the hole transfer dramatically. Consequently, we find that a charge hopping mechanism is operative already for short bridges with fewer than four adenines, in contrast to the charge-transfer models assuming static DNA structures, where only tunneling occurs. The solvent fluctuations introduce a significant correlation between neighboring sites, enhancing the charge-transfer rate, while the fluctuation of electronic couplings has only a minor impact on the charge-transfer characteristics. Our results emphasize the importance of an accurate description of solvent effects as well as proper sampling, and it is suggested that charge transfer in DNA is gated by the dynamics of solvent.

  8. Plasmid DNA manufacturing technology.

    PubMed

    Carnes, Aaron E; Williams, James A

    2007-01-01

    Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. This review summarizes recent patents and patent applications relating to plasmid manufacturing, in the context of a comprehensive description of the plasmid manufacturing intellectual property landscape. Strategies for plasmid manufacturers to develop or in-license key plasmid manufacturing technologies are described with the endpoint of efficiently producing kg quantities of plasmid DNA of a quality that meets anticipated European and FDA quality specifications for commercial plasmid products.

  9. The reversed terminator of octopine synthase gene on the Agrobacterium Ti plasmid has a weak promoter activity in prokaryotes.

    PubMed

    Shao, Jun-Li; Long, Yue-Sheng; Chen, Gu; Xie, Jun; Xu, Zeng-Fu

    2010-06-01

    Agrobacterium tumefaciens transfers DNA from its Ti plasmid to plant host cells. The genes located within the transferred DNA of Ti plasmid including the octopine synthase gene (OCS) are expressed in plant host cells. The 3'-flanking region of OCS gene, known as OCS terminator, is widely used as a transcriptional terminator of the transgenes in plant expression vectors. In this study, we found the reversed OCS terminator (3'-OCS-r) could drive expression of hygromycin phosphotransferase II gene (hpt II) and beta-glucuronidase gene in Escherichia coli, and expression of hpt II in A. tumefaciens. Furthermore, reverse transcription-polymerase chain reaction analysis revealed that an open reading frame (ORF12) that is located downstream to the 3'-OCS-r was transcribed in A. tumefaciens, which overlaps in reverse with the coding region of the OCS gene in octopine Ti plasmid.

  10. Plasmids spread very fast in heterogeneous bacterial communities.

    PubMed Central

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  11. Shortcut to adiabatic population transfer in quantum three-level systems: Effective two-level problems and feasible counterdiabatic driving

    NASA Astrophysics Data System (ADS)

    Li, Yi-Chao; Chen, Xi

    2016-12-01

    Shortcuts to adiabaticity in various quantum systems have attracted much attention with their wide applications in quantum information processing and quantum control. In this paper, we concentrate on a stimulated Raman shortcut-to-adiabatic passage in quantum three-level systems. To implement counterdiabatic driving but without additional coupling, we first reduce the quantum three-level systems to effective two-level problems at large intermediate-level detuning, or on resonance, apply counterdiabatic driving along with the unitary transformation and eventually modify the pump and Stokes pulses for achieving fast and high-fidelity population transfer. The required laser intensity and stability against parameter variation are further discussed, to demonstrate the advantage of shortcuts to adiabaticity.

  12. Plasmids of Azotobacter vinelandii.

    PubMed Central

    Maia, M; Sanchez, J M; Vela, G R

    1988-01-01

    Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty-five laboratory strains which could fix nitrogen did not have free, covalently closed circular plasmid DNA. The plasmids varied in size from 9 to 52 megadaltons, and each strain yielded only one plasmid. No discernible differences in ability to fix nitrogen were found between plasmid-bearing and cured cultures. PMID:3350795

  13. Invasion of E. coli biofilms by antibiotic resistance plasmids.

    PubMed

    Król, Jaroslaw E; Wojtowicz, Andrzej J; Rogers, Linda M; Heuer, Holger; Smalla, Kornelia; Krone, Stephen M; Top, Eva M

    2013-07-01

    In spite of the contribution of plasmids to the spread of antibiotic resistance in human pathogens, little is known about the transferability of various drug resistance plasmids in bacterial biofilms. The goal of this study was to compare the efficiency of transfer of 19 multidrug resistance plasmids into Escherichia coli recipient biofilms and determine the effects of biofilm age, biofilm-donor exposure time, and donor-to-biofilm attachment on this process. An E. coli recipient biofilm was exposed separately to 19 E. coli donors, each with a different plasmid, and transconjugants were determined by plate counting. With few exceptions, plasmids that transferred well in a liquid environment also showed the highest transferability in biofilms. The difference in transfer frequency between the most and least transferable plasmid was almost a million-fold. The 'invasibility' of the biofilm by plasmids, or the proportion of biofilm cells that acquired plasmids within a few hours, depended not only on the type of plasmid, but also on the time of biofilm exposure to the donor and on the ability of the plasmid donor to attach to the biofilm, yet not on biofilm age. The efficiency of donor strain attachment to the biofilm was not affected by the presence of plasmids. The most invasive plasmid was pHH2-227, which based on genome sequence analysis is a hybrid between IncU-like and IncW plasmids. The wide range in transferability in an E. coli biofilm among plasmids needs to be taken into account in our fight against the spread of drug resistance. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Horizontal Transfer, Not Duplication, Drives the Expansion of Protein Families in Prokaryotes

    PubMed Central

    Treangen, Todd J.; Rocha, Eduardo P. C.

    2011-01-01

    Gene duplication followed by neo- or sub-functionalization deeply impacts the evolution of protein families and is regarded as the main source of adaptive functional novelty in eukaryotes. While there is ample evidence of adaptive gene duplication in prokaryotes, it is not clear whether duplication outweighs the contribution of horizontal gene transfer in the expansion of protein families. We analyzed closely related prokaryote strains or species with small genomes (Helicobacter, Neisseria, Streptococcus, Sulfolobus), average-sized genomes (Bacillus, Enterobacteriaceae), and large genomes (Pseudomonas, Bradyrhizobiaceae) to untangle the effects of duplication and horizontal transfer. After removing the effects of transposable elements and phages, we show that the vast majority of expansions of protein families are due to transfer, even among large genomes. Transferred genes—xenologs—persist longer in prokaryotic lineages possibly due to a higher/longer adaptive role. On the other hand, duplicated genes—paralogs—are expressed more, and, when persistent, they evolve slower. This suggests that gene transfer and gene duplication have very different roles in shaping the evolution of biological systems: transfer allows the acquisition of new functions and duplication leads to higher gene dosage. Accordingly, we show that paralogs share most protein–protein interactions and genetic regulators, whereas xenologs share very few of them. Prokaryotes invented most of life's biochemical diversity. Therefore, the study of the evolution of biology systems should explicitly account for the predominant role of horizontal gene transfer in the diversification of protein families. PMID:21298028

  15. Selfish Operons: Horizontal Transfer May Drive the Evolution of Gene Clusters

    PubMed Central

    Lawrence, J. G.; Roth, J. R.

    1996-01-01

    A model is presented whereby the formation of gene clusters in bacteria is mediated by transfer of DNA within and among taxa. Bacterial operons are typically composed of genes whose products contribute to a single function. If this function is subject to weak selection or to long periods with no selection, the contributing genes may accumulate mutations and be lost by genetic drift. From a cell's perspective, once several genes are lost, the function can be restored only if all missing genes were acquired simultaneously by lateral transfer. The probability of transfer of multiple genes increases when genes are physically proximate. From a gene's perspective, horizontal transfer provides a way to escape evolutionary loss by allowing colonization of organisms lacking the encoded functions. Since organisms bearing clustered genes are more likely to act as successful donors, clustered genes would spread among bacterial genomes. The physical proximity of genes may be considered a selfish property of the operon since it affects the probability of successful horizontal transfer but may provide no physiological benefit to the host. This process predicts a mosaic structure of modern genomes in which ancestral chromosomal material is interspersed with novel, horizontally transferred operons providing peripheral metabolic functions. PMID:8844169

  16. Adiabatic quantum state transfer in tight-binding chains using periodic driving fields

    NASA Astrophysics Data System (ADS)

    Longhi, S.

    2014-09-01

    A method for high-fidelity coherent adiabatic transport in a zig-zag tight-binding chain, based on application of two external periodic driving fields, is theoretically proposed. The method turns out to be robust against imperfections and disorder of the static lattice Hamiltonian, is tolerant to next-nearest neighborhood interactions, and enables coherent transport in long chains without the need for a local control and timing of the trapping potential.

  17. Isolating and quantifying cross-beam energy transfer in direct-drive implosions on OMEGA and the National Ignition Facility

    SciTech Connect

    Davis, A. K.; Cao, D.; Michel, D. T.; Hohenberger, M.; Edgell, D. H.; Epstein, R.; Goncharov, V. N.; Hu, S. X.; Igumenshchev, I. V.; Marozas, J. A.; Maximov, A. V.; Myatt, J. F.; Radha, P. B.; Regan, S. P.; Sangster, T. C.; Froula, D. H.

    2016-04-20

    The angularly-resolved mass ablation rates and ablation front trajectories for Si-coated CH targets were measured in direct-drive inertial confinement fusion experiments to quantify crossbeam energy transfer (CBET) while constraining the hydrodynamic coupling. A polar-direct-drive laser configuration was used, where the equatorial laser beams were dropped from a symmetric direct-drive configuration to suppress CBET at the pole, while allowing it to persist at the equator. The combination of low- and high-CBET conditions in the same implosion allowed the effects of CBET on the ablation rate and ablation pressure to be decoupled from the other physics effects that influence laser-coupling. Hydrodynamic simulations performed without CBET reproduced the measured ablation rate and ablation front trajectory at the pole of the target, verifying that the other laser-coupling physics effects are well-modeled when CBET effects are negligible. The simulated mass ablation rates and ablation front trajectories were in excellent agreement with the measurements at all angles when a CBET model based on Randall’s equations [C. J. Randall et al., Phys. Fluids 24, 1474 (1981)] was included into the simulations with an optimized multiplier on the CBET gain factor. These measurements were performed on both OMEGA and the National Ignition Facility to access a wide range of plasma conditions, laser intensities, and laser beam geometries. Furthermore, the presence of the CBET gain multiplier required to match the data in all of the configurations tested suggests that additional physics effects, such as intensity variations due to diffraction, shortcomings of extending the 1-D Randall model to 3-D, or polarization effects, should be explored to explain the differences in observed and predicted drive.

  18. Isolating and quantifying cross-beam energy transfer in direct-drive implosions on OMEGA and the National Ignition Facility

    SciTech Connect

    Davis, A. K. Cao, D.; Michel, D. T.; Hohenberger, M.; Edgell, D. H.; Epstein, R.; Goncharov, V. N.; Hu, S. X.; Igumenshchev, I. V.; Marozas, J. A.; Maximov, A. V.; Myatt, J. F.; Radha, P. B.; Regan, S. P.; Sangster, T. C.; Froula, D. H.

    2016-05-15

    The angularly resolved mass ablation rates and ablation-front trajectories for Si-coated CH targets were measured in direct-drive inertial confinement fusion experiments to quantify cross-beam energy transfer (CBET) while constraining the hydrodynamic coupling. A polar-direct-drive laser configuration, where the equatorial laser beams were dropped and the polar beams were repointed from a symmetric direct-drive configuration, was used to limit CBET at the pole while allowing it to persist at the equator. The combination of low- and high-CBET conditions observed in the same implosion allowed for the effects of CBET on the ablation rate and ablation pressure to be determined. Hydrodynamic simulations performed without CBET agreed with the measured ablation rate and ablation-front trajectory at the pole of the target, confirming that the CBET effects on the pole are small. The simulated mass ablation rates and ablation-front trajectories were in excellent agreement with the measurements at all angles when a CBET model based on Randall's equations [C. J. Randall et al., Phys. Fluids 24, 1474 (1981)] was included into the simulations with a multiplier on the CBET gain factor. These measurements were performed on OMEGA and at the National Ignition Facility to access a wide range of plasma conditions, laser intensities, and laser beam geometries. The presence of the CBET gain multiplier required to match the data in all of the configurations tested suggests that additional physics effects, such as intensity variations caused by diffraction, polarization effects, or shortcomings of extending the 1-D Randall model to 3-D, should be explored to explain the differences in observed and predicted drive.

  19. Isolating and quantifying cross-beam energy transfer in direct-drive implosions on OMEGA and the National Ignition Facility

    SciTech Connect

    Davis, A. K.; Cao, D.; Michel, D. T.; Hohenberger, M.; Edgell, D. H.; Epstein, R.; Goncharov, V. N.; Hu, S. X.; Igumenshchev, I. V.; Marozas, J. A.; Maximov, A. V.; Myatt, J. F.; Radha, P. B.; Regan, S. P.; Sangster, T. C.; Froula, D. H.

    2016-04-20

    The angularly-resolved mass ablation rates and ablation front trajectories for Si-coated CH targets were measured in direct-drive inertial confinement fusion experiments to quantify crossbeam energy transfer (CBET) while constraining the hydrodynamic coupling. A polar-direct-drive laser configuration was used, where the equatorial laser beams were dropped from a symmetric direct-drive configuration to suppress CBET at the pole, while allowing it to persist at the equator. The combination of low- and high-CBET conditions in the same implosion allowed the effects of CBET on the ablation rate and ablation pressure to be decoupled from the other physics effects that influence laser-coupling. Hydrodynamic simulations performed without CBET reproduced the measured ablation rate and ablation front trajectory at the pole of the target, verifying that the other laser-coupling physics effects are well-modeled when CBET effects are negligible. The simulated mass ablation rates and ablation front trajectories were in excellent agreement with the measurements at all angles when a CBET model based on Randall’s equations [C. J. Randall et al., Phys. Fluids 24, 1474 (1981)] was included into the simulations with an optimized multiplier on the CBET gain factor. These measurements were performed on both OMEGA and the National Ignition Facility to access a wide range of plasma conditions, laser intensities, and laser beam geometries. Furthermore, the presence of the CBET gain multiplier required to match the data in all of the configurations tested suggests that additional physics effects, such as intensity variations due to diffraction, shortcomings of extending the 1-D Randall model to 3-D, or polarization effects, should be explored to explain the differences in observed and predicted drive.

  20. Isolating and quantifying cross-beam energy transfer in direct-drive implosions on OMEGA and the National Ignition Facility

    DOE PAGES

    Davis, A. K.; Cao, D.; Michel, D. T.; ...

    2016-04-20

    The angularly-resolved mass ablation rates and ablation front trajectories for Si-coated CH targets were measured in direct-drive inertial confinement fusion experiments to quantify crossbeam energy transfer (CBET) while constraining the hydrodynamic coupling. A polar-direct-drive laser configuration was used, where the equatorial laser beams were dropped from a symmetric direct-drive configuration to suppress CBET at the pole, while allowing it to persist at the equator. The combination of low- and high-CBET conditions in the same implosion allowed the effects of CBET on the ablation rate and ablation pressure to be decoupled from the other physics effects that influence laser-coupling. Hydrodynamic simulationsmore » performed without CBET reproduced the measured ablation rate and ablation front trajectory at the pole of the target, verifying that the other laser-coupling physics effects are well-modeled when CBET effects are negligible. The simulated mass ablation rates and ablation front trajectories were in excellent agreement with the measurements at all angles when a CBET model based on Randall’s equations [C. J. Randall et al., Phys. Fluids 24, 1474 (1981)] was included into the simulations with an optimized multiplier on the CBET gain factor. These measurements were performed on both OMEGA and the National Ignition Facility to access a wide range of plasma conditions, laser intensities, and laser beam geometries. Furthermore, the presence of the CBET gain multiplier required to match the data in all of the configurations tested suggests that additional physics effects, such as intensity variations due to diffraction, shortcomings of extending the 1-D Randall model to 3-D, or polarization effects, should be explored to explain the differences in observed and predicted drive.« less

  1. In vivo cloning strategy for Rhizobium plasmids.

    PubMed

    Hernández-Lucas, I; Mavingui, P; Finan, T; Chain, P; Martínez-Romero, E

    2002-10-01

    We have developed a simple system to clone indigenous Rhizobium plasmids into E. coli. The strategy consists of three matings: the first is to insert Tn5 in the plasmid to be cloned, the second incorporates the integrative vector into the inserted Tn5 in the native Rhizobium plasmid, and the last mating transfers the target plasmid directly into E. coli. This mating-based system was successfully used to clone plasmids of Rhizobium species with sizes ranging from 150 to 270 kb. In addition, a 500-kb fragment of a 600-kb megaplasmid was also cloned. This strategy could be used for cloning indigenous replicons of other gram-negative bacteria into a different host.

  2. Designing plasmid vectors.

    PubMed

    Tolmachov, Oleg

    2009-01-01

    Nonviral gene therapy vectors are commonly based on recombinant bacterial plasmids or their derivatives. The plasmids are propagated in bacteria, so, in addition to their therapeutic cargo, they necessarily contain a bacterial replication origin and a selection marker, usually a gene conferring antibiotic resistance. Structural and maintenance plasmid stability in bacteria is required for the plasmid DNA production and can be achieved by carefully choosing a combination of the therapeutic DNA sequences, replication origin, selection marker, and bacterial strain. The use of appropriate promoters, other regulatory elements, and mammalian maintenance devices ensures that the therapeutic gene or genes are adequately expressed in target human cells. Optimal immune response to the plasmid vectors can be modulated via inclusion or exclusion of DNA sequences containing immunostimulatory CpG sequence motifs. DNA fragments facilitating construction of plasmid vectors should also be considered for inclusion in the design of plasmid vectors. Techniques relying on site-specific or homologous recombination are preferred for construction of large plasmids (>15 kb), while digestion of DNA by restriction enzymes with subsequent ligation of the resulting DNA fragments continues to be the mainstream approach for generation of small- and medium-size plasmids. Rapid selection of a desired recombinant plasmid against a background of other plasmids continues to be a challenge. In this chapter, the emphasis is placed on efficient and flexible versions of DNA cloning protocols using selection of recombinant plasmids by restriction endonucleases directly in the ligation mixture.

  3. Fusion and Compatibility of Camphor and Octane Plasmids in Pseudomonas

    PubMed Central

    Chou, George I. N.; Katz, Dvorah; Gunsalus, I. C.

    1974-01-01

    The octane (OCT) plasmid in Pseudomonas putida derived from the ω-hydroxylase-carrying strain of Coon and coworkers is transferable to the camphor (CAM) plasmid-bearing strain by conjugation or by transduction. While the majority of the Cam +Oct+ exconjugants segregate Cam+ or Oct+ cells, exconjugants with stable Cam +Oct+ phenotype (CAM-OCT) can be detected at a low frequency. The transductants are all of the CAM-OCT phenotype. In the stable Cam +Oct+ strains, the OCT plasmid resembles the CAM plasmid with respect to curing by mitomycin C, transfer in conjugation, and reaction to ts (temperature-sensitive) mutation specifically affecting CAM plasmid replication. Therefore, it is suggested that certain regions of homology exist between the CAM and OCT plasmids that enable them to recombine to form a single plasmid, and to overcome the incompatibility barrier that prevents their coexisting. PMID:4527812

  4. The Influence of Biofilms in the Biology of Plasmids.

    PubMed

    Cook, Laura C C; Dunny, Gary M

    2014-10-01

    The field of plasmid biology has historically focused on bacteria growing in liquid culture. Surface-attached communities of bacterial biofilms have recently been understood to be the normal environment of bacteria in the natural world. Thus, studies examining plasmid replication, maintenance, and transfer in biofilms are essential for a true understanding of bacterial plasmid biology. This article reviews the current knowledge of the interplay between bacterial biofilms and plasmids, focusing on the role of plasmids in biofilm development and the role of biofilms in plasmid maintenance, copy-number control, and transfer. The studies examined herein highlight the importance of biofilms as an important ecological niche in which bacterial plasmids play an essential role.

  5. Spread of CTX-M-15 Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates through Household Contact and Plasmid Transfer

    PubMed Central

    Kojima, Yasuko; Harada, Sohei; Aoki, Kotaro; Sawa, Tomoka; Hasegawa, Kei; Saji, Tsutomu; Yamaguchi, Keizo; Tateda, Kazuhiro

    2014-01-01

    We document the household spread of extended-spectrum β-lactamase-producing Escherichia coli. One isolate belonged to sequence type 1193 and caused urinary tract infection in a 4-month-old female, and the other isolate belonged to sequence type 131 and colonized three family members, including the index patient. These isolates carried similar Inc-I1-Iγ plasmids, harboring blaCTX-M-15. PMID:24554749

  6. Spread of CTX-M-15 extended-spectrum β-lactamase-producing Escherichia coli isolates through household contact and plasmid transfer.

    PubMed

    Kojima, Yasuko; Harada, Sohei; Aoki, Kotaro; Ishii, Yoshikazu; Sawa, Tomoka; Hasegawa, Kei; Saji, Tsutomu; Yamaguchi, Keizo; Tateda, Kazuhiro

    2014-05-01

    We document the household spread of extended-spectrum β-lactamase-producing Escherichia coli. One isolate belonged to sequence type 1193 and caused urinary tract infection in a 4-month-old female, and the other isolate belonged to sequence type 131 and colonized three family members, including the index patient. These isolates carried similar Inc-I1-Iγ plasmids, harboring blaCTX-M-15.

  7. Acquisition through horizontal gene transfer of plasmid pSMA198 by Streptococcus macedonicus ACA-DC 198 points towards the dairy origin of the species.

    PubMed

    Papadimitriou, Konstantinos; Anastasiou, Rania; Maistrou, Eleni; Plakas, Thomas; Papandreou, Nikos C; Hamodrakas, Stavros J; Ferreira, Stéphanie; Supply, Philip; Renault, Pierre; Pot, Bruno; Tsakalidou, Effie

    2015-01-01

    Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. However, S. macedonicus is closely related to commensal opportunistic pathogens of the Streptococcus bovis/Streptococcus equinus complex. We analyzed the pSMA198 plasmid isolated from the dairy strain Streptococcus macedonicus ACA-DC 198 in order to provide novel clues about the main ecological niche of this bacterium. pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and to the best of our knowledge it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 sequence revealed a high degree of similarity with plasmids isolated from Lactococcus lactis strains deriving from milk or its products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Additionally, cloning of the pSMA198 ori in L. lactis revealed a 100% stability of replication over 100 generations. Both pSMA198 and the chromosome of S. macedonicus exhibit a high percentage of potential pseudogenes, indicating that they have co-evolved under the same gene decay processes. We identified chromosomal regions in S. macedonicus that may have originated from pSMA198, also supporting a long co-existence of the two replicons. pSMA198 was also found in divergent biotypes of S. macedonicus and in strains isolated from dispersed geographic locations (e.g. Greece and Switzerland) showing that pSMA198's acquisition is not a recent event. Here we propose that S. macedonicus acquired plasmid pSMA198 from L. lactis via an ancestral genetic exchange event that took place most probably in milk or dairy products. We provide important evidence that point towards the dairy origin of this species.

  8. Plasmid-encoding extended-spectrum β-lactamase CTX-M-55 in a clinical Shigella sonnei strain, China.

    PubMed

    Qu, Fen; Ying, Zhe; Zhang, Chuanling; Chen, Zhenhong; Chen, Suming; Cui, Enbo; Bao, Chunmei; Yang, Huiying; Wang, Jie; Liu, Changting; Mao, Yuanli; Zhou, Dongsheng

    2014-01-01

    To characterize a clinical Shigella sonnei strain harboring a conjugatable blaCTX-M-55-borne plasmid. S. sonnei strain #1081 was isolated from a dysentery patient in China. A CTX-M-55-encoding plasmid harbored in this strain was transformed to Escherichia coli, and then its complete nucleotide sequence was determined by next generation sequencing. The MIC values of bacterial strains were tested by using Vitec(®) 2 (Biomerieux, Marcy l'Etoile, France). Strain #1081 conferred the resistance to multiple beta-lactam antibiotics. blaCTX-M-55 was the only known antibiotic resistance gene and located in a 3090-bp ISEcp1-blaCTX-M-55-orf477 transposition unit carried by a conjugatable plasmid p1081-CTXM in #1081. The ISEcp1-mediated transposition provided a sole promoter, which was located adjacently upstream of the inverted repeat right element of ISEcp1, to drive the expression of CTX-M-55. Plasmid p1081-CTXM was a close variant of the IncI2-type plasmid pHN1122-1 that was harbored in a faecal E. coli strain recovered from a dog in China, indicating the potential transfer of CTX-M-55-encoding plasmids from faecal flora E. coli to human pathogen S. sonnei.

  9. Mutagenesis of dimeric plasmids by the transposon. gamma. delta. (Tn1000)

    SciTech Connect

    Liu, L.; Berg, C.M. )

    1990-05-01

    The Escherichia coli F factor mediates conjugal transfer of a plasmid such as pBR322 primarily by replicative transposition of transposon {gamma}{delta} (Tn1000) from F to that plasmid to form a cointegrate intermediate. Although resolution of this cointegrate always yields a plasmid containing a single {gamma}{delta} insertion, the occasional recovery of transposon-free plasmids after connuugal transfer has led to alternative hypotheses for F mobilization. The authors show here that {gamma}{delta}-free plasmids are found after F-mediated conjugal transfer only when the donor plasmid is a dimer and the recipient is Rec{sup +}.

  10. Broad host range plasmids.

    PubMed

    Jain, Aayushi; Srivastava, Preeti

    2013-11-01

    Plasmids are and will remain important cloning vehicles for biotechnology. They have also been associated with the spread of a number of diseases and therefore are a subject of environmental concern. With the advent of sequencing technologies, the database of plasmids is increasing. It will be of immense importance to identify the various bacterial hosts in which the plasmid can replicate. The present review article describes the features that confer broad host range to the plasmids, the molecular basis of plasmid host range evolution, and applications in recombinant DNA technology and environment.

  11. Evaluation of the Effects of Cross-Beam Energy Transfer in NIF Polar-Drive Exploding-Pusher Experiments

    NASA Astrophysics Data System (ADS)

    McKenty, P. W.; Marshall, F. J.; Hohenberger, M.; Craxton, R. S.; Marozas, J. A.; Delettrez, J. A.; Shvydky, A.; Froula, D. H.; Michel, D. T.; Edgell, D. H.; Seka, W.; Olson, P. A.; To, S.; Cao, D.; Moses, G.; Le Pape, S.; MacKinnon, A. J.; Ma, T.

    2013-10-01

    Polar-drive (PD) target implosions have been designed and fielded for neutron diagnostic development on the National Ignition Facility (NIF). Experimental results evaluating the overall hydrodynamic assembly have previously indicated a significant discrepancy with DRACO predictions of the in-flight shell evolution. New physics models, addressing nonlocal electron thermal transport and cross-beam energy transfer within the incoming laser light, have been implemented into DRACO. Results detailing comparisons of experiments with simulations using these models will be presented that indicate significantly better agreement and may provide insight into the application of these models in other inertial confinement fusion experiments. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DE-NA0001944. A. M. Cok, R. S. Craxton, and P. W. McKenty, Phys. Plasmas 15, 082705 (2008).

  12. Improved Wavelength Detuning Cross-Beam Energy Transfer Mitigation Strategy for Polar Direct Drive at the National Ignition Facility

    NASA Astrophysics Data System (ADS)

    Marozas, J. A.; Collins, T. J. B.; McKenty, P. W.; Zuegel, J. D.

    2015-11-01

    Cross-beam energy transfer (CBET) reduces absorbed light and implosion velocity, alters time-resolved scattered-light spectra, and redistributes absorbed and scattered light. These effects reduce target performance in both symmetric direct-drive and polar-direct-drive (PDD) experiments on the OMEGA Laser System and the National Ignition Facility (NIF). The CBET package (Adaawam) incorporated into the 2-D hydrodynamics code DRACO is an integral part of the 3-D ray-trace package (Mazinisin). The CBET exchange occurs primarily over the equatorial region in PDD, where successful mitigation strategies concentrate. Detuning the initial laser wavelength (dλ0) reduces the CBET interaction volume, which can be combined with other mitigation domains (e.g., spatial and temporal). By judiciously selecting the ring and/or port +/-dλ0 in each hemisphere, using new DRACO diagnostic abilities, improved wavelength detuning strategies trade-off overall energy absorption for improved hemispherical energy balance control. These balanced-wavelength detuning strategies improve performance for high-convergence implosions. Simulations (2-D DRACO) predict improved implosion performance and control in both the shell trajectory and morphology for planned intermediate PDD experiments on the NIF. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DE-NA0001944.

  13. Driving Force Dependence of Electron Transfer from Electronically Excited [Ir(COD)(μ-Me2pz)]2 to Photo-Acid Generators.

    PubMed

    Sattler, Wesley; Rachford, Aaron A; LaBeaume, Paul J; Coley, Suzanne M; Thackeray, James W; Cameron, James F; Müller, Astrid M; Winkler, Jay R; Gray, Harry B

    2017-09-28

    We report the rates of electron transfer (ET) reactions of electronically excited [Ir(COD)(μ-Me2pz)]2 with onium salt photoacid generators (PAGs). The reduction potentials of the PAGs span a large electrochemical window that allows determination of the driving force dependence of the ET reactions. Rate constants of ET from electronically excited [Ir(COD)(μ-Me2pz)]2 to onium PAGs are determined by the reaction driving force until the diffusion limit in acetonitrile is reached.

  14. Rapid Stress System Drives Chemical Transfer of Fear from Sender to Receiver

    PubMed Central

    de Groot, Jasper H. B.; Smeets, Monique A. M.; Semin, Gün R.

    2015-01-01

    Humans can register another person’s fear not only with their eyes and ears, but also with their nose. Previous research has demonstrated that exposure to body odors from fearful individuals elicited implicit fear in others. The odor of fearful individuals appears to have a distinctive signature that can be produced relatively rapidly, driven by a physiological mechanism that has remained unexplored in earlier research. The apocrine sweat glands in the armpit that are responsible for chemosignal production contain receptors for adrenalin. We therefore expected that the release of adrenalin through activation of the rapid stress response system (i.e., the sympathetic-adrenal medullary system) is what drives the release of fear sweat, as opposed to activation of the slower stress response system (i.e., hypothalamus-pituitary-adrenal axis). To test this assumption, sweat was sampled while eight participants prepared for a speech. Participants had higher heart rates and produced more armpit sweat in the fast stress condition, compared to baseline and the slow stress condition. Importantly, exposure to sweat from participants in the fast stress condition induced in receivers (N = 31) a simulacrum of the state of the sender, evidenced by the emergence of a fearful facial expression (facial electromyography) and vigilant behavior (i.e., faster classification of emotional facial expressions). PMID:25723720

  15. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

    DOE PAGES

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; ...

    2015-03-02

    Some of the most damaging tree diseases are caused by pathogens that induce cankers, a stem deformation often lethal. To investigate the cause of this adaptation, we sequenced the genomes of poplar pathogens that do and do not cause cankers. We found a unique cluster of genes that produce secondary metabolites and are co-activated when the canker pathogen is grown on poplar wood and leaves. The gene genealogy is discordant with the species phylogeny, showing a signature of horizontal transfer from fungi associated with wood decay. Furthermore, genes encoding hemicellulose-degrading enzymes are up-regulated on poplar wood chips, with some havingmore » been acquired horizontally. In conclusion, we propose that adaptation to colonize poplar woody stems is the result of acquisition of these genes.« less

  16. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

    SciTech Connect

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M.; Levasseur, Anthony; Lindquist, Erika A.; Majoor, Eline; Ohm, Robin A.; Pangilinan, Jasmyn L.; Pribowo, Amadeus; Saddler, John N.; Sakalidis, Monique L.; de Vries, Ronald P.; Grigoriev, Igor V.; Goodwin, Stephen B.; Tanguay, Philippe; Hamelin, Richard C.

    2015-03-02

    Some of the most damaging tree diseases are caused by pathogens that induce cankers, a stem deformation often lethal. To investigate the cause of this adaptation, we sequenced the genomes of poplar pathogens that do and do not cause cankers. We found a unique cluster of genes that produce secondary metabolites and are co-activated when the canker pathogen is grown on poplar wood and leaves. The gene genealogy is discordant with the species phylogeny, showing a signature of horizontal transfer from fungi associated with wood decay. Furthermore, genes encoding hemicellulose-degrading enzymes are up-regulated on poplar wood chips, with some having been acquired horizontally. In conclusion, we propose that adaptation to colonize poplar woody stems is the result of acquisition of these genes.

  17. Radiative transfer and radiative driving of outflows in active galactic nuclei and starbursts

    NASA Astrophysics Data System (ADS)

    Novak, G. S.; Ostriker, J. P.; Ciotti, L.

    2012-12-01

    To facilitate the study of black hole fuelling, star formation and feedback in galaxies, we outline a method for treating the radial forces on interstellar gas due to absorption of photons by dust grains. The method gives the correct behaviour in all of the relevant limits [dominated by the central point source; dominated by the distributed isotropic source; optically thin; optically thick to ultraviolet (UV)/optical; optically thick to infrared (IR)] and reasonably interpolates between the limits when necessary. The method is explicitly energy conserving so that UV/optical photons that are absorbed are not lost, but are rather redistributed to the IR where they may scatter out of the galaxy. We implement the radiative transfer algorithm in a two-dimensional hydrodynamical code designed to study feedback processes in the context of early-type galaxies. We find that the dynamics and final state of simulations are measurably but only moderately affected by radiative forces on dust, even when assumptions about the dust-to-gas ratio are varied from zero to a value appropriate for the Milky Way. In simulations with high gas densities designed to mimic ultraluminous IR galaxies with a star formation rate of several hundred solar masses per year, dust makes a more substantial contribution to the dynamics and outcome of the simulation. We find that, despite the large opacity of dust to UV radiation, the momentum input to the flow from radiation very rarely exceeds L/c due to two factors: the low opacity of dust to the re-radiated IR and the tendency for dust to be destroyed by sputtering in hot gas environments. We also develop a simplification of our radiative transfer algorithm that respects the essential physics but is much easier to implement and requires a fraction of the computational cost.

  18. Virulence plasmid diversity in Clostridium perfringens type D isolates.

    PubMed

    Sayeed, Sameera; Li, Jihong; McClane, Bruce A

    2007-05-01

    Clostridium perfringens type D isolates are important in biodefense and also cause natural enterotoxemias in sheep, goats, and occasionally cattle. In these isolates, the gene (etx) encoding epsilon-toxin is thought to reside on poorly characterized large plasmids. Type D isolates sometimes also produce other potentially plasmid-encoded toxins, including C. perfringens enterotoxin and beta2 toxin, encoded by the cpe and cbp2 genes, respectively. In the current study we demonstrated that the etx, cpe, and cpb2 genes are carried on plasmids in type D isolates and characterized the toxin-encoding plasmids to obtain insight into their genetic organization, potential transferability, and diversity. Southern blotting of pulsed-field gels showed that the etx gene of type D isolates can be present on at least five different plasmids, whose sizes range from 48 to 110 kb. The etx plasmids also typically carried IS1151 and tcp open reading frames (ORFs) known to mediate conjugative transfer of C. perfringens plasmid pCW3. PCR studies revealed that other than their tcp ORFs, etx plasmids of type D isolates do not carry substantial portions of the conserved or variable regions in the cpe plasmids of type A isolates. Southern blotting also demonstrated that in type D isolates the cpe and cpb2 genes are sometimes present on the etx plasmid. Collectively, these findings confirmed that the virulence of type D isolates is heavily plasmid dependent and indicated that (i) a single type D isolate can carry multiple virulence plasmids, (ii) a single type D virulence plasmid can carry up to three different toxin genes, and (iii) many etx plasmids should be capable of conjugative transfer.

  19. [Isolation of the R'his plasmids of Vibrio cholerae].

    PubMed

    Rusina, O Iu; Tiganova, I G; Aleshkin, G I; Andreeva, I V; Skavronskaia, A G

    1987-06-01

    V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.

  20. Acquisition through Horizontal Gene Transfer of Plasmid pSMA198 by Streptococcus macedonicus ACA-DC 198 Points towards the Dairy Origin of the Species

    PubMed Central

    Papadimitriou, Konstantinos; Anastasiou, Rania; Maistrou, Eleni; Plakas, Thomas; Papandreou, Nikos C.; Hamodrakas, Stavros J.; Ferreira, Stéphanie; Supply, Philip; Renault, Pierre; Pot, Bruno; Tsakalidou, Effie

    2015-01-01

    Background Streptococcus macedonicus is an intriguing streptococcal species whose most frequent source of isolation is fermented foods similarly to Streptococcus thermophilus. However, S. macedonicus is closely related to commensal opportunistic pathogens of the Streptococcus bovis/Streptococcus equinus complex. Methodology/Principal Findings We analyzed the pSMA198 plasmid isolated from the dairy strain Streptococcus macedonicus ACA-DC 198 in order to provide novel clues about the main ecological niche of this bacterium. pSMA198 belongs to the narrow host range pCI305/pWV02 family found primarily in lactococci and to the best of our knowledge it is the first such plasmid to be reported in streptococci. Comparative analysis of the pSMA198 sequence revealed a high degree of similarity with plasmids isolated from Lactococcus lactis strains deriving from milk or its products. Phylogenetic analysis of the pSMA198 Rep showed that the vast majority of closely related proteins derive from lactococcal dairy isolates. Additionally, cloning of the pSMA198 ori in L. lactis revealed a 100% stability of replication over 100 generations. Both pSMA198 and the chromosome of S. macedonicus exhibit a high percentage of potential pseudogenes, indicating that they have co-evolved under the same gene decay processes. We identified chromosomal regions in S. macedonicus that may have originated from pSMA198, also supporting a long co-existence of the two replicons. pSMA198 was also found in divergent biotypes of S. macedonicus and in strains isolated from dispersed geographic locations (e.g. Greece and Switzerland) showing that pSMA198’s acquisition is not a recent event. Conclusions/Significance Here we propose that S. macedonicus acquired plasmid pSMA198 from L. lactis via an ancestral genetic exchange event that took place most probably in milk or dairy products. We provide important evidence that point towards the dairy origin of this species. PMID:25584532

  1. Bacteriophages limit the existence conditions for conjugative plasmids.

    PubMed

    Harrison, Ellie; Wood, A Jamie; Dytham, Calvin; Pitchford, Jonathan W; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A

    2015-06-02

    Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. Conjugative plasmids are infectious loops of DNA capable of transmitting DNA between bacterial cells and between species. Because plasmids often carry extra genes that allow bacteria to live in otherwise-inhospitable environments, their dynamics are central to understanding bacterial adaptive evolution. The plasmid-bacterium interaction has typically been studied in isolation, but in natural bacterial communities, bacteriophages, viruses that infect bacteria, are ubiquitous. Using experiments, mathematical models, and computer simulations we show that bacteriophages drive plasmid dynamics through their ecological and evolutionary effects on bacteria and ultimately

  2. Selective contacts drive charge extraction in quantum dot solids via asymmetry in carrier transfer kinetics

    NASA Astrophysics Data System (ADS)

    Mora-Sero, Ivan; Bertoluzzi, Luca; Gonzalez-Pedro, Victoria; Gimenez, Sixto; Fabregat-Santiago, Francisco; Kemp, Kyle W.; Sargent, Edward H.; Bisquert, Juan

    2013-08-01

    Colloidal quantum dot solar cells achieve spectrally selective optical absorption in a thin layer of solution-processed, size-effect tuned, nanoparticles. The best devices built to date have relied heavily on drift-based transport due to the action of an electric field in a depletion region that extends throughout the thickness of the quantum dot layer. Here we study for the first time the behaviour of the best-performing class of colloidal quantum dot films in the absence of an electric field, by screening using an electrolyte. We find that the action of selective contacts on photovoltage sign and amplitude can be retained, implying that the contacts operate by kinetic preferences of charge transfer for either electrons or holes. We develop a theoretical model to explain these experimental findings. The work is the first to present a switch in the photovoltage in colloidal quantum dot solar cells by purposefully formed selective contacts, opening the way to new strategies in the engineering of colloidal quantum dot solar cells.

  3. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

    PubMed Central

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M.; Levasseur, Anthony; Lindquist, Erika A.; Majoor, Eline; Ohm, Robin A.; Pangilinan, Jasmyn L.; Pribowo, Amadeus; Saddler, John N.; Sakalidis, Monique L.; de Vries, Ronald P.; Grigoriev, Igor V.; Goodwin, Stephen B.; Tanguay, Philippe; Hamelin, Richard C.

    2015-01-01

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems. PMID:25733908

  4. Diagnosing Cross-Beam Energy Transfer Using Beamlets of Unabsorbed Light from Direct-Drive Implosions

    NASA Astrophysics Data System (ADS)

    Edgell, D. H.; Follett, R. K.; Goncharov, V. N.; Igumenshchev, I. V.; Katz, J.; Myatt, J. F.; Seka, W.; Froula, D. H.

    2015-11-01

    A new diagnostic is now being fielded to record the unabsorbed laser light from implosions on OMEGA. Unabsorbed light from each OMEGA beam is imaged as a distinct ``spot'' in time-integrated images. Each spot is, in essence, the end point of a beamlet of light that originates from a specific region of a beam profile and follows a path determined by refraction. The intensity of light in the beamlet varies along that path because of absorption and cross-beam energy transfer (CBET) with other beamlets. This diagnostic allows for the detailed investigation of the effects of CBET on specific locations of the beam profile. A pinhole can be used to isolate specific spots, allowing the time-resolved spectrum of the beamlet to be measured. A fully 3-D CBET hydrodynamics code postprocessor is used to model the intensity and wavelength of each beamlet as it traverses the coronal plasma to the diagnostic. The model predicts that if a single beam in a symmetric implosion is turned off, the recorded intensity of nearby spots will decrease by ~ 15% as a result of loss of CBET from the dropped beam. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DE-NA0001944.

  5. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

    PubMed

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M; Levasseur, Anthony; Lindquist, Erika A; Majoor, Eline; Ohm, Robin A; Pangilinan, Jasmyn L; Pribowo, Amadeus; Saddler, John N; Sakalidis, Monique L; de Vries, Ronald P; Grigoriev, Igor V; Goodwin, Stephen B; Tanguay, Philippe; Hamelin, Richard C

    2015-03-17

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.

  6. Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene.

    PubMed Central

    Fuqua, C; Burbea, M; Winans, S C

    1995-01-01

    The Agrobacterium Ti plasmid tra regulon was previously found to be positively regulated by the TraR protein in the presence of a diffusible N-acyl homoserine lactone designated Agrobacterium autoinducer (AAI). TraR and AAI are similar to LuxR from Vibrio fischeri and the Vibrio autoinducer (VAI), which regulate target bioluminescence (lux) genes in a cell density-dependent manner. We now show that tra genes are also regulated by a second protein, designated TraM, which acts to antagonize TraR-dependent activation. The traM gene is closely linked to traR, and the two genes are transcribed convergently. The predicted TraM proteins of two different Ti plasmids are 77% identical but are not significantly similar to other protein sequences in the database, and thus TraM may represent a novel regulatory protein. Null mutations in traM cause strongly increased conjugation, tra gene transcription, and AAI production. A functional copy of traM introduced into traM mutants decreased conjugation, tra gene transcription, and AAI synthesis. TraM inhibits transcription of traA, traI, and traM. Although traM was first identified by its octopine-inducible promoter, we now show that induction by octopine requires traR, strongly suggesting that TraR is the direct traM activator. PMID:7868612

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:26347724

  9. A plasmid from an Antarctic haloarchaeon uses specialized membrane vesicles to disseminate and infect plasmid-free cells.

    PubMed

    Erdmann, Susanne; Tschitschko, Bernhard; Zhong, Ling; Raftery, Mark J; Cavicchioli, Ricardo

    2017-08-21

    The major difference between viruses and plasmids is the mechanism of transferring their genomic information between host cells. Here, we describe the archaeal plasmid pR1SE from an Antarctic species of haloarchaea that transfers via a mechanism similar to a virus. pR1SE encodes proteins that are found in regularly shaped membrane vesicles, and the vesicles enclose the plasmid DNA. The released vesicles are capable of infecting a plasmid-free strain, which then gains the ability to produce plasmid-containing vesicles. pR1SE can integrate and replicate as part of the host genome, resolve out with fragments of host DNA incorporated or portions of the plasmid left behind, form vesicles and transfer to new hosts. The pR1SE mechanism of transfer of DNA could represent the predecessor of a strategy used by viruses to pass on their genomic DNA and fulfil roles in gene exchange, supporting a strong evolutionary connection between plasmids and viruses.An archaeal plasmid that can be transported in membrane vesicles, similar to a virus, and encodes proteins that can insert into host membranes and membrane vesicles, provides insights into the evolutionary link between plasmids and viruses.

  10. Reduction of electron deficient guanine radical species in plasmid DNA by tyrosine derivatives.

    PubMed

    Tsoi, Mandi; Do, Trinh T; Tang, Vicky J; Aguilera, Joseph A; Milligan, Jamie R

    2010-06-07

    Guanine bases are the most easily oxidized sites in DNA and therefore electron deficient guanine radical species are major intermediates in the direct effect of ionizing radiation (ionization of the DNA itself) on DNA as a consequence of hole migration to guanine. As a model for this process we have used gamma-irradiation in the presence of thiocyanate ions to generate single electron oxidized guanine radicals in a plasmid target in aqueous solution. The stable species formed from these radicals can be detected and quantified by the formation of strand breaks in the plasmid after a post-irradiation incubation using a suitable enzyme. If a tyrosine derivative is also present during irradiation, the production of guanine oxidation products is decreased by electron transfer from tyrosine to the intermediate guanyl radical species. By using cationic tyrosine containing ligands we are able to observe this process when the tyrosine is electrostatically bound to the plasmid. The driving force dependence of this reaction was determined by comparing the reactivity of tyrosine with its 3-nitro analog. The results imply that the electron transfer reaction is coupled to a proton transfer. The experimental conditions used in this model system provide a reasonable approximation to those involved in the radioprotection of DNA by tightly bound proteins in chromatin.

  11. The conjugative plasmid of a bean-nodulating Sinorhizobium fredii strain is assembled from sequences of two Rhizobium plasmids and the chromosome of a Sinorhizobium strain

    PubMed Central

    2011-01-01

    Background Bean-nodulating Rhizobium etli originated in Mesoamerica, while soybean-nodulating Sinorhizobium fredii evolved in East Asia. S. fredii strains, such as GR64, have been isolated from bean nodules in Spain, suggesting the occurrence of conjugative transfer events between introduced and native strains. In R. etli CFN42, transfer of the symbiotic plasmid (pRet42d) requires cointegration with the endogenous self-transmissible plasmid pRet42a. Aiming at further understanding the generation of diversity among bean nodulating strains, we analyzed the plasmids of S. fredii GR64: pSfr64a and pSfr64b (symbiotic plasmid). Results The conjugative transfer of the plasmids of strain GR64 was analyzed. Plasmid pSfr64a was self-transmissible, and required for transfer of the symbiotic plasmid. We sequenced pSfr64a, finding 166 ORFs. pSfr64a showed three large segments of different evolutionary origins; the first one presented 38 ORFs that were highly similar to genes located on the chromosome of Sinorhizobium strain NGR234; the second one harbored 51 ORFs with highest similarity to genes from pRet42d, including the replication, but not the symbiosis genes. Accordingly, pSfr64a was incompatible with the R. etli CFN42 symbiotic plasmid, but did not contribute to symbiosis. The third segment contained 36 ORFs with highest similarity to genes localized on pRet42a, 20 of them involved in conjugative transfer. Plasmid pRet42a was unable to substitute pSfr64a for induction of pSym transfer, and its own transfer was significantly diminished in GR64 background. The symbiotic plasmid pSfr64b was found to differ from typical R. etli symbiotic plasmids. Conclusions S. fredii GR64 contains a chimeric transmissible plasmid, with segments from two R. etli plasmids and a S. fredii chromosome, and a symbiotic plasmid different from the one usually found in R. etli bv phaseoli. We infer that these plasmids originated through the transfer of a symbiotic-conjugative-plasmid cointegrate

  12. Yeast DNA plasmids.

    PubMed

    Gunge, N

    1983-01-01

    The study of yeast DNA plasmids has been initiated with the discovery of the 2-micron DNA in Saccharomyces cerevisiae. This multiple copy plasmid, organized into chromatin structure in vivo, probably exists in the nucleus and provides a good system to obtain information on eukaryotic DNA replication. Yeast transformation with the 2-micron DNA or artificially constructed chimeric plasmids had contributed significantly to the study of the molecular biology of yeast and eukaryotes, allowing the isolation and characterization of various genes, ars, centromeres, and telomeres, and also serving as a tool to study the expression of various heterologous genes. Encouraged by these fruitful results, new yeast plasmids have been screened among phylogenetically distant yeasts. The linear DNA plasmids (pGKl1 and pGKl2) from Kluyveromyces lactis are the first case of yeast plasmids associated with biological function (killer phenotype). This plasmid system would be ideal as a model to study the structure and function of eukaryotic linear chromosomes. The extracellular secretion of protein toxin suggests the plasmids to be an excellent candidate for a secretion vector. The importance of yeasts as suitable materials for the study of eukaryotic cell biology would be much enhanced by the advent of new transformation systems with diverse host yeasts of genetically and phylogenetically distinct properties.

  13. Ecological and genetic determinants of plasmid distribution in Escherichia coli.

    PubMed

    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.

  14. A wave-based model for cross-beam energy transfer in direct-drive inertial confinement fusion

    NASA Astrophysics Data System (ADS)

    Myatt, J. F.; Follett, R. K.; Shaw, J. G.; Edgell, D. H.; Froula, D. H.; Igumenshchev, I. V.; Goncharov, V. N.

    2017-05-01

    Cross-beam energy transfer (CBET) is thought to be responsible for a 30% reduction in hydrodynamic coupling efficiency on OMEGA and up to 50% at the ignition scale for direct-drive (DD) implosions. These numbers are determined by ray-based models that have been developed and integrated within the radiation-hydrodynamics codes LILAC (1-D) and DRACO (2-D). However, ray-based modeling of CBET in an inhomogeneous plasma assumes a steady-state plasma response, does not include the effects of beam speckle, and treats ray caustics in an ad hoc manner. The validity of the modeling for ignition-scale implosions has not yet been determined. To address the physics shortcomings, which have important implications for DD inertial confinement fusion, a new wave-based model has been developed. It solves the time-enveloped Maxwell equations in three dimensions, including polarization effects, plasma inhomogeneity, and open-boundary conditions with the ability to prescribe beams incident at arbitrary angles. Beams can be made realistic with respect to laser speckle, polarization smoothing, and laser bandwidth. This, coupled to a linearized low-frequency plasma response that does not assume a steady state, represents the most-complete model of CBET to date.

  15. The Effect of Cross-Beam Energy Transfer on Two-Plasmon Decay in Direct-Drive Implosions

    NASA Astrophysics Data System (ADS)

    Froula, D. H.; Follett, R. K.; Henchen, R. J.; Davis, A. K.; Goncharov, V. N.; Edgell, D. H.; Solodov, A. A.; Michel, D. T.; Myatt, J. F.; Shaw, J. G.; Stoeckl, C.

    2016-10-01

    Mitigation of cross-beam energy transfer (CBET) in direct-drive implosions was shown to increase the hot electrons generated by two-plasmon decay. Reducing the diameter of the laser spots by 30% significantly reduces CBET and the laser absorption was measured to increase from 75% to nearly 90%. The reduced CBET leads to higher intensity at the quarter-critical density surface, increasing the hot-electron production by a factor of 7 . Adding a thin layer (0.6 to 1.1 μm) of Si to the target ablator reduced the hot-electron fraction by a factor of 2 . Spatially resolved Thomson-scattering measurements show an 15 % increase in the electron temperature and an increase in the Si fraction at the quarter-critical surface when the Si layer is added. Three-dimensional laser-plasma interaction simulations of hot-electron production using the code LPSE show that in addition to the reduced gain (smaller ILn Te), the observed reduction in hot electrons results from increased electron-ion collision frequencies and reduced Landau damping of ion-acoustic waves. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DE-NA0001944.

  16. Functional Subsets of the VirB Type IV Transport Complex Proteins Involved in the Capacity of Agrobacterium tumefaciens To Serve as a Recipient in virB-Mediated Conjugal Transfer of Plasmid RSF1010

    PubMed Central

    Liu, Zhenying; Binns, Andrew N.

    2003-01-01

    The virB-encoded type IV transport complex of Agrobacterium tumefaciens mediates the transfer of DNA and proteins into plant cells, as well as the conjugal transfer of IncQ plasmids, such as RSF1010, between Agrobacterium strains. While several studies have indicated that there are physical interactions among the 11 VirB proteins, the functional significance of the interactions has been difficult to establish since all of the proteins are required for substrate transfer. Our previous studies, however, indicated that although all of the VirB proteins are required for the capacity of a strain to serve as an RSF1010 donor, only a subset of these proteins in the recipient is necessary to increase the conjugal frequency by 3 to 4 logs. The roles of particular groups of VirB proteins in this increased recipient activity were examined in the study reported here. Examination of the expression of subgroups of virB genes revealed that translation of virB6 is necessary for expression of downstream open reading frames. Expression of limited subsets of the VirB proteins in a recipient strain lacking the Ti plasmid revealed that the VirB7 to VirB10 proteins yield a subcomplex that is functional in the recipient assay but that the VirB1 to VirB4 proteins, as a group, dramatically increase this activity in strains expressing VirB7 to VirB10. Finally, the membrane distribution and cross-linking patterns of VirB10, but not of VirB8 or VirB9, in a strain expressing only VirB7 to VirB10 are significantly altered compared to the patterns of the wild type. These characteristics are, however, restored to the wild-type status by coexpression of VirB1 to VirB3. Taken together, these results define subsets of type IV transport complex proteins that are critical in allowing a strain to participate as a recipient in virB-mediated conjugal RSF1010 transfer. PMID:12754223

  17. Parameters controlling interbacterial plasmid spreading in a gnotoxenic chicken gut system: influence of plasmid and bacterial mutations.

    PubMed Central

    Sansonetti, P; Lafont, J P; Jaffé-Brachet, A; Guillot, J F; Chaslus-Dancla, E

    1980-01-01

    Conjugative transfer of R plasmids R64 and R64drd-11 has been compared in vitro and in vivo without selective pressure by antibiotics in a simplified experimental system; the ecosystem was the bowel of germfree chickens, with the host bacteria almost isogenic, and the plasmids differing only in their conjugative transfer frequency. The spread of repressed and derepressed (drd) R plasmids in recipient bacterial populations was very extensive. The repressed phenotype had only a transient effect during the first 4 h. The level of implantation of the donor bacterial population seems to be of minor importance. Only with a poor recipient (con strain) could the spread of R plasmids be reduced and a steady state with a predominantly sensitive bacterial population be established. It is suggested that this steady state results from an equilibrium between the frequencies of R plasmid transfer and loss. PMID:6999980

  18. The Agrobacterium Ti Plasmids.

    PubMed

    Christie, Peter J; Gordon, Jay E

    2014-12-01

    Agrobacterium tumefaciens is a plant pathogen with the capacity to deliver a segment of oncogenic DNA carried on a large plasmid called the tumor-inducing or Ti plasmid to susceptible plant cells. A. tumefaciens belongs to the class Alphaproteobacteria, whose members include other plant pathogens (Agrobacterium rhizogenes), plant and insect symbionts (Rhizobium spp. and Wolbachia spp., respectively), human pathogens (Brucella spp., Bartonella spp., Rickettsia spp.), and nonpathogens (Caulobacter crescentus, Rhodobacter sphaeroides). Many species of Alphaproteobacteria carry large plasmids ranging in size from ∼100 kb to nearly 2 Mb. These large replicons typically code for functions essential for cell physiology, pathogenesis, or symbiosis. Most of these elements rely on a conserved gene cassette termed repABC for replication and partitioning, and maintenance at only one or a few copies per cell. The subject of this review is the ∼200-kb Ti plasmids carried by infectious strains of A. tumefaciens. We will summarize the features of this plasmid as a representative of the repABC family of megaplasmids. We will also describe novel features of this plasmid that enable A. tumefaciens cells to incite tumor formation in plants, sense and respond to an array of plant host and bacterial signal molecules, and maintain and disseminate the plasmid among populations of agrobacteria. At the end of this review, we will describe how this natural genetic engineer has been adapted to spawn an entire industry of plant biotechnology and review its potential for use in future therapeutic applications of plant and nonplant species.

  19. A Wave-Based Model for Cross-Beam Energy Transfer in Direct-Drive Inertial Confinement Fusion Implosions

    NASA Astrophysics Data System (ADS)

    Myatt, J. F.

    2016-10-01

    Cross-beam energy transfer (CBET) is thought to be responsible for an 30 % reduction in hydrodynamic coupling efficiency on OMEGA and up to 50% at the ignition scale for direct-drive (DD) implosions. These numbers are determined by ray-based models that have been developed and integrated within the radiation-hydrodynamics codes LILAC (1-D) and DRACO (2-D). However, ray-based modeling of CBET in an inhomogeneous plasma assumes a steady-state plasma response, does not include the effects of beam speckle, and ray caustics are treated in an ad hoc manner. Nevertheless, simulation results are in good qualitative agreement with implosion experiments on OMEGA (when combined with a model for nonlocal heat transport). The validity of the modeling for ignition-scale implosions has not yet been determined. To address the physics shortcomings, which have important implications for DD inertial confinement fusion, a new wave-based model has been constructed. It solves the time-enveloped Maxwell equations in three-dimensions, including polarization effects, plasma inhomogeneity, and open-boundary conditions with the ability to prescribe beams incident at arbitrary angles. Beams can be made realistic with respect to laser speckle, polarization smoothing, and laser bandwidth. This, coupled to a linearized low-frequency plasma response that does not assume a steady state, represents the most-complete model of CBET to date. New results will be presented and the implications for CBET modeling and mitigation will be described. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DENA0001944, in collaboration with J. G. Shaw, R. K. Follett, and D. H. Edgell (LLE).

  20. Domain Wall Displacement Detection Magnetooptical Drive for Mobile Use, with 15 Gbit/inch2 Recording Density and 24 Mbps Data Transfer Rate

    NASA Astrophysics Data System (ADS)

    Nagura, Chihiro; Torii, Shinnosuke; Ishii, Kazuyoshi; Nagatsuka, Osamu; Nishikawa, Koichiro; Miyaoka, Yasuyuki; Suzuki, Kazunori; Koyama, Osamu

    2004-07-01

    We have developed a domain wall displacement detection (DWDD) magnetooptical drive for the 2-inch-diameter disc with 3 GB capacity. The newly developed magnetic head and the drive circuit realize the high efficiency of 1.84 Oe/mA and the high speed switching of 13.5 ns. An optical pickup (OPU) with wavelength of 660 nm and numerical aperture (NA) of 0.6 is sufficiently compact to approach the innermost disc radius of 11 mm, and the overall weight is 3.2 g. The volume and power consumption of the entire drive system are 35.5 cc and 0.78 W respectively, and they are adequate for use in mobile storage devices. Furthermore, we have confirmed a complete read/write performance with 15 Gbit/inch2 recording density and 24 Mbps data transfer rate.

  1. DNA Sequence Analysis of Plasmids from Multidrug Resistant Salmonella enterica Serotype Heidelberg Isolates

    PubMed Central

    David, Donna E.; Tang, Hailin; Xu, Joshua; Nayak, Rajesh; Kaldhone, Pravin; Logue, Catherine M.; Foley, Steven L.

    2012-01-01

    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. PMID:23251446

  2. Chlamydial plasmids and bacteriophages.

    PubMed

    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.

  3. Occurrence and persistence of indigenous transconjugants carrying conjugative plasmids in soil.

    PubMed

    Inoue, Daisuke; Soda, Satoshi; Tsutsui, Hirofumi; Yamazaki, Yuji; Murashige, Katsushi; Sei, Kazunari; Fujita, Masanori; Ike, Michihiko

    2009-09-01

    The transfer of the self-transmissible plasmids, RP4 and pJP4, from introduced bacteria to indigenous bacteria was examined in soil and slurry microcosms. The introduced plasmids persisted in indigenous transconjugants despite the low survival of introduced donors. The potential of the transconjugants for growth and conjugation affects the persistence of introduced plasmids in soil.

  4. Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine.

    PubMed Central

    Haefeli, C; Franklin, C; Hardy, K

    1984-01-01

    A silver-resistant strain of Pseudomonas stutzeri was isolated from a silver mine. It harbored three plasmids, the largest of which (pKK1; molecular weight, 49.4 X 10(6)) specified silver resistance. Plasmid pKK1 was apparently nonconjugative but could be transferred to Pseudomonas putida by mobilization with plasmid R68.45. Images PMID:6715284

  5. Prevalence of ColE1-like plasmids and kanamycinr resistance genes in Salmonella enterica serotypes

    USDA-ARS?s Scientific Manuscript database

    Multi-antibiotic resistant Salmonella enterica serotypes are increasing in prevalence and concern in human and animal health. Many strains carry resistance determinants on plasmids; current practices focus heavily on large plasmids, and the role that small plasmids play in resistance gene transfer ...

  6. Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae.

    PubMed

    Bossé, Janine T; Li, Yanwen; Atherton, Tom G; Walker, Stephanie; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Holden, Matthew T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

    2015-08-05

    The complete nucleotide sequence of a 7.7kb mobilisable plasmid (pM3446F), isolated from a florfenicol resistant isolate of Actinobacillus pleuropneumoniae, showed extended similarity to plasmids found in other members of the Pasteurellaceae containing the floR gene as well as replication and mobilisation genes. Mobilisation into other Pasteurellaceae species confirmed that this plasmid can be transferred horizontally.

  7. Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10

    SciTech Connect

    Hill, K.E.; Weightman, A.J.; Fry, J.C. )

    1992-04-01

    This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 {times} 10{sup {minus}8} to 4.5 {times} 10{sup {minus}3} per recipient at 20C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of {beta}- and {gamma}-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.

  8. Plasmid-determined resistance to fosfomycin in Serratia marcescens.

    PubMed Central

    Mendoza, C; Garcia, J M; Llaneza, J; Mendez, F J; Hardisson, C; Ortiz, J M

    1980-01-01

    Multiple-antibiotic-resistant strains of Serratia marcescens isolated from hospitalized patients were examined for their ability to transfer antibiotic resistance to Escherichia coli by conjugation. Two different patterns of linked transferable resistance were found among the transconjugants. The first comprised resistance to carbenicillin, streptomycin, and fosfomycin; the second, and more common, pattern included resistance to carbenicillin, streptomycin, kanamycin, gentamicin, tetracycline, chloramphenicol, sulfonamide, and fosfomycin. The two types of transconjugant strains carried a single plasmid of either 57 or 97 megadaltons in size. Both of these plasmids are present in parental S. marcescens strains resistant to fosfomycin. The 57-megadalton plasmid was transformed into E. coli. Images PMID:7004337

  9. Plasmid Introduction in Metal-Stressed, Subsurface-Derived Microcosms: Plasmid Fate and Community Response

    PubMed Central

    Smets, Barth F.; Morrow, Jayne B.; Arango Pinedo, Catalina

    2003-01-01

    The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 μM CdCl2), permitting long-term community monitoring. The broad-host-range IncPα plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cdr or Nir density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Nir transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed. PMID:12839785

  10. Plasmid-mediate transfer of FLO1 into industrial Saccharomyces cerevisiae PE-2 strain creates a strain useful for repeat-batch fermentations involving flocculation-sedimentation.

    PubMed

    Gomes, Daniel G; Guimarães, Pedro M R; Pereira, Francisco B; Teixeira, José A; Domingues, Lucília

    2012-03-01

    The flocculation gene FLO1 was transferred into the robust industrial strain Saccharomyces cerevisiae PE-2 by the lithium acetate method. The recombinant strain showed a fermentation performance similar to that of the parental strain. In 10 repeat-batch cultivations in VHG medium with 345 g glucose/L and cell recycling by flocculation-sedimentation, an average final ethanol concentration of 142 g/L and an ethanol productivity of 2.86 g/L/h were achieved. Due to the flocculent nature of the recombinant strain it is possible to reduce the ethanol production cost because of lower centrifugation and distillation costs.

  11. Electrotransfer of Plasmid Vector DNA into Muscle

    NASA Astrophysics Data System (ADS)

    Miyazaki, Satsuki; Miyazaki, Jun-Ichi

    Wolff et al. (1990) first reported that plasmid DNA injected into skeletal muscle is taken up by muscle cells and the genes in the plasmid are expressed for more than two months thereafter, although the transfected DNA does not usually undergo chromosomal integration (Wolff et al., 1991, 1992). However, the relatively low expression levels attained by this method have hampered its applications for uses other than as a DNA vaccine (Davis et al., 1995). There are a number of reports analyzing the conditions that affect the efficiency of gene transfer by intramuscular DNA injection and assessing the fine structures of expression plasmid vectors that may affect expression levels (Davis et al., 1993; Liang et al., 1996; Norman et al., 1997). Furthermore, various attempts were done to improve the efficiency of gene transfer by intramus cular DNA injection. Consequently, regenerating muscle was shown to produce 80-fold or more protein than did normal muscle, following injection of an expression plas-mid. Muscle regeneration was induced by treatment with cardiotoxin or bupivacaine (Wells, 1993; Vitadello et al., 1994). We previously demonstrated that by combining a strong promoter and bupivacaine pretreatment intramuscular injection of an IL-5 expression plasmid results in IL-5 production in muscle at a level sufficient to induce marked proliferation of eosinophils in the bone marrow and eosinophil infiltration of various organs (Tokui et al., 1997). It was also reported that a single intramuscular injection of an erythropoietin expression plasmid produced physiologically significant elevations in serum erythropoietin levels and increased hematocrits in adult mice (Tripathy et al., 1996). Hematocrits in these animals remained elevated at >60% for at least 90 days after a single injection. However, improvements to this method have not been sufficient to extend its applications including clinical use.

  12. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  13. Horizontal Transfer of the Salmonella enterica Serovar Infantis Resistance and Virulence Plasmid pESI to the Gut Microbiota of Warm-Blooded Hosts

    PubMed Central

    Aviv, Gili; Rahav, Galia

    2016-01-01

    ABSTRACT Salmonella enterica serovar Infantis is one of the prevalent salmonellae worldwide. Recently, we showed that the emergence of S. Infantis in Israel was facilitated by the acquisition of a unique megaplasmid (pESI) conferring multidrug resistance and increased virulence phenotypes. Here we elucidate the ecology, transmission properties, and regulation of pESI. We show that despite its large size (~280 kb), pESI does not impose a significant metabolic burden in vitro and that it has been recently fixed in the domestic S. Infantis population. pESI conjugation and the transcription of its pilus (pil) genes are inhibited at the ambient temperature (27°C) and by ≥1% bile but increased under temperatures of 37 to 41°C, oxidative stress, moderate osmolarity, and the microaerobic conditions characterizing the intestinal environment of warm-blooded animals. The pESI-encoded protein TraB and the oxygen homeostasis regulator Fnr were identified as transcriptional regulators of pESI conjugation. Using the mouse model, we show that following S. Infantis infection, pESI can be horizontally transferred to the gut microbiota, including to commensal Escherichia coli strains. Possible transfer, but not persistence, of pESI was also observed into Gram-positive mouse microbiota species, especially Lactobacillus reuteri. Moreover, pESI was demonstrated to further disseminate from gut microbiota to S. enterica serovar Typhimurium, in the context of gastrointestinal infection. These findings exhibit the ability of a selfish clinically relevant megaplasmid to distribute to and from the microbiota and suggest an overlooked role of the microbiota as a reservoir of mobile genetic elements and intermediator in the spread of resistance and virulence genes between commensals and pathogenic bacteria. PMID:27601577

  14. Amelioration of the cost of conjugative plasmid carriage in Eschericha coli K12.

    PubMed Central

    Dahlberg, Cecilia; Chao, Lin

    2003-01-01

    Although plasmids can provide beneficial functions to their host bacteria, they might confer a physiological or energetic cost. This study examines how natural selection may reduce the cost of carrying conjugative plasmids with drug-resistance markers in the absence of antibiotic selection. We studied two plasmids, R1 and RP4, both of which carry multiple drug resistance genes and were shown to impose an initial fitness cost on Escherichia coli. To determine if and how the cost could be reduced, we subjected plasmid-containing bacteria to 1100 generations of evolution in batch cultures. Analysis of the evolved populations revealed that plasmid loss never occurred, but that the cost was reduced through genetic changes in both the plasmids and the bacteria. Changes in the plasmids were inferred by the demonstration that evolved plasmids no longer imposed a cost on their hosts when transferred to a plasmid-free clone of the ancestral E. coli. Changes in the bacteria were shown by the lowered cost when the ancestral plasmids were introduced into evolved bacteria that had been cured of their (evolved) plasmids. Additionally, changes in the bacteria were inferred because conjugative transfer rates of evolved R1 plasmids were lower in the evolved host than in the ancestral host. Our results suggest that once a conjugative bacterial plasmid has invaded a bacterial population it will remain even if the original selection is discontinued. PMID:14704155

  15. Physical and genetic analysis of the ColD plasmid.

    PubMed

    Frey, J; Ghersa, P; Palacios, P G; Belet, M

    1986-04-01

    The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis. Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s). A detailed map of the ColD plasmid was established for 10 restriction enzymes. Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi). These genes were all located contiguously on a 2,400-base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF). The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP. The location of the mobilization functions was determined by deletion analysis. The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication.

  16. Physical and genetic analysis of the ColD plasmid.

    PubMed Central

    Frey, J; Ghersa, P; Palacios, P G; Belet, M

    1986-01-01

    The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis. Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s). A detailed map of the ColD plasmid was established for 10 restriction enzymes. Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi). These genes were all located contiguously on a 2,400-base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF). The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP. The location of the mobilization functions was determined by deletion analysis. The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication. Images PMID:3007432

  17. Community-wide plasmid gene mobilization and selection

    PubMed Central

    Sentchilo, Vladimir; Mayer, Antonia P; Guy, Lionel; Miyazaki, Ryo; Green Tringe, Susannah; Barry, Kerrie; Malfatti, Stephanie; Goessmann, Alexander; Robinson-Rechavi, Marc; van der Meer, Jan R

    2013-01-01

    Plasmids have long been recognized as an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from the host's chromosome and their frequent self-transfer. It is thought that plasmids accumulate, rearrange and distribute nonessential genes, which may provide an advantage for host proliferation under selective conditions. In order to test this hypothesis independently of biases from culture selection, we study the plasmid metagenome from microbial communities in two activated sludge systems, one of which receives mostly household and the other chemical industry wastewater. We find that plasmids from activated sludge microbial communities carry among the largest proportion of unknown gene pools so far detected in metagenomic DNA, confirming their presumed role of DNA innovators. At a system level both plasmid metagenomes were dominated by functions associated with replication and transposition, and contained a wide variety of antibiotic and heavy metal resistances. Plasmid families were very different in the two metagenomes and grouped in deep-branching new families compared with known plasmid replicons. A number of abundant plasmid replicons could be completely assembled directly from the metagenome, providing insight in plasmid composition without culturing bias. Functionally, the two metagenomes strongly differed in several ways, including a greater abundance of genes for carbohydrate metabolism in the industrial and of general defense factors in the household activated sludge plasmid metagenome. This suggests that plasmids not only contribute to the adaptation of single individual prokaryotic species, but of the prokaryotic community as a whole under local selective conditions. PMID:23407308

  18. Phytoplasma plasmid DNA extraction.

    PubMed

    Andersen, Mark T; Liefting, Lia W

    2013-01-01

    Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA.

  19. Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids.

    PubMed

    Walsh, P M; McKay, L L

    1982-05-01

    We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.

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

    PubMed Central

    Klümper, Uli; Riber, Leise; Dechesne, Arnaud; Sannazzarro, Analia; Hansen, Lars H; Sørensen, Søren J; Smets, Barth F

    2015-01-01

    Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes 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 donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids. PMID:25333461

  1. Plasmids in different strains of Streptomyces ambofaciens: free and integrated form of plasmid pSAM2.

    PubMed

    Pernodet, J L; Simonet, J M; Guérineau, M

    1984-01-01

    Five strains of Streptomyces ambofaciens were examined for their plasmid content. Among these strains, four belong to the same lineage (strains B) and the other was isolated independently (strain A). A large plasmid (ca. 80 kb), called pSAM1 in this paper and already described, was present in all B strains, and absent in strain A. A second plasmid, not described before, was found as covalently closed circular DNA in two of the four B strains. This plasmid with a size of 11.1 kb was called pSAM2. A restriction map for 14 enzymes was established. Hybridization experiments showed that a unique sequence homologous to this plasmid is integrated in a larger replicon, which is not pSAM1 and is probably the chromosome, in all B strains and not in strain A. It seems probable that the integrated sequence is the origin of the free plasmid found in two strains of the B family. It is noteworthy that the integrated form and the free plasmid may be found together. Transformation experiments proved that pSAM2 may be maintained autonomously in S. ambofaciens strain A and in S. lividans. pSAM2 is a self-transmissible plasmid, able to elicit the lethal zygosis reaction. pSAM2 was compared to the plasmids SLP1, pIJ110 and pIJ408, which all come from integrated sequences in three Streptomyces species and are found as autonomous plasmids after transfer to S. lividans. If pSAM2 resembles these plasmids in its origin, it does not appear to be related directly to them. Concerning their plasmid content, the two isolates of S. ambofaciens are very different.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Plasmid interference for curing antibiotic resistance plasmids in vivo.

    PubMed

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M; Partridge, Sally R; Iredell, Jonathan R

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

  3. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    PubMed

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  4. Chemotherapy of Bacterial Plasmids

    DTIC Science & Technology

    1979-01-29

    multiresistance to chemotherapeutic drugs, mediated drug resistance are the emergence of strains determined by R-plasmids, causes treatment failures of Haemophilus ... influenzae , resistant to ampicillin [8] of hospital infections, foremost in patients with a or chloramphenicol [9] and of Neisseria gonorrhoeae

  5. Plasmids of Legionella Species.

    DTIC Science & Technology

    1982-06-18

    these organisms could support the replication of other plasmids. In recent years, there have been epidemics of typhoid fever in Vietnam and Mexico due...and M. Pollack. 1973. Chloram- phenicol-resistant typhoid fever in Vietnam associated with R factor. Lancet ii:983-985. IA I~ ! l -- +t.-t, lo. re

  6. Plasmids as Tools for Containment.

    PubMed

    García, José L; Díaz, Eduardo

    2014-10-01

    Active containment systems are a major tool for reducing the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats for a large number of biotechnological applications (e.g., bioremediation, bioleaching, biopesticides, biofuels, biotransformations, live vaccines, etc.). While biological containment reduces the survival of the introduced organism outside the target habitat and/or upon completion of the projected task, gene containment strategies reduce the lateral spread of the key genetic determinants to indigenous microorganisms. In fundamental research, suicide circuits become relevant tools to address the role of gene transfer, mainly plasmid transfer, in evolution and how this transfer contributes to genome plasticity and to the rapid adaptation of microbial communities to environmental changes. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. As many new genomes are being sequenced, novel lethal genes and regulatory elements are available, e.g., new toxin-antitoxin modules, and they could be used to increase further the current containment efficiencies and to expand containment to other organisms. Although the current containment systems can increase the predictability of genetically modified organisms in the environment, containment will never be absolute, due to the existence of mutations that lead to the appearance of surviving subpopulations. In this sense, orthogonal systems (xenobiology) appear to be the solution for setting a functional genetic firewall that will allow absolute containment of recombinant organisms.

  7. Plasmid interference for curing antibiotic resistance plasmids in vivo

    PubMed Central

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M.; Partridge, Sally R.

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored. PMID:28245276

  8. Plasmid-Mediated Antimicrobial Resistance in Staphylococci and Other Firmicutes.

    PubMed

    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.

  9. An updated view of plasmid conjugation and mobilization in Staphylococcus

    PubMed Central

    Ramsay, Joshua P.; Kwong, Stephen M.; Murphy, Riley J. T.; Yui Eto, Karina; Price, Karina J.; Nguyen, Quang T.; O'Brien, Frances G.; Grubb, Warren B.; Coombs, Geoffrey W.; Firth, Neville

    2016-01-01

    ABSTRACT The horizontal gene transfer facilitated by mobile genetic elements impacts almost all areas of bacterial evolution, including the accretion and dissemination of antimicrobial-resistance genes in the human and animal pathogen Staphylococcus aureus. Genome surveys of staphylococcal plasmids have revealed an unexpected paucity of conjugation and mobilization loci, perhaps suggesting that conjugation plays only a minor role in the evolution of this genus. In this letter we present the DNA sequences of historically documented staphylococcal conjugative plasmids and highlight that at least 3 distinct and widely distributed families of conjugative plasmids currently contribute to the dissemination of antimicrobial resistance in Staphylococcus. We also review the recently documented “relaxase-in trans” mechanism of conjugative mobilization facilitated by conjugative plasmids pWBG749 and pSK41, and discuss how this may facilitate the horizontal transmission of around 90% of plasmids that were previously considered non-mobilizable. Finally, we enumerate unique sequenced S. aureus plasmids with a potential mechanism of mobilization and predict that at least 80% of all non-conjugative S. aureus plasmids are mobilizable by at least one mechanism. We suggest that a greater research focus on the molecular biology of conjugation is essential if we are to recognize gene-transfer mechanisms from our increasingly in silico analyses. PMID:27583185

  10. Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:27923922

  11. Detection and Characterization of Conjugative Degradative Plasmids in Xenobiotic-Degrading Sphingomonas Strains

    PubMed Central

    Basta, Tamara; Keck, Andreas; Klein, Joachim; Stolz, Andreas

    2004-01-01

    A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained. PMID:15175300

  12. Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains.

    PubMed

    Basta, Tamara; Keck, Andreas; Klein, Joachim; Stolz, Andreas

    2004-06-01

    A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.

  13. Dynamic protein conformations preferentially drive energy transfer along the active chain of the photosystem II reaction centre

    NASA Astrophysics Data System (ADS)

    Zhang, Lu; Silva, Daniel-Adriano; Zhang, Houdao; Yue, Alexander; Yan, Yijing; Huang, Xuhui

    2014-06-01

    One longstanding puzzle concerning photosystem II, a core component of photosynthesis, is that only one of the two symmetric branches in its reaction centre is active in electron transfer. To investigate the effect of the photosystem II environment on the preferential selection of the energy transfer pathway (a prerequisite for electron transfer), we have constructed an exciton model via extensive molecular dynamics simulations and quantum mechanics/molecular mechanics calculations based on a recent X-ray structure. Our results suggest that it is essential to take into account an ensemble of protein conformations to accurately compute the site energies. We identify the cofactor CLA606 of active chain as the most probable site for the energy excitation. We further pinpoint a number of charged protein residues that collectively lower the CLA606 site energy. Our work provides insights into the understanding of molecular mechanisms of the core machinery of the green-plant photosynthesis.

  14. Dynamic protein conformations preferentially drive energy transfer along the active chain of the photosystem II reaction centre.

    PubMed

    Zhang, Lu; Silva, Daniel-Adriano; Zhang, Houdao; Yue, Alexander; Yan, YiJing; Huang, Xuhui

    2014-06-23

    One longstanding puzzle concerning photosystem II, a core component of photosynthesis, is that only one of the two symmetric branches in its reaction centre is active in electron transfer. To investigate the effect of the photosystem II environment on the preferential selection of the energy transfer pathway (a prerequisite for electron transfer), we have constructed an exciton model via extensive molecular dynamics simulations and quantum mechanics/molecular mechanics calculations based on a recent X-ray structure. Our results suggest that it is essential to take into account an ensemble of protein conformations to accurately compute the site energies. We identify the cofactor CLA606 of active chain as the most probable site for the energy excitation. We further pinpoint a number of charged protein residues that collectively lower the CLA606 site energy. Our work provides insights into the understanding of molecular mechanisms of the core machinery of the green-plant photosynthesis.

  15. Dynamic protein conformations preferentially drive energy transfer along the active chain of the photosystem II reaction centre

    PubMed Central

    Zhang, Lu; Silva, Daniel-Adriano; Zhang, Houdao; Yue, Alexander; Yan, YiJing; Huang, Xuhui

    2014-01-01

    One longstanding puzzle concerning photosystem II, a core component of photosynthesis, is that only one of the two symmetric branches in its reaction centre is active in electron transfer. To investigate the effect of the photosystem II environment on the preferential selection of the energy transfer pathway (a prerequisite for electron transfer), we have constructed an exciton model via extensive molecular dynamics simulations and quantum mechanics/molecular mechanics calculations based on a recent X-ray structure. Our results suggest that it is essential to take into account an ensemble of protein conformations to accurately compute the site energies. We identify the cofactor CLA606 of active chain as the most probable site for the energy excitation. We further pinpoint a number of charged protein residues that collectively lower the CLA606 site energy. Our work provides insights into the understanding of molecular mechanisms of the core machinery of the green-plant photosynthesis. PMID:24954746

  16. Identification and properties of a novel clt locus in the Streptomyces phaeochromogenes plasmid pJV1.

    PubMed Central

    Servín-González, L

    1996-01-01

    A novel clt locus required for efficient transfer of the Streptomyces phaeochromogenes plasmid pJV1 was identified and mapped. The clt region was functional in both orientations, and its absence caused a severe reduction in plasmid transfer. Chromosome mobilization, on the other hand, was not affected by absence of the clt locus. The clt region showed structural, but not sequence, similarity to transfer origins of gram-negative plasmids. PMID:8763967

  17. Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

    PubMed

    Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

    2015-08-25

    Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

  18. Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella

    PubMed Central

    Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

    2015-01-01

    Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host–plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σS and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σS and, together, RprA and σS orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer. PMID:26307765

  19. Horizontal transfer and gene conversion as an important driving force in shaping the landscape of mitochondrial introns.

    PubMed

    Wu, Baojun; Hao, Weilong

    2014-04-16

    Group I introns are highly dynamic and mobile, featuring extensive presence-absence variation and widespread horizontal transfer. Group I introns can invade intron-lacking alleles via intron homing powered by their own encoded homing endonuclease gene (HEG) after horizontal transfer or via reverse splicing through an RNA intermediate. After successful invasion, the intron and HEG are subject to degeneration and sequential loss. It remains unclear whether these mechanisms can fully address the high dynamics and mobility of group I introns. Here, we found that HEGs undergo a fast gain-and-loss turnover comparable with introns in the yeast mitochondrial 21S-rRNA gene, which is unexpected, as the intron and HEG are generally believed to move together as a unit. We further observed extensively mosaic sequences in both the introns and HEGs, and evidence of gene conversion between HEG-containing and HEG-lacking introns. Our findings suggest horizontal transfer and gene conversion can accelerate HEG/intron degeneration and loss, or rescue and propagate HEG/introns, and ultimately result in high HEG/intron turnover rate. Given that up to 25% of the yeast mitochondrial genome is composed of introns and most mitochondrial introns are group I introns, horizontal transfer and gene conversion could have served as an important mechanism in introducing mitochondrial intron diversity, promoting intron mobility and consequently shaping mitochondrial genome architecture.

  20. Proposed model for the high rate of rearrangement and rapid migration observed in some IncA/C plasmid lineages

    USDA-ARS?s Scientific Manuscript database

    IncA/C plasmids are a class of plasmids from Enterobacteraciae that are relatively large (49 to >180 kbp), are readily transferred by conjugation, and carry multiple antimicrobial resistance genes. Reconstruction of the phylogeny of these plasmids has been difficult because of the high rate of remo...

  1. blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

    PubMed

    Call, Douglas R; Singer, Randall S; Meng, Da; Broschat, Shira L; Orfe, Lisa H; Anderson, Janet M; Herndon, David R; Kappmeyer, Lowell S; Daniels, Joshua B; Besser, Thomas E

    2010-02-01

    Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of blaCMY-2, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in floR. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the blaCMY-2 plasmids from E. coli and S. enterica are genetically distinct from those originating from Yersinia pestis and Photobacterium damselae and distal to one originating from Yersinia ruckeri. While the overall similarity of these plasmids supports the likelihood of recent movements among E. coli and S. enterica hosts, their greater divergence from Y. pestis or Y. ruckeri suggests less recent plasmid transfer among these pathogen groups.

  2. Complementation of Conjugation Functions of Streptomyces lividans Plasmid pIJ101 by the Related Streptomyces Plasmid pSB24.2

    PubMed Central

    Pettis, Gregg S.; Prakash, Shubha

    1999-01-01

    A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24.2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems. PMID:10419972

  3. Survival and Evolution of a Large Multidrug Resistance Plasmid in New Clinical Bacterial Hosts.

    PubMed

    Porse, Andreas; Schønning, Kristian; Munck, Christian; Sommer, Morten O A

    2016-11-01

    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 of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli. We use experimental evolution, mathematical modelling and population 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 of costly regions from the plasmid backbone, effectively expanding the host-range of the plasmid. Although these adaptations were also beneficial to plasmid persistence in a naïve K. pneumoniae host, they were never observed in this species, indicating that differential evolvability can limit opportunities of plasmid adaptation. While insertion sequences are well known to supply plasmids with adaptive traits, our findings suggest that they also play an important role in plasmid evolution by maintaining the plasticity necessary to alleviate plasmid-host constrains. Further, the observed evolutionary strategy 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. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Single molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae

    PubMed Central

    Conlan, Sean; Thomas, Pamela J.; Deming, Clayton; Park, Morgan; Lau, Anna F.; Dekker, John P.; Snitkin, Evan S.; Clark, Tyson A.; Luong, Khai; Song, Yi; Tsai, Yu-Chih; Boitano, Matthew; Gupta, Jyoti; Brooks, Shelise Y.; Schmidt, Brian; Young, Alice C.; Thomas, James W.; Bouffard, Gerard G.; Blakesley, Robert W.; Mullikin, James C.; Korlach, Jonas; Henderson, David K.; Frank, Karen M.; Palmore, Tara N.; Segre, Julia A.

    2014-01-01

    Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common healthcare-associated infections nearly impossible to treat. We performed comprehensive surveillance and genomic sequencing to identify carbapenem-resistant Enterobacteriaceae in the NIH Clinical Center patient population and hospital environment in order to to articulate the diversity of carbapenemase-encoding plasmids and survey the mobility of and assess the mobility of these plasmids between bacterial species. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem-resistance genes on a wide array of plasmids. Klebsiella pneumoniae and Enterobacter cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, overriding the epidemiological scenario of plasmid transfer between organisms within this patient. We did, however, find evidence supporting horizontal transfer of carbapenemase-encoding plasmids between Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii in the hospital environment. Our comprehensive sequence data, with full plasmid identification, challenges assumptions about horizontal gene transfer events within patients and identified wider possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Pantoea species, from unrelated patients and the hospital environment. PMID:25232178

  5. Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications.

    PubMed

    Schmeer, Marco; Buchholz, Tatjana; Schleef, Martin

    2017-10-01

    Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. For direct gene transfer into humans, good manufacturing practice (GMP)-grade plasmid DNA is mandatory. The same holds true if the drug substance contains a genetically modified cell, for example chimeric antigen receptor (CAR) T cells, where these cells as well as the contained plasmids are used. According to the responsible regulatory agencies, they have to be produced under full GMP. On the other hand, for GMP production of, for example, mRNA or viral vectors (lentiviral vectors, adeno-associated virus vectors, etc.), in many cases, High Quality Grade plasmid DNA is accepted as a starting material. The manufacturing process passes through different production steps. To ensure the right conditions are used for the plasmid, a pilot run must be conducted at the beginning. In this step, a followed upscaling with respect to reproducibility and influences on product quality is performed. Subsequently, a cell bank of the transformed productions strain is established and characterized. This cell bank is used for the cultivation process. After cell harvesting and lysis, several chromatography steps are conducted to receive a pure plasmid product. Depending on the respective required quality grade, the plasmid product is subject to several quality controls. The last step consists of formulation and filling of the product.

  6. Plasmid incidence in bacteria from deep subsurface sediments

    SciTech Connect

    Fredrickson, J.K.; Hicks, R.J.; Li, S.W.; Brockman, F.J. )

    1988-12-01

    Bacteria were isolated from deep terrestrial subsurface sediments underlying the coastal plain of South Carolina. A total of 163 isolates from deep sediments, surface soil, and return drill muds were examined for plasmid DNA content and resistance to the antibiotics penicillin, ampicillin, carbenicillin, streptomycin, kanamycin, and tetracycline. MICs of Cu{sup 2+}, Cr{sup 3+}, and Hg{sup 2+} for each isolate were also determined. The overall frequency of plasmid occurrence in the subsurface bacteria was 33%. Resistance was most frequent to penicillin (70% of all isolates), ampicillin (49%), and carbenicillin (32%) and was concluded to be related to the concentrations of the individual antibiotics in the disks used for assaying resistance and to the production of low levels of {beta}-lactamase. The frequencies of resistance to penicillin and ampicillin were significantly greater for isolates bearing plasmids than for plasmidless isolates; however, resistance was not transferable to penicillin-sensitive Escherichia coli. Hybridization of subsurface bacterial plasmids and chromosomal DNA with a whole-TOL-plasmid (pWWO) probe revealed some homology of subsurface bacterial plasmid and chromosomal DNAs, indicating a potential for those bacterial to harbor catabolic genes on plasmids or chromosomes. The incidences of antibiotic resistance and MICs of metals for subsurface bacteria were significantly different from those drill mud bacteria, ruling out the possibility that bacteria from sediments were derived from drill muds.

  7. Tubular cationized pullulan hydrogels as local reservoirs for plasmid DNA.

    PubMed

    San Juan, Aurélie; Ducrocq, Grégory; Hlawaty, Hanna; Bataille, Isabelle; Guénin, Erwann; Letourneur, Didier; Feldman, Laurent J

    2007-12-01

    In the present study, we measured the ability of various cationized pullulan tubular hydrogels to retain plasmid DNA, and tested the ability of retained plasmid DNA to transfect vascular smooth muscle cells (VSMCs). Cationized pullulans were obtained by grafting at different charge densities ethylamine (EA) or diethylaminoethylamine (DEAE) on the pullulan backbone. Polymers were characterized by elemental analysis, acid-base titration, size exclusion chromatography, Fourier-transform infrared spectroscopy, and proton nuclear magnetic resonance. The complexation of cationized pullulans in solution with plasmid DNA was evidenced by fluorescence quenching with PicoGreen. Cationized pullulans were then chemically crosslinked with phosphorus oxychloride to obtain tubular cationized pullulan hydrogels. Native pullulan tubes did not retain loaded plasmid DNA. In contrast, the ability of cationized pullulan tubes to retain plasmid DNA was dependent on both the amine content and the type of amine. The functional integrity of plasmid DNA in cationized pullulan tubes was demonstrated by in vitro transfection of VSMCs. Hence, cationized pullulan hydrogels can be designed as tubular structures with high affinity for plasmid DNA, which may provide new biomaterials to enhance the efficiency of local arterial gene transfer strategies.

  8. Comparison between plasmids of Salmonella and other enterobacteria isolated from the same patients.

    PubMed

    Palomares, J C; Perea, E J

    1982-01-01

    The ecology of R plasmids was studied in the intestinal flora of 19 patients with salmonellosis without antibiotic treatment. The plasmids found in the Salmonella strains and the accompanying non-pathogenic Enterobacteriaceae were characterized in each patient. We determined the transferability by conjugation, the fi character and the incompatibility group and did enzyme restriction analysis of these plasmids. The results obtained showed that S. typhimurium is the species of this genus with the highest incidence of R plasmids, and Escherichia coli among the non-pathogenic Enterobacteriaceae. The plasmids found in Salmonella are different from the plasmids found in the other Enterobacteriaceae in fi character (50% fi+ in Salmonella and 5% in the other Enterobacteriaceae) and incompatibility group (33% belong to the FII group in Salmonella plasmids and none on the other Enterobacteriaceae), thereby expressing a different origin.

  9. Driving forces for metamorphic vein filling during bauxite dehydration: insights from Li and Al transfer illustrated by LIBS compositional profiles (Western Alps)

    NASA Astrophysics Data System (ADS)

    Verlaguet, Anne; Brunet, Fabrice; Goffé, Bruno; Menut, Denis; Findling, Nathaniel; Poinssot, Christophe

    2015-04-01

    In subduction zones, the significant amounts of aqueous fluid released in the course of the successive dehydration reactions occurring during prograde metamorphism are expected to strongly influence the rock rheology, as well as kinetics of metamorphic reactions and mass transfer efficiency. Mineralized veins, ubiquitous in metamorphic rocks, can be seen as preserved witnesses of fluid and mass redistribution that partly accommodate the rock deformation (lateral segregation). However, the driving forces and mechanisms of mass transfer towards fluid-filled open spaces remain somewhat unclear. The aim of this study is to investigate the vein-forming processes and the modalities of mass transfer during local fluid-rock interactions, and their links with fluid production and rock deformation, with new insights from Laser Induced Breakdown Spectroscopy (LIBS) profiles. This study focuses on karstic pockets (metre scale) of Triassic metabauxites embedded in thick carbonate units, that have been isolated from large-scale fluid flow during HP-LT Alpine metamorphism (W. Vanoise, French Alps). These rocks display several generations of metamorphic veins containing various Al-bearing minerals, which give particular insights into mass transfer processes. It is proposed that the internally-derived fluid (~13 vol% produced by successive dehydration reactions) has promoted the opening of fluid-filled open spaces (euhedral habits of vein minerals) and served as medium for diffusive mass transfer from rock to vein. Based on mineralogical and textural features, two vein types can be distinguished: (1) some veins are filled with newly formed products of either prograde (chloritoid) or retrograde (chlorite) metamorphic reactions; in this case, fluid-filled open spaces seem to offer energetically favourable nucleation/growth sites; (2) the second vein type is filled with cookeite (Li-Al-rich chlorite) or pyrophyllite, which were present in the host-rock prior to the vein formation. In

  10. A New Shuttle Plasmid That Stably Replicates in Clostridium acetobutylicum.

    PubMed

    Lee, Sang-Hyun; Kwon, Min-A; Choi, Sunwha; Kim, Sooah; Kim, Jungyeon; Shin, Yong-An; Kim, Kyoung Heon

    2015-10-01

    We have developed a new shuttle plasmid, designated as pLK1-MCS that can replicate in both Clostridium acetobutylicum and Escherichia coli, by combining the pUB110 and pUC19 plasmids. Plasmid pLK1-MCS replicated more stably than previously reported plasmids containing either the pIM13 or the pAMβ1 replicon in the absence of antibiotic selective pressure. The transfer frequency of pLK1-MCS into C. acetobutylicum was similar to the transfer frequency of other shuttle plasmids. We complemented C. acetobutylicum ML1 (that does not produce solvents such as acetone, butanol, and ethanol owing to loss of the megaplasmid pSOL1 harboring the adhE1-ctfAB-adc operon) by introducing pLK1-MCS carrying the adhE1-ctfAB-adc operon into C. acetobutylicum ML1. The transformed cells were able to resume anaerobic solvent production, indicating that the new shuttle plasmid has the potential for practical use in microbial biotechnology.

  11. Capacity of aquatic bacteria to act as recipients of plasmid DNA.

    PubMed Central

    Genthner, F J; Chatterjee, P; Barkay, T; Bourquin, A W

    1988-01-01

    A total of 68 gram-negative freshwater bacterial isolates were screened for their ability to receive and express plasmids from Pseudomonas aeruginosa donors. The plate mating technique identified 26 of the isolates as recipient active for the self-transmissible wide-host-range plasmid R68; 10 were recipient active by R68 mobilization for the wide-host-range plasmid cloning vector R1162. Frequencies of transfer were compared by using three conjugal transfer procedures: broth, plate, and filter mating. For every recipient tested, a solid environment was superior to a liquid environment for transfer. The broth mating technique failed to demonstrate R68 transfer in 63% of the recipient-active isolates. Filter mating, in general, yielded the highest transfer frequencies. The more-rapid plate mating procedure, however, was just as sensitive for testing the capacity of natural isolates to participate in conjugal plasmid transfer. PMID:3345074

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

    PubMed

    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.

  13. Diverse Broad-Host-Range Plasmids from Freshwater Carry Few Accessory Genes

    PubMed Central

    Sen, Diya; Yano, Hirokazu; Bauer, Matthew L.; Rogers, Linda M.; Van der Auwera, Geraldine A.

    2013-01-01

    Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid's ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities. PMID:24096417

  14. Characterization of plasmids that encode streptomycin-resistance in bacterial epiphytes of apple.

    PubMed

    Huang, T C; Burr, T J

    1999-05-01

    Streptomycin resistance in strains of Pseudomonas syringae pv. papulans, Pantoea agglomerans and a yellow-pigmented, non-fluorescent Pseudomonas sp. (Py), isolated from apple orchards in New York and Washington states, is predominantly associated with strA-strB genes carried on conjugal plasmids (R plasmids). None of 128 resistant Erwinia amylovora strains from the eastern and western USA hybridized with a strA-strB probe, SMP3. Resistant Py strains transfered R plasmids to Ps. syringae pv. papulans and to Py in vitro at frequencies of 10(-1)-10(-2) per recipient cell whereas Ps. syringae pv. papulans transferred its plasmids at frequencies of 10(-2) to below detectable levels. Transfer of R plasmids to P. agglomerans was not detected and resistant P. agglomerans did not transfer their R plasmids to any recipients. R plasmids were found to be highly diverse as measured by DNA fingerprint analysis. Transfer-deficient transposon mutants of R plasmid pCPP519 were generated, and 3.9 kb EcoRI and 3.0 kb SmaI fragments that hybridized with a Tn5 probe were cloned and sequenced. The deduced amino acid sequences of the 3.9 kb fragment were similar to proteins involved in replication, nicking at oriT, and piliation in other bacteria.

  15. The A to Z of A/C plasmids.

    PubMed

    Harmer, Christopher J; Hall, Ruth M

    2015-07-01

    Plasmids belonging to incompatibility groups A and C (now A/C) were among the earliest to be associated with antibiotic resistance in Gram-negative bacteria. A/C plasmids are large, conjugative plasmids with a broad host range. The prevalence of A/C plasmids in collections of clinical isolates has revealed their importance in the dissemination of extended-spectrum β-lactamases and carbapenemases. They also mobilize SGI1-type resistance islands. Revived interest in the family has yielded many complete A/C plasmid sequences, revealing that RA1, designated A/C1, is different from the remainder, designated A/C2. There are two distinct A/C2 lineages. Backbones of 128-130 kb include over 120 genes or ORFs encoding proteins of at least 100 amino acids, but very few have been characterized. Genes potentially required for replication, stability and transfer have been identified, but only the replication system of RA1 and the regulation of transfer have been studied. There is enormous variety in the antibiotic resistance genes carried by A/C2 plasmids but they are usually clustered in larger regions at various locations in the backbone. The ARI-A and ARI-B resistance islands are always at a specific location but have variable content. ARI-A is only found in type 1 A/C2 plasmids, which disseminate blaCMY-2 and blaNDM-1 genes, whereas ARI-B, carrying the sul2 gene, is found in both type 1 and type 2. This review summarizes current knowledge of A/C plasmids, and highlights areas of research to be considered in the future.

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

    SciTech Connect

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

    1986-02-01

    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.

  17. High Emigration Propensity and Low Mortality on Transfer Drives Female-Biased Dispersal of Pyriglena leucoptera in Fragmented Landscapes.

    PubMed

    Awade, Marcelo; Candia-Gallardo, Carlos; Cornelius, Cintia; Metzger, Jean Paul

    2017-01-01

    Dispersal is a biological process performed in three stages: emigration, transfer and immigration. Intra-specific variation on dispersal behavior, such as sex-bias, is very common in nature, particularly in birds and mammals. However, dispersal is difficult to measure in the field and many hypotheses concerning the causes of sex-biased dispersal remain without empirical confirmation. An important limitation of most empirical studies is that inferences about sex-biased dispersal are based only on emigration proneness or immigration success data. Thus, we still do not know whether sex-biased immigration in fragmented landscapes occurs during emigration, transfer or in both stages. We conducted translocation and radiotracking experiments to assess i) whether inter-patch dispersal movements of a rainforest bird (Pyriglena leucoptera) is sex-biased and ii) how dispersal stages and the perceptual range of the individuals are integrated to generate dispersal patterns. Our results showed that inter-patch dispersal is sex-biased at all stages for P. leucoptera, as females not only exhibit a higher emigration propensity but are subjected to a lower risk of predation when moving through the matrix. Moreover, our data support a perceptual range of 80 m and our results showed that dispersal success decreases considerably when inter-patch distances exceeds this perceptual range. In this case, birds have a higher probability of travelling over longer routes and, as a consequence, the risk of predation increases, specially for males. Overall, results supported that assuming dispersal as a single-stage process to describe dispersal behavior may be misleading. In this way, our study advanced our understanding of processes and patterns related to inter-patch dispersal of neotropical forest birds, shedding light on potential implications for population dynamics and for the management of fragmented landscapes.

  18. High Emigration Propensity and Low Mortality on Transfer Drives Female-Biased Dispersal of Pyriglena leucoptera in Fragmented Landscapes

    PubMed Central

    Awade, Marcelo; Candia-Gallardo, Carlos; Cornelius, Cintia; Metzger, Jean Paul

    2017-01-01

    Dispersal is a biological process performed in three stages: emigration, transfer and immigration. Intra-specific variation on dispersal behavior, such as sex-bias, is very common in nature, particularly in birds and mammals. However, dispersal is difficult to measure in the field and many hypotheses concerning the causes of sex-biased dispersal remain without empirical confirmation. An important limitation of most empirical studies is that inferences about sex-biased dispersal are based only on emigration proneness or immigration success data. Thus, we still do not know whether sex-biased immigration in fragmented landscapes occurs during emigration, transfer or in both stages. We conducted translocation and radiotracking experiments to assess i) whether inter-patch dispersal movements of a rainforest bird (Pyriglena leucoptera) is sex-biased and ii) how dispersal stages and the perceptual range of the individuals are integrated to generate dispersal patterns. Our results showed that inter-patch dispersal is sex-biased at all stages for P. leucoptera, as females not only exhibit a higher emigration propensity but are subjected to a lower risk of predation when moving through the matrix. Moreover, our data support a perceptual range of 80 m and our results showed that dispersal success decreases considerably when inter-patch distances exceeds this perceptual range. In this case, birds have a higher probability of travelling over longer routes and, as a consequence, the risk of predation increases, specially for males. Overall, results supported that assuming dispersal as a single-stage process to describe dispersal behavior may be misleading. In this way, our study advanced our understanding of processes and patterns related to inter-patch dispersal of neotropical forest birds, shedding light on potential implications for population dynamics and for the management of fragmented landscapes. PMID:28107517

  19. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    NASA Astrophysics Data System (ADS)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  20. Quantifying the Growth of Cross-Beam Energy Transfer in Polar-Direct-Drive Implosions at the Omega Laser and National Ignition Facilities

    NASA Astrophysics Data System (ADS)

    Davis, A. K.

    2015-11-01

    Direct-drive inertial confinement fusion requires multiple overlapping laser beams that can drive the cross-beam energy transfer (CBET) instability. This instability is of primary concern because it can reduce the laser energy coupling and can affect the symmetry in a polar-direct-drive (PDD) configuration. An experiment was designed to determine the CBET growth by measuring the angularly resolved mass ablation rate and ablation-front trajectory in a PDD configuration. Adding a thin layer of Si over a CH shell generates two peaks in x-ray self-emission images that are measured with a time-resolved pinhole imager. The inner peak is related to the position of the ablation front and the outer peak corresponds to the position of the interface of the two layers in the plasma. The emergence of the second peak is used to measure the time for the laser to burn through the outer layer, giving the average mass ablation rate of the material. The mass ablation rate was measured by varying the thickness of the outer silicon layer. The shell trajectory and mass ablation rate measured in PDD on the pole, where CBET has little effect, were compared with simulations to validate the electron thermal-transport model. Excellent agreement was obtained when using a 2-D nonlocal transport model, and these observables could not be reproduced with flux-limited models. A similar comparison was performed on the equator where the CBET growth is large. Without the CBET model, the shell velocity and mass ablation rate were significantly overestimated by the simulation. Adding the CBET model reduced the drive on the equator and reproduced the experimental results. This material is based upon work supported by the Department of Energy National Nuclear Security Administration under Award Number DE-NA0001944. In collaboration with, D. Cao, D. T. Michel, M. Hohenberger, R. Epstein, V. N. Goncharov, S. X. Hu, I. V. Igumenshchev, J. A. Marozas, D. D. Meyerhofer, P. B. Radha, S. P. Regan, T. C

  1. Plasmid diversity and phylogenetic consistency in the Lyme disease agent Borrelia burgdorferi.

    PubMed

    Casjens, Sherwood R; Gilcrease, Eddie B; Vujadinovic, Marija; Mongodin, Emmanuel F; Luft, Benjamin J; Schutzer, Steven E; Fraser, Claire M; Qiu, Wei-Gang

    2017-02-15

    Bacteria from the genus Borrelia are known to harbor numerous linear and circular plasmids. We report here a comparative analysis of the nucleotide sequences of 236 plasmids present in fourteen independent isolates of the Lyme disease agent B. burgdorferi. We have sequenced the genomes of 14 B. burgdorferi sensu stricto isolates that carry a total of 236 plasmids. These individual isolates carry between seven and 23 plasmids. Their chromosomes, the cp26 and cp32 circular plasmids, as well as the lp54 linear plasmid, are quite evolutionarily stable; however, the remaining plasmids have undergone numerous non-homologous and often duplicative recombination events. We identify 32 different putative plasmid compatibility types among the 236 plasmids, of which 15 are (usually) circular and 17 are linear. Because of past rearrangements, any given gene, even though it might be universally present in these isolates, is often found on different linear plasmid compatibility types in different isolates. For example, the arp gene and the vls cassette region are present on plasmids of four and five different compatibility types, respectively, in different isolates. A majority of the plasmid types have more than one organizationally different subtype, and the number of such variants ranges from one to eight among the 18 linear plasmid types. In spite of this substantial organizational diversity, the plasmids are not so variable that every isolate has a novel version of every plasmid (i.e., there appears to be a limited number of extant plasmid subtypes). Although there have been many past recombination events, both homologous and nonhomologous, among the plasmids, particular organizational variants of these plasmids correlate with particular chromosomal genotypes, suggesting that there has not been rapid horizontal transfer of whole linear plasmids among B. burgdorferi lineages. We argue that plasmid rearrangements are essentially non-revertable and are present at a frequency of

  2. Plasmid Rolling-Circle Replication.

    PubMed

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  3. Mechanisms of Theta Plasmid Replication.

    PubMed

    Lilly, Joshua; Camps, Manel

    2015-02-01

    Plasmids are autonomously replicating pieces of DNA. This article discusses theta plasmid replication, which is a class of circular plasmid replication that includes ColE1-like origins of replication popular with expression vectors. All modalities of theta plasmid replication initiate synthesis with the leading strand at a predetermined site and complete replication through recruitment of the host's replisome, which extends the leading strand continuously while synthesizing the lagging strand discontinuously. There are clear differences between different modalities of theta plasmid replication in mechanisms of DNA duplex melting and in priming of leading- and lagging-strand synthesis. In some replicons duplex melting depends on transcription, while other replicons rely on plasmid-encoded trans-acting proteins (Reps); primers for leading-strand synthesis can be generated through processing of a transcript or in other replicons by the action of host- or plasmid-encoded primases. None of these processes require DNA breaks. The frequency of replication initiation is tightly regulated to facilitate establishment in permissive hosts and to achieve a steady state. The last section of the article reviews how plasmid copy number is sensed and how this feedback modulates the frequency of replication.

  4. Comparative genomics of pAKD4, the prototype IncP-1δplasmid with a complete backbone

    PubMed Central

    Sen, Diya; Yano, Hirokazu; Suzuki, Haruo; Król, Jaroslaw E.; Rogers, Linda; Brown, Celeste J.; Top, Eva M.

    2010-01-01

    Plasmids of the incompatibility group IncP-1 are important agents of horizontal gene transfer and contribute to the spread of antibiotic resistance and xenobiotic degradation within bacterial communities. Even though some prototype plasmids have been studied in much detail, the diversity of this plasmid group was still greatly underestimated until recently, as only two of the five currently known divergent sub-groups had been described. To further improve our insight into the diversity and evolutionary history of this family of broad-host-range plasmids, we compared the complete nucleotide sequence of a new IncP-1δ plasmid pAKD4 to the genomes of other IncP-1 plasmids. Plasmid pAKD4 was previously isolated by exogenous plasmid isolation from an agricultural soil in Norway. Its 56,803 bp nucleotide sequence shows high similarity in gene sequence and gene order to both plasmids pEST4011 and pIJB1, the only other IncP-1δ plasmids sequenced so far. While all three plasmids have a typical IncP-1 backbone comprising replication, transfer and stable inheritance/control genes, the low sequence similarity in some regions and presence/absence of some backbone genes compared to other IncP-1 plasmids cluster them in a divergent sub-group. Therefore this study validates the presence of a real IncP-1δ clade with multiple plasmids. Moreover, since both pEST4011 and pIJB1 are missing a portion of their transfer genes, pAKD4 represents the first completely sequenced self-transferable plasmid with a complete IncP-1δ backbone. We therefore propose it to be the prototype IncP-1δ plasmid. PMID:20018208

  5. Plasmid acquisition in microgravity

    NASA Technical Reports Server (NTRS)

    Juergensmeyer, Margaret A.; Juergensmeyer, Elizabeth A.; Guikema, James A.

    1995-01-01

    In microgravity, bacteria often show an increased resistance to antibiotics. Bacteria can develop resistance to an antibiotic after transformation, the acquisition of DNA, usually in the form of a plasmid containing a gene for resistance to one or more antibiotics. In order to study the capacity of bacteria to become resistant to antibiotics in microgravity, we have modified the standard protocol for transformation of Escherichia coli for use in the NASA-flight-certified hardware package, The Fluid Processing Apparatus (FPA). Here we report on the ability of E. coli to remain competent for long periods of time at temperatures that are readily available on the Space Shuttle, and present some preliminary flight results.

  6. Plasmid acquisition in microgravity

    NASA Technical Reports Server (NTRS)

    Juergensmeyer, Margaret A.; Juergensmeyer, Elizabeth A.; Guikema, James A.

    1995-01-01

    In microgravity, bacteria often show an increased resistance to antibiotics. Bacteria can develop resistance to an antibiotic after transformation, the acquisition of DNA, usually in the form of a plasmid containing a gene for resistance to one or more antibiotics. In order to study the capacity of bacteria to become resistant to antibiotics in microgravity, we have modified the standard protocol for transformation of Escherichia coli for use in the NASA-flight-certified hardware package, The Fluid Processing Apparatus (FPA). Here we report on the ability of E. coli to remain competent for long periods of time at temperatures that are readily available on the Space Shuttle, and present some preliminary flight results.

  7. Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection.

    PubMed

    Cohen, Richard N; van der Aa, Marieke A E M; Macaraeg, Nichole; Lee, Ai Ping; Szoka, Francis C

    2009-04-17

    Nuclear uptake of plasmid DNA is one of the many cellular barriers that limit the efficiency of non-viral gene delivery systems. We have determined the number of plasmids that reach the nucleus of a transfected cell using an internally standardized quantitative PCR (qPCR) assay. We isolated nuclei using two different protocols: a density gradient technique and a detergent-based method. The density gradient procedure yielded nuclei with substantially less adhering plasmids on the outside of the nuclei. Using the density gradient protocol we determined that cells transfected with Lipofectamine lipoplexes or polyethylenimine polyplexes contained between 75 and 50,000 plasmids/nucleus, depending on the applied plasmid dose. Any increase above 3000 plasmids/nucleus resulted in only marginal increases in transgene expression. Furthermore, lipoplex-delivered plasmids were more efficiently expressed, on the basis of protein expression per plasmid number in the nucleus, than polyplex-delivered plasmids. This indicates that polymer may remain bound to some plasmids in the nucleus. Lastly, by sorting transfected cells into high- and low-expressing sub-populations, we observe that a sub-population of cells contain 3x greater plasmids/nucleus but express nearly 100x more transgene than other cells within a single transfection reaction. Taken together these results suggest the importance of considering the processes downstream from nuclear entry for strategies to improve the efficiency of gene transfer reagents.

  8. Dynamics of the IncW genetic backbone imply general trends in conjugative plasmid evolution.

    PubMed

    Fernández-López, Raúl; Garcillán-Barcia, M Pilar; Revilla, Carlos; Lázaro, Miguel; Vielva, Luis; de la Cruz, Fernando

    2006-11-01

    Plasmids cannot be understood as mere tools for genetic exchange: they are themselves subject to the forces of evolution. Their genomic and phylogenetic features have been less studied in this respect. Focusing on the IncW incompatibility group, which includes the smallest known conjugative plasmids, we attempt to unveil some common trends in plasmid evolution. The functional modules of IncW genetic backbone are described, with emphasis on their architecture and relationships to other plasmid groups. Some plasmid regions exhibit strong phylogenetic mosaicism, in striking contrast to others of unusual synteny conservation. The presence of genes of unknown function that are widely distributed in plasmid genomes is also emphasized, exposing the existence of ill-defined yet conserved plasmid functions. Conjugation is an essential hallmark of IncW plasmid biology and special attention is given to the organization and evolution of its transfer modules. Genetic exchange between plasmids and their hosts is analysed by following the evolution of the type IV secretion system. Adaptation of the trw conjugative machinery to pathogenicity functions in Bartonella is discussed as an example of how plasmids can change their host modus vivendi. Starting from the phage paradigm, our analysis articulates novel concepts that apply to plasmid evolution.

  9. Ornamental fish as a source of plasmid-mediated quinolone resistance genes and antibiotic resistance plasmids.

    PubMed

    Dobiasova, Hana; Kutilova, Iva; Piackova, Veronika; Vesely, Tomas; Cizek, Alois; Dolejska, Monika

    2014-07-16

    Growing ornamental fish industry is associated with public health concerns including extensive antibiotic use accompanied by increasing antibiotic resistance. The aim of this study was to analyze Aeromonas isolates from imported tropical ornamental fish and coldwater koi carps bred in the Czech Republic to assess the potential risk of ornamental fish as a source of plasmid-mediated quinolone resistance genes (PMQR) and antibiotic resistance plasmids. A collection of Aeromonas spp. with reduced susceptibility to ciprofloxacin (MIC ≥ 0.05 mg/L) was selected for the detection of PMQR genes. Isolates harbouring PMQR genes were further analyzed for the additional antibiotic resistance, integron content, clonality, biofilm production and transferability of PMQR genes by conjugation and transformation. Comparative analysis of plasmids carrying PMQR genes was performed. Fifteen (19%, n=80) isolates from koi carps and 18 (24%, n=76) isolates from imported ornamental fish were positive for qnrS2, aac(6')-Ib-cr or qnrB17 genes. PMQR-positive isolates from imported ornamental fish showed higher MIC levels to quinolones, multiresistance and diverse content of antibiotic resistance genes and integrons compared to the isolates from the carps. Related IncU plasmids harbouring qnrS2 and aac(6')-Ib-cr genes were found in Aeromonas spp. from imported ornamental fish and koi carps from various geographical areas. Ornamental fish may represent a potential source of multiresistant bacteria and mobile genetic elements for the environment and for humans.

  10. Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci

    PubMed Central

    Pollet, Rebecca M.; Ingle, James D.; Hymes, Jeff P.; Eakes, Thomas C.; Eto, Karina Yui; Kwong, Stephen M.; Ramsay, Joshua P.; Firth, Neville

    2016-01-01

    ABSTRACT Antimicrobial resistance in Staphylococcus aureus presents 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 the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region 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 the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, 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-like oriT. These data indicate that the conjugative relaxase in trans mechanism 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. IMPORTANCE Understanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus

  11. Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci.

    PubMed

    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

    2016-01-04

    Antimicrobial resistance in Staphylococcus aureus presents 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 the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region 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 the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, 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-like oriT. These data indicate that the conjugative relaxase in trans mechanism 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. Understanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus multiresistance

  12. Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology

    PubMed Central

    Orlek, Alex; Stoesser, Nicole; Anjum, Muna F.; Doumith, Michel; Ellington, Matthew J.; Peto, Tim; Crook, Derrick; Woodford, Neil; Walker, A. Sarah; Phan, Hang; Sheppard, Anna E.

    2017-01-01

    Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of ‘accessory genes,’ such as antibiotic resistance genes, as well as ‘backbone’ loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made. PMID:28232822

  13. Plasmid Classification in an Era of Whole-Genome Sequencing: Application in Studies of Antibiotic Resistance Epidemiology.

    PubMed

    Orlek, Alex; Stoesser, Nicole; Anjum, Muna F; Doumith, Michel; Ellington, Matthew J; Peto, Tim; Crook, Derrick; Woodford, Neil; Walker, A Sarah; Phan, Hang; Sheppard, Anna E

    2017-01-01

    Plasmids are extra-chromosomal genetic elements ubiquitous in bacteria, and commonly transmissible between host cells. Their genomes include variable repertoires of 'accessory genes,' such as antibiotic resistance genes, as well as 'backbone' loci which are largely conserved within plasmid families, and often involved in key plasmid-specific functions (e.g., replication, stable inheritance, mobility). Classifying plasmids into different types according to their phylogenetic relatedness provides insight into the epidemiology of plasmid-mediated antibiotic resistance. Current typing schemes exploit backbone loci associated with replication (replicon typing), or plasmid mobility (MOB typing). Conventional PCR-based methods for plasmid typing remain widely used. With the emergence of whole-genome sequencing (WGS), large datasets can be analyzed using in silico plasmid typing methods. However, short reads from popular high-throughput sequencers can be challenging to assemble, so complete plasmid sequences may not be accurately reconstructed. Therefore, localizing resistance genes to specific plasmids may be difficult, limiting epidemiological insight. Long-read sequencing will become increasingly popular as costs decline, especially when resolving accurate plasmid structures is the primary goal. This review discusses the application of plasmid classification in WGS-based studies of antibiotic resistance epidemiology; novel in silico plasmid analysis tools are highlighted. Due to the diverse and plastic nature of plasmid genomes, current typing schemes do not classify all plasmids, and identifying conserved, phylogenetically concordant genes for subtyping and phylogenetics is challenging. Analyzing plasmids as nodes in a network that represents gene-sharing relationships between plasmids provides a complementary way to assess plasmid diversity, and allows inferences about horizontal gene transfer to be made.

  14. Application of a plasmid classification system to determine prevalence of replicon families among multidrug resistant enterococci

    USDA-ARS?s Scientific Manuscript database

    The presence and transfer of plasmids from commensal bacteria to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. However, prevalence of plasmids from commensal bacteria in food animals such as the enterococci remains largely unknown. In this study, the prevale...

  15. Effect of Plasmid pIJ1008 from Rhizobium leguminosarum on Symbiotic Function of Rhizobium meliloti.

    PubMed

    Bedmar, E J; Brewin, N J; Phillips, D A

    1984-04-01

    Plasmid pIJ1008, which carries determinants for uptake hydrogenase (Hup) activity, was transferred from Rhizobium leguminosarum to Rhizobium meliloti without impairing the capacity of the latter species to form root nodules on alfalfa. The plasmid was still present in rhizobia reisolated from the root nodules of 12 different alfalfa cultivars, but only low levels of Hup activity were detected in alfalfa.

  16. An in vivo assay for conjugation-mediated recombination yields novel results for Streptomyces plasmid pIJ101.

    PubMed

    Ducote, Matthew J; Pettis, Gregg S

    2006-05-01

    Efficient transmission of circular plasmids in Streptomyces spp. proceeds by an uncharacterized mechanism that requires a cis-acting locus of transfer (clt) and often only a single plasmid-encoded protein. For circular plasmids from other bacteria, site- and strand-specific nicking takes place at the cis-acting oriT locus via the plasmid-encoded relaxase protein prior to single-strand transfer. Using an assay originally designed to demonstrate that conjugative transfer of plasmids containing tandem oriT loci results in the formation of a single composite oriT locus, we show here that an analogous construct involving the pIJ101 clt locus apparently does not undergo such a conjugation-mediated event during plasmid transfer. Our results, which imply that streptomycete plasmids are transferred by a functionally distinct mechanism compared to oriT-containing plasmids, are complementary to other recent evidences that support a novel double-stranded model for streptomycete circular plasmid transfer.

  17. Transformation of Actinobacillus actinomycetemcomitans by electroporation, utilizing constructed shuttle plasmids.

    PubMed Central

    Sreenivasan, P K; LeBlanc, D J; Lee, L N; Fives-Taylor, P

    1991-01-01

    Actinobacillus actinomycetemcomitans, a periodontal pathogen, has been strongly implicated in human periodontal disease. Advances in the molecular analysis of A. actinomycetemcomitans virulence factors have been limited due to the unavailability of systems for genetic transfer, transposon mutagenesis, and gene complementation. Slow progress can be traced almost exclusively to the lack of gene vector systems and methods for the introduction of DNA into A. actinomycetemcomitans. An electrotransformation system that allowed at least five strains of A. actinomycetemcomitans to be transformed with stable shuttle plasmids which efficiently replicated in both Escherichia coli and A. actinomycetemcomitans was developed. One plasmid, a potential shuttle vector designated pDL282, is 5.7 kb in size, has several unique restriction enzyme sites, and codes for resistance to spectinomycin and ampicillin. E. coli and A. actinomycetemcomitans were transformed with equal efficiencies of approximately 10(5) transformants per micrograms of DNA. Similar transformation efficiencies were obtained whether the plasmid DNA was isolated from A. actinomycetemcomitans or E. coli. In addition, frozen competent cells of A. actinomycetemcomitans yielded comparable efficiencies of transformation. Restriction enzyme analysis of pDL282 isolated after transformation confirmed the presence of intact donor plasmids. A plasmid isolated from A. pleuropneumoniae was also capable of transforming some isolates of A. actinomycetemcomitans, although generally at a lower frequency. The availability of these shuttle plasmids and an efficient transformation procedure should significantly facilitate the molecular analysis of virulence factors of A. actinomycetemcomitans. PMID:1937823

  18. Pocket-size solid-state iPOD and flash drives for gigabyte storage, display and transfer of digital medical images: Review and work initiated

    PubMed Central

    Sankaran, A.

    2009-01-01

    A locally assembled image viewer system with pocket-size iPOD (80 GB) and flash (2 GB) drives for gigabyte storage, display and transfer of digital medical images, oriented towards training purposes, is described. Both the iPOD and flash drive enable storage of thousands of images from diverse medical-imaging equipments. The iPOD, in addition, can display with sufficient resolution any of these images and serves as a transportable preview device. Through the use of a computer, these devices can access/ store/ display the images/ photos from a CD, digital camera or the internet. A TV image viewing unit is also provided. The operational features and the advantages of these devices are discussed in detail. The quality assurance (QA) of the displays has been successfully carried out with standard test patterns. The image quality has been tested with dynamic and static medical images. The system will be highly useful for storage and remote display of multitude of images from several modalities in the hospital, as well as other images, from the point of view of education and training. It has good potential for use in clinical diagnosis as well. Other recent advancements using iPHONE and improved but expensive computers, integrated with picture archiving and communication system (PACS) as well as radiology and hospital information system (RHIS) for versatile applications in modern radiology, are also highlighted.This system, assembled with indigenous equipments, is much less expensive and specially suited for teaching radiologists, physicists and technologists, particularly in developing countries. PMID:20098565

  19. Pocket-size solid-state iPOD and flash drives for gigabyte storage, display and transfer of digital medical images: Review and work initiated.

    PubMed

    Sankaran, A

    2009-07-01

    A locally assembled image viewer system with pocket-size iPOD (80 GB) and flash (2 GB) drives for gigabyte storage, display and transfer of digital medical images, oriented towards training purposes, is described. Both the iPOD and flash drive enable storage of thousands of images from diverse medical-imaging equipments. The iPOD, in addition, can display with sufficient resolution any of these images and serves as a transportable preview device. Through the use of a computer, these devices can access/ store/ display the images/ photos from a CD, digital camera or the internet. A TV image viewing unit is also provided. The operational features and the advantages of these devices are discussed in detail. The quality assurance (QA) of the displays has been successfully carried out with standard test patterns. The image quality has been tested with dynamic and static medical images. The system will be highly useful for storage and remote display of multitude of images from several modalities in the hospital, as well as other images, from the point of view of education and training. It has good potential for use in clinical diagnosis as well. Other recent advancements using iPHONE and improved but expensive computers, integrated with picture archiving and communication system (PACS) as well as radiology and hospital information system (RHIS) for versatile applications in modern radiology, are also highlighted.This system, assembled with indigenous equipments, is much less expensive and specially suited for teaching radiologists, physicists and technologists, particularly in developing countries.

  20. Plasmids of Carotenoid-Producing Paracoccus spp. (Alphaproteobacteria) - Structure, Diversity and Evolution

    PubMed Central

    Maj, Anna; Dziewit, Lukasz; Czarnecki, Jakub; Wlodarczyk, Miroslawa; Baj, Jadwiga; Skrzypczyk, Grazyna; Giersz, Dorota; Bartosik, Dariusz

    2013-01-01

    Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. PMID:24260361

  1. Tn5-mediated transposition of plasmid DNA after transduction to Myxococcus xanthus.

    PubMed Central

    Downard, J S

    1988-01-01

    After coliphage P1-mediated transfer of Tn5-containing plasmid DNA from Escherichia coli to Myxococcus xanthus, transductants were identified which contained plasmid sequences integrated at many sites on the bacterial chromosome. The unaltered plasmid DNA sequences in these transductants were apparently flanked by intact Tn5 or IS50 sequences. These results suggest that Tn5-mediated transposition has occurred and provide a method for integrating plasmid DNA into the M. xanthus chromosome without the requirement for homologous recombination. Images PMID:2844730

  2. War Wound Treatment Complications Due to Transfer of an IncN Plasmid Harboring blaOXA-181 from Morganella morganii to CTX-M-27-Producing Sequence Type 131 Escherichia coli

    PubMed Central

    Snesrud, Erik; Ong, Ana C.; Appalla, Lakshmi; Koren, Michael; Kwak, Yoon I.; Waterman, Paige E.; Lesho, Emil P.

    2015-01-01

    A 22-year-old male developed a recurrent sacral abscess associated with embedded shrapnel following a blast injury. Cultures grew extended-spectrum β-lactamase (ESBL)-producing, carbapenem-susceptible Escherichia coli. Ertapenem was administered, but the infection recurred after each course of antibiotics. Initial surgical interventions were unsuccessful, and subsequent cultures yielded E. coli and Morganella morganii, both nonsusceptible to carbapenems. The isolates were Carba NP test negative, gave ambiguous results with the modified Hodge test, and amplified the blaOXA48-like gene by real-time PCR. All E. coli isolates were sequence type 131 (ST131), carried nine resistance genes (including blaCTX-M-27) on an IncF plasmid, and were identical by genome sequencing, except for 150 kb of plasmid DNA in carbapenem-nonsusceptible isolates only. Sixty kilobases of this was shared by M. morganii and represented an IncN plasmid harboring blaOXA-181. In M. morganii, the gene was flanked by IS3000 and ISKpn19, but in all but one of the E. coli isolates containing blaOXA-181, a second copy of ISKpn19 had inserted adjacent to IS3000. To the best of our knowledge, this is the first report of blaOXA-181 in the virulent ST131 clonal group and carried by the promiscuous IncN family of plasmids. The tendency of M. morganii to have high MICs of imipenem, a blaOXA-181 substrate profile that includes penicillins but not extended-spectrum cephalosporins, and weak carbapenemase activity almost resulted in the presence of blaOXA-181 being overlooked. We highlight the importance of surveillance for carbapenem resistance in all species, even those with intrinsic resistances, and the value of advanced molecular techniques in detecting subtle genetic changes. PMID:25870058

  3. Previously undescribed plasmids recovered from activated sludge confer tetracycline resistance and phenotypic changes to Acinetobacter oleivorans DR1.

    PubMed

    Hong, Hyerim; Ko, Hyeok-Jin; Choi, In-Geol; Park, Woojun

    2014-02-01

    We used culture-dependent and culture-independent methods to extract previously undescribed plasmids harboring tetracycline (TC) resistance genes from activated sludge. The extracted plasmids were transformed into naturally competent Acinetobacter oleivorans DR1 to recover a non-Escherichia coli-based plasmid. The transformed cells showed 80-100-fold higher TC resistance than the wild-type strain. Restriction length polymorphism performed using 30 transformed cells showed four different types of plasmids. Illumina-based whole sequencing of the four plasmids identified three previously unreported plasmids and one previously reported plasmid. All plasmids carried TC resistance-related genes (tetL, tetH), tetracycline transcriptional regulators (tetR), and mobilization-related genes. As per expression analysis, TC resistance genes were functional in the presence of TC. The recovered plasmids showed mosaic gene acquisition through horizontal gene transfer. Membrane fluidity, hydrophobicity, biofilm formation, motility, growth rate, sensitivity to stresses, and quorum sensing signals of the transformed cells were different from those of the wild-type cells. Plasmid-bearing cells seemed to have an energy burden for maintaining and expressing plasmid genes. Our data showed that acquisition of TC resistance through plasmid uptake is related to loss of biological fitness. Thus, cells acquiring antibiotic resistance plasmids can survive in the presence of antibiotics, but must pay ecological costs.

  4. Thioredoxin-like proteins in F and other plasmid systems.

    PubMed

    Hemmis, Casey W; Schildbach, Joel F

    2013-09-01

    Bacterial conjugation is the process by which a conjugative plasmid transfers from donor to recipient bacterium. During this process, single-stranded plasmid DNA is actively and specifically transported from the cytoplasm of the donor, through a large membrane-spanning assembly known as the pore complex, and into the cytoplasm of the recipient. In Gram negative bacteria, construction of the pore requires localization of a subset of structural and catalytically active proteins to the bacterial periplasm. Unlike the cytoplasm, the periplasm contains proteins that promote disulfide bond formation within or between cysteine-containing proteins. To ensure proper protein folding and assembly, bacteria employ periplasmic redox systems for thiol oxidation, disulfide bond/sulfenic acid reduction, and disulfide bond isomerization. Recent data suggest that plasmid-based proteins belonging to the disulfide bond formation family play an integral role in the conjugative process by serving as mediators in folding and/or assembly of pore complex proteins. Here we report the identification of 165 thioredoxin-like family members across 89 different plasmid systems. Using phylogenetic analysis, all but nine family members were categorized into thioredoxin-like subfamilies. In addition, we discuss the diversity, conservation, and putative roles of thioredoxin-like proteins in plasmid systems, which include homologs of DsbA, DsbB, DsbC, DsbD, DsbG, and CcmG from Escherichia coli, TlpA from Bradyrhizobium japonicum, Com1 from Coxiella burnetii, as well as TrbB and TraF from plasmid F, and the absolute conservation of a disulfide isomerase in plasmids containing homologs of the transfer proteins TraH, TraN, and TraU.

  5. Plasma-activated air mediates plasmid DNA delivery in vivo

    PubMed Central

    Edelblute, Chelsea M; Heller, Loree C; Malik, Muhammad A; Bulysheva, Anna; Heller, Richard

    2016-01-01

    Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets. PMID:27110584

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

    PubMed Central

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

    2015-01-01

    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. PMID:25628616

  7. 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

    USDA-ARS?s Scientific Manuscript database

    IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. While antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types...

  8. Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules.

    PubMed

    Nyberg, Lena K; Quaderi, Saair; Emilsson, Gustav; Karami, Nahid; Lagerstedt, Erik; Müller, Vilhelm; Noble, Charleston; Hammarberg, Susanna; Nilsson, Adam N; Sjöberg, Fei; Fritzsche, Joachim; Kristiansson, Erik; Sandegren, Linus; Ambjörnsson, Tobias; Westerlund, Fredrik

    2016-07-27

    The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.

  9. Nitrogen-fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids.

    PubMed Central

    Martínez, E; Palacios, R; Sánchez, F

    1987-01-01

    Rhizobium phaseoli CFN299 forms nitrogen-fixing nodules in Phaseolus vulgaris (bean) and in Leucaena esculenta. It has three plasmids of 185, 225, and 410 kilobases. The 410-kilobase plasmid contains the nitrogenase structural genes. We have transferred these plasmids to the plasmid-free strain Agrobacterium tumefaciens GMI9023. Transconjugants containing different combinations of the R. phaseoli plasmids were obtained, and they were exhaustively purified before nodulation was assayed. Only transconjugants harboring the 410-kilobase plasmid nodulate P. vulgaris and L. esculenta. Nodules formed by all such transconjugants are able to reduce acetylene. Transconjugants containing the whole set of plasmids from CFN299 nodulate better and fix more nitrogen than the transconjugants carrying only the Sym plasmid. Microscopic analysis of nodules induced by A. tumefaciens transconjugants reveals infected cells and vascular bundles. None of the A. tumefaciens transconjugants, not even the one with the whole set of plasmids from CFN299, behaves in symbiosis like the original R. phaseoli strain; the transconjugants produce fewer nodules and have lower acetylene reduction (25% as compared to the original R. phaseoli strain) and more amyloplasts per nodule. More than 2,000 bacterial isolates from nodules of P. vulgaris and L. esculenta formed by the transconjugants were analyzed by different criteria. Not a single rhizobium could be detected. Our results show that R. phaseoli plasmids may be expressed in the A. tumefaciens background and direct the formation of effective, differentiated nodules. Images PMID:3584072

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

    PubMed

    Krupovic, Mart; Gonnet, Mathieu; Hania, Wajdi Ben; Forterre, Patrick; Erauso, Gaël

    2013-01-01

    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.

  11. Insights into Dynamics of Mobile Genetic Elements in Hyperthermophilic Environments from Five New Thermococcus Plasmids

    PubMed Central

    Krupovic, Mart; Gonnet, Mathieu; Hania, Wajdi Ben; Forterre, Patrick; Erauso, Gaël

    2013-01-01

    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. PMID:23326305

  12. Prevalence of ColE1-like plasmids and kanamycin resistance genes in Salmonella enterica serovars.

    PubMed

    Chen, Chin-Yi; Lindsey, Rebecca L; Strobaugh, Terence P; Frye, Jonathan G; Meinersmann, Richard J

    2010-10-01

    Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kan(r)) phenotypes, 102 Kan(r) Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kan(r) Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3')-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group.

  13. Characterization of Extended-Spectrum β-Lactamase-Carrying Plasmids in Clinical Isolates of Klebsiella pneumoniae from Taiwan.

    PubMed

    Chang, Chung-Yu; Lin, Heng-Jia; Chang, Lin-Li; Ma, Ling; Siu, L Kristopher; Tung, Yi-Ching; Lu, Po-Liang

    2017-01-01

    We analyzed the replicon types, sizes, and restriction fragment length polymorphism (RFLP) typing of plasmids carrying extended-spectrum β-lactamase (ESBL) genes in Klebsiella pneumoniae isolates from Taiwan. Fifty-one Escherichia coli transconjugant strains with plasmids from ESBL-producing K. pneumoniae from the Taiwan Surveillance of Antimicrobial Resistance III Program in 2002 were included. All the 51 plasmids carried a blaCTX-M gene, the majority of which were blaCTX-M-3 (28/51 [54.9%]). Plasmids ranged in size from 126 to 241 kb by S1 nuclease digestion and subsequent pulsed-field gel electrophoresis, and the most common plasmid size (37.3%) was 161-170 kb. The most common replicon type of plasmids was incompatibility group (Inc)A/C (60.8%). The IncA/C plasmids all carried blaCTX-M (blaCTX-M-3, -14, -15), and some also carried blaSHV (blaSHV-5, -12) genes. All 51 plasmids could be typed with PstI, and 27 (52.9%) belonged to 10 clusters. Thirty-eight of the 51 plasmids were typable with BamHI, and 21 plasmids (55.3%) fell into 7 clusters. Plasmids in the same cluster belonged to the same incompatibility group, with the exception of cluster C6. In conclusion, IncA/C plasmids are the main plasmid type responsible for the dissemination of ESBL genes of K. pneumoniae from Taiwan. RFLP with PstI possessed better discriminatory power than that with BamHI and PCR-based replicon typing for ESBL-carrying plasmids in K. pneumoniae in this study. Greater than 50% of plasmids fell into clusters, and >60% of cluster-classified plasmids were present in clonally unrelated isolates, indicating that horizontal transfer of plasmids plays an important role in the spread of ESBL genes.

  14. Combined IL-15 and IL-12 drives the generation of CD34(+)-derived natural killer cells with superior maturation and alloreactivity potential following adoptive transfer.

    PubMed

    Cany, Jeannette; van der Waart, Anniek B; Spanholtz, Jan; Tordoir, Marleen; Jansen, Joop H; van der Voort, Robbert; Schaap, Nicolaas M; Dolstra, Harry

    2015-07-01

    Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against cancer, including acute myeloid leukemia (AML). Previously, we reported a cytokine-based culture method for the generation of NK cell products with high cell number and purity. In this system, CD34(+) hematopoietic progenitor cells (HPC) were expanded and differentiated into NK cells under stroma-free conditions in the presence of IL-15 and IL-2. We show that combining IL-15 with IL-12 drives the generation of more mature and highly functional NK cells. In particular, replacement of IL-2 by IL-12 enhanced the cytolytic activity and IFNγ production of HPC-NK cells toward cultured and primary AML cells in vitro, and improved antileukemic responses in NOD/SCID-IL2Rγnull (NSG) mice bearing human AML cells. Phenotypically, IL-12 increased the frequency of HPC-NK cells expressing NKG2A and killer immunoglobulin-like receptor (KIR), which were more responsive to target cell stimulation. In addition, NK15/12 cell products demonstrated superior maturation potential, resulting in >70% positivity for CD16 and/or KIR within 2 weeks after infusion into NSG mice. We predict that higher functionality and faster in vivo maturation will favor HPC-NK cell alloreactivity toward malignant cells in patients, making this cytokine combination an attractive strategy to generate clinical HPC-NK cell products for cancer adoptive immunotherapy.

  15. Plasmid-mediated quinolone resistance

    PubMed Central

    Jacoby, George A.; Strahilevitz, Jacob; Hooper, David C.

    2014-01-01

    Summary Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6′)-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat. PMID:25584197

  16. Plasmid-mediated quinolone resistance.

    PubMed

    Jacoby, George A; Strahilevitz, Jacob; Hooper, David C

    2014-10-01

    Three mechanisms for plasmid-mediated quinolone resistance (PMQR) have been discovered since 1998. Plasmid genes qnrA, qnrB, qnrC, qnrD, qnrS, and qnrVC code for proteins of the pentapeptide repeat family that protects DNA gyrase and topoisomerase IV from quinolone inhibition. The qnr genes appear to have been acquired from chromosomal genes in aquatic bacteria, are usually associated with mobilizing or transposable elements on plasmids, and are often incorporated into sul1-type integrons. The second plasmid-mediated mechanism involves acetylation of quinolones with an appropriate amino nitrogen target by a variant of the common aminoglycoside acetyltransferase AAC(6')-Ib. The third mechanism is enhanced efflux produced by plasmid genes for pumps QepAB and OqxAB. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. The plasmid-mediated mechanisms provide only low-level resistance that by itself does not exceed the clinical breakpoint for susceptibility but nonetheless facilitates selection of higher-level resistance and makes infection by pathogens containing PMQR harder to treat.

  17. The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans.

    PubMed Central

    Giebelhaus, L A; Frost, L; Lanka, E; Gormley, E P; Davies, J E; Leskiw, B

    1996-01-01

    Escherichia coli cells and Streptomyces mycelia are able to form close contacts in the absence of a conjugative system which might facilitate intergeneric plasmid transfer without the genes required for mating pair formation (Tra2) of the RP4 plasmid. The same Tra2 genes found to be essential for RP4 plasmid transfer, RSF1010 mobilization, and donor-specific phage propagation in E. coli were also required for intergeneric transfer between E. coli and Streptomyces lividans. PMID:8892846

  18. Molecular survey of the dissemination of two blaKPC-harboring IncFIA plasmids in New Jersey and New York hospitals.

    PubMed

    Chen, Liang; Chavda, Kalyan D; Melano, Roberto G; Hong, Tao; Rojtman, Albert D; Jacobs, Michael R; Bonomo, Robert A; Kreiswirth, Barry N

    2014-01-01

    Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains have spread worldwide and become a major threat in health care facilities. Transmission of blaKPC, the plasmid-borne KPC gene, can be mediated by clonal spread and horizontal transfer. Here, we report the complete nucleotide sequences of two novel blaKPC-3-harboring IncFIA plasmids, pBK30661 and pBK30683. pBK30661 is 74 kb in length, with a mosaic plasmid structure; it exhibits homologies to several other plasmids but lacks the plasmid transfer operon (tra) and the origin of transfer (oriT) that are required for plasmid transfer. pBK30683 is a conjugative plasmid with a cointegrated plasmid structure, comprising a 72-kb element that highly resembles pBK30661 (>99.9% nucleotide identities) and an extra 68-kb element that harbors tra and oriT. A PCR scheme was designed to detect the distribution of blaKPC-harboring IncFIA (pBK30661-like and pBK30683-like) plasmids in a collection of clinical Enterobacteriaceae isolates from 10 hospitals in New Jersey and New York. KPC-harboring IncFIA plasmids were found in 20% of 491 K. pneumoniae isolates, and all carried blaKPC-3. pBK30661-like plasmids were identified mainly in the epidemic sequence type 258 (ST258) K. pneumoniae clone, while pBK30683-like plasmids were widely distributed in ST258 and other K. pneumoniae sequence types and among non-K. pneumoniae Enterobacteriaceae species. This suggests that both clonal spread and horizontal plasmid transfer contributed to the dissemination of blaKPC-harboring IncFIA plasmids in our area. Further studies are needed to understand the distribution of this plasmid group in other health care regions and to decipher the origins of pBK30661-like and pBK30683-like plasmids.

  19. Ribonucleases, antisense RNAs and the control of bacterial plasmids.

    PubMed

    Saramago, Margarida; Bárria, Cátia; Arraiano, Cecília M; Domingues, Susana

    2015-03-01

    In the last decade regulatory RNAs have emerged as powerful tools to regulate the expression of genes both in prokaryotes and in eukaryotes. RNases, by degrading these RNA molecules, control the right amount of regulatory RNAs, which is fundamental for an accurate regulation of gene expression in the cell. Remarkably the first antisense RNAs identified were plasmid-encoded and their detailed study was crucial for the understanding of prokaryotic antisense RNAs. In this review we highlight the role of RNases in the precise modulation of antisense RNAs that control plasmid replication, maintenance and transfer.

  20. Chemical adjuvants for plasmid DNA vaccines.

    PubMed

    Greenland, John R; Letvin, Norman L

    2007-05-10

    Plasmid DNA vaccines are a promising modality for immunization against a variety of human pathogens. Immunization via multiple routes with plasmid DNA can elicit potent cellular immune responses, and these immunogens can be administered repeatedly without inducing anti-vector immunity. Nonetheless, the immunogenicity of plasmid DNA vaccines has been limited by problems associated with delivery. A number of adjuvants have been designed to improve plasmid DNA immunogenicity, either by directly stimulating the immune system or by enhancing plasmid DNA expression. Chemical adjuvants for enhancing plasmid DNA expression include liposomes, polymers, and microparticles, all of which have shown promise for enhancing the expression and immunogenicity of plasmid DNA vaccines in animal models. Micro- and nanoparticles have not been shown to enhance immune responses to plasmid DNA vaccines. However, formulation of plasmid DNA with some non-particulate polymeric adjuvants has led to a statistically significant enhancement of immune responses. Further development of these technologies will significantly improve the utility of plasmid DNA vaccination.

  1. A Transmissible Plasmid Controlling Camphor Oxidation in Pseudomonas putida

    PubMed Central

    Rheinwald, J. G.; Chakrabarty, A. M.; Gunsalus, I. C.

    1973-01-01

    Earlier papers demonstrated an extensive genetic exchange among fluorescent Pseudomonads; this one documents for genes specifying enzymes of peripheral dissimilation an extrachromosomal array, segregation, and frequent interstrain transfer. An hypothesis is presented of a general mechanism for the formation and maintenance of metabolic diversity. The example used, the path of oxidative cleavage of the carbocyclic rings of the bicyclic monoterpene D- and L-camphor, terminates in acetate release and isobutyrate chain debranching. By transduction, two gene linkage groups are shown for the reactions before and after isobutyrate. The group for reactions before isobutyrate is plasmid borne, contransferable by conjugation, mitomycin curable, and shows a higher segregation rate from cells that are multiplasmid rather than carrying a single plasmid. The genes that code for isobutyrate and essential anaplerotic and amphibolic metabolism are chromosomal. By conjugation plasmid-borne genes are transferred at a higher frequency than are chromosomal, and are transferred in homologous crosses more frequently than between heterologous species. Most isobutyrate-positive fluorescent pseudomonad strains will accept and express the camphor plasmid. PMID:4351810

  2. Evolutionary Paths That Expand Plasmid Host-Range: Implications for Spread of Antibiotic Resistance.

    PubMed

    Loftie-Eaton, Wesley; Yano, Hirokazu; Burleigh, Stephen; Simmons, Ryan S; Hughes, Julie M; Rogers, Linda M; Hunter, Samuel S; Settles, Matthew L; Forney, Larry J; Ponciano, José M; Top, Eva M

    2016-04-01

    The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Trans-complementable copy-number mutants of plasmid ColE1.

    PubMed

    Twigg, A J; Sherratt, D

    1980-01-10

    Plasmid ColE1, like many other small non-conjugative plasmids, is present in multiple copies (about 15 per chromsome equivalent) in Escherichia coli cells. Because of their high copy number, the replication of such plasmids has been described as 'relaxed', even though there is good evidence that it is strictly controlled: ColE1 derivatives have characteristic but different copy numbers and ColE1 copy-number mutants have been characterised. No plasmid-specified protein is essential for the replication of ColE1 and related plasmids, as extensive replication can occur in chloramphenicol-treated cells, in plasmid-free chloramphenicol-treated cells transfected with a hybrid ColE1/phage replicon and in vitro in extracts derived from plasmid-free cells. Nevertheless, it is possible that a plasmid-specified protein is involved in ColE1 replication control in viable cells. Here we show that deletion of a given non-essential region from ColE1-like plasmids results in a raised copy number. Such plasmids are stably maintained and have their copy number returned to normal when a complementing plasmid is present in the same cell, indicating that a plasmid-specified diffusible gene product regulates the plasmid content of ColE1-containing cells. Deletion of the equivalent region from the cloning vector pBR322 gives a derivative which has a raised copy number and which has also lost its origin for conjugal transfer; unlike pBR322, it cannot be mobilised.

  4. Vibrio cholerae conjugative plasmid pSJ15 contains transposable prophage dVcA1.

    PubMed Central

    Johnson, S R; Romig, W R

    1981-01-01

    Evidence is presented that defective prophage dVcA1 in Vibrio cholerae strain 162 was transposed to the hybrid P::Tn1 plasmid pSJ5. Properties of the resulting conjugative plasmid, pSJ15, indicated that bacteriophage VcA1, like coliphage Mu, can insert at many sites. By analogy with other Hfr-like donors, the high-frequency, polarized chromosomal transfer mediated by plasmid pSJ15 in strain 162 appeared to depend on plasmid integration through the homologous dVcA1 sequences in both replicons. When strain 162(pSJ15) donors were mated to the nonlysogenic El Tor strain RJ1, many potential ampicillin-resistant transconjugants were zygotically induced. However, surviving transconjugants (i) were immune to phage VcA1, (ii) cotransferred immunity and ampicillin resistance to nonlysogenic recipients, and (iii) did not preferentially transfer any chromosomal markers. Recombinant plasmids that transferred wild-type VcA1 prophages were readily isolated from strain RJ1 (VcA1+) lysogens that contained plasmid pSJ15. Physical measurements revealed that plasmid pSJ15 and the recombinant plasmids were about one VcA1 genome (22 to 24 megadaltons) larger than the 51-megadalton pSJ5 plasmid. Similar Hfr-like donors were constructed by introducing plasmid pSJ15 into different strain RJ1 (VcA1+) lysogens. Transfer properties of these donors indicated that the VcA1 prophage was integrated at several sites in the strain RJ1 chromosome. Images PMID:6260754

  5. A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation

    PubMed Central

    Thoma, Lina; Dobrowinski, Hyazinth; Finger, Constanze; Guezguez, Jamil; Linke, Dirk

    2015-01-01

    ABSTRACT Conjugative DNA transfer in mycelial Streptomyces is a unique process involving the transfer of a double-stranded plasmid from the donor into the recipient and the subsequent spreading of the transferred plasmid within the recipient mycelium. This process is associated with growth retardation of the recipient and manifested by the formation of circular inhibition zones, named pocks. To characterize the unique Streptomyces DNA transfer machinery, we replaced each gene of the conjugative 12.1-kbp Streptomyces venezuelae plasmid pSVH1, with the exception of the rep gene required for plasmid replication, with a hexanucleotide sequence. Only deletion of traB, encoding the FtsK-like DNA translocase, affected efficiency of the transfer dramatically and abolished pock formation. Deletion of spdB3, spd79, or spdB2 had a minor effect on transfer but prevented pock formation and intramycelial plasmid spreading. Biochemical characterization of the encoded proteins revealed that the GntR-type regulator TraR recognizes a specific sequence upstream of spdB3, while Orf108, SpdB2, and TraR bind to peptidoglycan. SpdB2 promoted spheroplast formation by T7 lysozyme and formed pores in artificial membranes. Bacterial two-hybrid analyses and chemical cross-linking revealed that most of the pSVH1-encoded proteins interacted with each other, suggesting a multiprotein DNA translocation complex of TraB and Spd proteins which directs intramycelial plasmid spreading. PMID:26015502

  6. Complete sequence of three plasmids from Bacillus thuringiensis INTA-FR7-4 environmental isolate and comparison with related plasmids from the Bacillus cereus group.

    PubMed

    Amadio, Ariel F; Benintende, Graciela B; Zandomeni, Rubén O

    2009-11-01

    Bacillus thuringiensis is an insect pathogen used worldwide as a bioinsecticide. It belongs to the Bacillus cereus sensu lato group as well as Bacillus anthracis and B. cereus. Plasmids from this group of organisms have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that affect mammals and insects. Some plasmids, like pAW63 and pBT9727, encode a functional conjugation machinery allowing them to be transferred to a recipient cell. They also share extensive homology with the non-functional conjugation apparatus of pXO2 from B. anthracis. In this study we report the complete sequence of three plasmids from an environmental B. thuringiensis isolate from Argentina, obtained by a shotgun sequencing method. We obtained the complete nucleotide sequence of plasmids pFR12 (12,095bp), pFR12.5 (12,459bp) and pFR55 (55,712bp) from B. thuringiensis INTA-FR7-4. pFR12 and pFR12.5 were classified as cryptic as they do not code for any obvious functions besides replication and mobilization. Both small plasmids were classified as RCR plasmids due to similarities with the replicases they encode. Plasmid pFR55 showed a structural organization similar to that observed for plasmids pAW63, pBT9727 and pXO2. pFR55 also shares a tra region with these plasmids, containing genes related to T4SS and conjugation. A comparison between pFR55 and conjugative plasmids led to the postulation that pFR55 is a conjugative plasmid. Genes related to replication functions in pFR55 are different to those described for plasmids with known complete sequences. pFR55 is the first completely sequenced plasmid with a replication machinery related to that of ori44. The analysis of the complete sequence of plasmids from an environmental isolate of B. thuringiensis permitted the identification of a near complete conjugation apparatus in pFR55, resembling those of plasmids pAW63, pBT9727 and pXO2. The availability of this sequence is a step forward in the study

  7. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

    PubMed Central

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J.; Fox, Catherine A.

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  8. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    PubMed

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-04-07

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  9. 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.

    PubMed

    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.

  10. iDriving (Intelligent Driving)

    SciTech Connect

    Malikopoulos, Andreas

    2012-09-17

    iDriving identifies the driving style factors that have a major impact on fuel economy. An optimization framework is used with the aim of optimizing a driving style with respect to these driving factors. A set of polynomial metamodels is constructed to reflect the responses produced in fuel economy by changing the driving factors. The optimization framework is used to develop a real-time feedback system, including visual instructions, to enable drivers to alter their driving styles in responses to actual driving conditions to improve fuel efficiency.

  11. Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

    PubMed

    Lim, Suk-Kyung; Tanimoto, Koichi; Tomita, Haruyoshi; Ike, Yasuyoshi

    2006-10-01

    The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information

  12. Transcription of ColE1Ap mbeC induced by conjugative plasmids from twelve different incompatibility groups.

    PubMed Central

    Selvaratnam, S; Gealt, M A

    1993-01-01

    Although nonconjugative mobilizable plasmids require helping functions of conjugative plasmids in order to be mobilized into recipients, at least some genes from the nonconjugative plasmids may be induced to assist in the DNA transfer process. Conjugative plasmids from 12 different incompatibility groups mobilized the nonconjugative plasmid ColE1Ap between Escherichia coli strains. Introduction of any of the conjugative plasmids into the ColE1Ap-containing strain resulted in an induction of mbeC, the product of which is a component of the mobilization relaxation complex. Each of the conjugative plasmids caused protein to bind specifically to mbe promoter DNA, suggesting a direct regulatory interaction. Images PMID:8226641

  13. sRNA-Xcc1, an integron-encoded transposon- and plasmid-transferred trans-acting sRNA, is under the positive control of the key virulence regulators HrpG and HrpX of Xanthomonas campestris pathovar campestris.

    PubMed

    Chen, Xiao-Lin; Tang, Dong-Jie; Jiang, Rui-Ping; He, Yong-Qiang; Jiang, Bo-Le; Lu, Guang-Tao; Tang, Ji-Liang

    2011-01-01

    sRNA-Xcc1 is a trans-acting sRNA recently identified from the plant pathogenic bacterium Xanthomonas campestris pathovar campestris (Xcc). Here, the phylogenetic distribution, predicted secondary structure and regulation of expression of sRNA-Xcc1 were analyzed. The analysis showed (1) a total 81 sRNA-Xcc1 homologs that are found in some bacterial strains that are taxonomically unrelated, belonging to the α-, β-, γ- and δ-proteobacteria (2) that some sRNA-Xcc1 homologs are located in a plasmid-borne transposon or near a transposase coding gene, (3) that sRNA-Xcc1 is encoded by a integron gene cassette in Xcc and sRNA-Xcc1 homologs occur in integron gene cassettes of some uncultured bacteria and (4) that sRNA-Xcc1 homologs have a highly conserved sequence motif and a stable consensus secondary structure. These findings strongly support the idea that sRNA-Xcc1 represents a novel family of sRNAs which may be originally captured by integrons from natural environments and then spread among different bacterial species via horizontal gene transfer, possibly by means of transposons and plasmids. The expression analysis results demonstrated that the transcription of sRNA-Xcc1 is under the positive control of the key virulence regulators HrpG and HrpX, indicating that sRNA-Xcc1 may be involved in the virulence regulation of Xcc.

  14. Recombinant plasmid associated cell aggregation and high-frequency conjugation of Streptococcus lactis ML3.

    PubMed

    Walsh, P M; McKay, L L

    1981-06-01

    Lactose-positive (Lac+) transconjugants resulting from matings between Streptococcus lactic ML3 and S. lactis LM2301 possess a single plasmid of approximately 60 megadaltons (Mdal) which is nearly twice the size of the lactose plasmid of the donor. The majority of these Lac+ transconjugants aggregated in broth and were able to transfer lactose-fermenting ability at a frequency higher than 10(-1) per donor on milk agar plates or in broth. Lac+ transconjugants which did not clump conjugated at a much lower frequency. Lactose-negative derivatives of Lac+ clumping transconjugants did not aggregate in broth and were missing the 60-Mdal plasmid. The ability to aggregates in broth was very unstable. Strains could lose the ability to clump but retain lactose-fermenting ability. The majority of these Lac+ nonclumping derivatives of clumping transconjugants contained a plasmid of approximately 33 Mdal, the size of the lactose plasmid of the original donor ML3. These strains transferred lactose-fermenting ability at a frequency of approximately 10(-6) per donor, resulting in both Lac+ clumping transconjugants which contained a 60-Mdal plasmid and Lac+ nonclumping transconjugants which possessed a 33-Mdal plasmid. Our results suggest that the genes responsible for cell aggregation and high-frequency conjugation are on the segment of deoxyribonucleic acid which recombined with the 33-Mdal lactose plasmid in S. lactis ML3.

  15. Characterization of plasmids in extensively drug-resistant acinetobacter strains isolated in India and Pakistan.

    PubMed

    Jones, Lim S; Carvalho, Maria J; Toleman, Mark A; White, P Lewis; Connor, Thomas R; Mushtaq, Ammara; Weeks, Janis L; Kumarasamy, Karthikeyan K; Raven, Katherine E; Török, M Estée; Peacock, Sharon J; Howe, Robin A; Walsh, Timothy R

    2015-02-01

    The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae.

  16. Characterization of Plasmids in Extensively Drug-Resistant Acinetobacter Strains Isolated in India and Pakistan

    PubMed Central

    Carvalho, Maria J.; Toleman, Mark A.; White, P. Lewis; Connor, Thomas R.; Mushtaq, Ammara; Weeks, Janis L.; Kumarasamy, Karthikeyan K.; Raven, Katherine E.; Török, M. Estée; Peacock, Sharon J.; Howe, Robin A.; Walsh, Timothy R.

    2014-01-01

    The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae. PMID:25421466

  17. Conjugative multi-resistant plasmids in Haihe River and their impacts on the abundance and spatial distribution of antibiotic resistance genes.

    PubMed

    Dang, Bingjun; Mao, Daqing; Xu, Yan; Luo, Yi

    2017-03-15

    In this study, five classes of antibiotic resistance genes (ARGs) were quantified in sediment samples of Haihe River, China, with abundance ranging from 1.39 × 10(4) to 1.58 × 10(10) copies/g dry weight. Meanwhile, antibiotic resistant conjugative plasmids were also isolated from these samples through filter mating assays. In total, 202 transconjugants were isolated and tested for their antibiotic resistance phenotypes, among which 26 different types of conjugative plasmids were observed. The majority of these plasmids showed a multi-resistant phenotype and the most prevalent resistance was tetracycline resistance and sulfonamide resistance. Furthermore, we tested the transfer frequencies of these plasmids, determined their genotypes and then compared the plasmid-borne ARGs with their corresponding abundance in Haihe River. Most of the isolated plasmids exhibited high transfer frequencies to the recipient strain Escherichia coli J53. Plasmids isolated from the urban areas of Haihe River have higher transfer frequencies than the rural areas. Results from comprehensive analysis of plasmid genotypes, ARG abundance and plasmid sequencing confirmed that most of the plasmid-borne ARGs were the dominant genes in the Haihe River. Therefore, conjugative plasmids isolated from the Haihe River plays a crucial role in the dissemination, abundance and spatial distribution of ARGs in Haihe River, especially some unfrequent ARGs like blaGES-1. This study will help to increase the knowledge on the conjugative plasmid-mediated ARG propagation in the environment.

  18. Structural plasmid evolution as a result of coupled recombinations at bom and cer sites.

    PubMed

    Zakharova, M V; Beletskaya, I V; Bolovin, D V; Yurkova, T V; Semenova, L M; Solonin, A S

    2003-12-01

    We have studied the recombination of plasmids bearing bom and cer sites. The bom ( basis of mobilization) site is required for conjugative transfer, while the cer ( Col E1 resolution) site is involved in the resolution of plasmid multimers, which increases plasmid stability. We constructed a pair of parent plasmids in such a way as to allow us select clones containing recombinant plasmids directly. Clone selection was based on the McrA sensitivity of recipient host DNA modified by M. Ecl18kI, which is encoded by one of the parent plasmids. The recombinant plasmid contains segments originating from both parental DNAs, which are bounded by bom and cer sites. Its structure is in accordance with our previously proposed model for recombination mediated by bom and cer sequences. The frequency of recombinant plasmid formation coincided with the frequency of recombination at the bom site. We also show that bom-mediated recombination in trans, unlike in cis, is independent of other genetic determinants on the conjugative plasmids.

  19. Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola

    PubMed Central

    Van Ham, Roeland C. H. J.; González-Candelas, Fernando; Silva, Francisco J.; Sabater, Beatriz; Moya, Andrés; Latorre, Amparo

    2000-01-01

    Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids. PMID:10984505

  20. Modular architecture of the conjugative plasmid pSVH1 from Streptomyces venezuelae.

    PubMed

    Reuther, Jens; Wohlleben, Wolfgang; Muth, Günther

    2006-05-01

    The conjugative rolling circle replication (RCR) type plasmid pSVH1 from the chloramphenicol producer Streptomyces venezuelae was characterized by DNA sequence analysis and insertion/deletion analysis. Nucleotide sequence of the 12,652 bp pSVH1 revealed 11 open reading frames with high coding probability for which putative functions could be assigned. Beside the replication initiator gene rep for RCR, pSVH1 contained only genes involved in conjugative transfer. The transfer gene traB encoding the septal DNA translocator TraB is regulated by the GntR-type transcriptional regulator TraR. Six spd genes involved in intra-mycelial plasmid spreading are organized in two operons, consisting of two and three translationally coupled genes. Subcloning experiments demonstrated that the transfer gene traB represents a kill function and localized the pSVH1 minimal replicon consisting of rep and the dso origin to a 2072-bp fragment. Plasmid pSVH1 showed a modular architecture. Its replication region resembled that of the Streptomyces natalensis plasmid pSNA1, while the transfer and spread regions involved in conjugative plasmid transfer were highly similar to the corresponding regions of the Streptomyces ghanaensis plasmid pSG5.

  1. Conjugation of plasmids of Neisseria gonorrhoeae to other Neisseria species: potential reservoirs for the beta-lactamase plasmid.

    PubMed

    Genco, C A; Knapp, J S; Clark, V L

    1984-09-01

    The discovery that penicillinase production in Neisseria gonorrhoeae was plasmid mediated and the spread of the beta-lactamase encoding plasmids in gonococcal isolates since 1976, raise the possibility that a nonpathogenic indigenous bacterium could serve as a reservoir for these plasmids. We initiated studies to define the ability of commensal Neisseria species and Branhamella catarrhalis strains, as well as strains of the pathogen Neisseria meningitidis, to serve as recipients in conjugation with Neisseria gonorrhoeae. We found that with N. gonorrhoeae as the donor, 3 of 5 Neisseria cinerea, 2 of 5 Neisseria flava, 0 of 1 Neisseria flavescens, 1 of 3 Neisseria subflava, 0 of 6 B. catarrhalis, 0 of 7 Neisseria lactamica, 1 of 5 Neisseria mucosa, 1 of 7 Neisseria perflava/sicca, and 0 of 13 N. meningitidis strains gave detectable conjugation frequencies (greater than 10(-8). N. cinerea was the only species found to maintain the gonococcal conjugal plasmid (pLE2451). A N. cinerea transconjugant containing pLE2451 was observed to transfer both the beta-lactamase plasmid and pLE2451 to N. gonorrhoeae at high frequency.

  2. Linear Plasmids and the Rate of Sequence Evolution in Plant Mitochondrial Genomes

    PubMed Central

    Warren, Jessica M.; Simmons, Mark P.; Wu, Zhiqiang; Sloan, Daniel B.

    2016-01-01

    The mitochondrial genomes of flowering plants experience frequent insertions of foreign sequences, including linear plasmids that also exist in standalone forms within mitochondria, but the history and phylogenetic distribution of plasmid insertions is not well known. Taking advantage of the increased availability of plant mitochondrial genome sequences, we performed phylogenetic analyses to reconstruct the evolutionary history of these plasmids and plasmid-derived insertions. Mitochondrial genomes from multiple land plant lineages (including liverworts, lycophytes, ferns, and gymnosperms) include fragmented remnants from ancient plasmid insertions. Such insertions are much more recent and widespread in angiosperms, in which approximately 75% of sequenced mitochondrial genomes contain identifiable plasmid insertions. Although conflicts between plasmid and angiosperm phylogenies provide clear evidence of repeated horizontal transfers, we were still able to detect significant phylogenetic concordance, indicating that mitochondrial plasmids have also experienced sustained periods of (effectively) vertical transmission in angiosperms. The observed levels of sequence divergence in plasmid-derived genes suggest that nucleotide substitution rates in these plasmids, which often encode their own viral-like DNA polymerases, are orders of magnitude higher than in mitochondrial chromosomes. Based on these results, we hypothesize that the periodic incorporation of mitochondrial genes into plasmids contributes to the remarkable heterogeneity in substitution rates among genes that has recently been discovered in some angiosperm mitochondrial genomes. In support of this hypothesis, we show that the recently acquired ψtrnP-trnW gene region in a maize linear plasmid is evolving significantly faster than homologous sequences that have been retained in the mitochondrial chromosome in closely related grasses. PMID:26759362

  3. Impaired Driving

    MedlinePlus

    Impaired driving is dangerous. It's the cause of more than half of all car crashes. It means operating a ... texting Having a medical condition which affects your driving For your safety and the safety of others, ...

  4. p518, a small floR plasmid from a South American isolate of Actinobacillus pleuropneumoniae.

    PubMed

    da Silva, Giarlã Cunha; Rossi, Ciro César; Santana, Mateus Ferreira; Langford, Paul R; Bossé, Janine T; Bazzolli, Denise Mara Soares

    2017-05-01

    A small (3.9kb) plasmid (p518), conferring resistance to florfenicol (MIC >8μg/mL) and chloramphenicol (MIC >8μg/mL) was isolated from an Actinobacillus pleuropneumoniae clinical isolate from Southeastern Brazil. To date, this is the smallest florfenicol resistance plasmid isolated from a member of the Pasteurellaceae. The complete nucleotide of this plasmid revealed a unique gene arrangement compared to previously reported florfenicol resistance plasmids found in other members of the Pasteurellaceae. In addition to the floR gene and a lysR gene, common to various florfenicol resistance plasmids, p518 also encodes strA and a partial strB sequence. An origin of replication (oriV) similar to that in the broad host range plasmid, pLS88, was identified in p518, and transformation into Escherichia coli MFDpir confirmed the ability to replicate in other species. Mobilisation genes appear to have been lost, with only a partial mobC sequence remaining, and attempts to transfer p518 from a conjugal donor strain (E. coli MFDpir) were not successful, suggesting this plasmid is not mobilisable. Similarly, attempts to transfer p518 into a competent A. pleuropneumoniae strain, MIDG2331, by natural transformation were also not successful. These results suggest that p518 may be only transferred by vertical descent. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Homology of cryptic plasmid of Neisseria gonorrhoeae with plasmids from Neisseria meningitidis and Neisseria lactamica.

    PubMed

    Ison, C A; Bellinger, C M; Walker, J

    1986-10-01

    DNA probe hybridisation was used to examine the relation between the cryptic plasmid from Neisseria gonorrhoeae and plasmids carried by pharyngeal isolates of Neisseria meningitidis and Neisseria lactamica. The complete gonococcal cryptic plasmid and HinfI derived digestion fragments subcloned into Escherichia coli were used to probe Southern blots of plasmid extracts. Homology was found to a plasmid of approximate molecular weight 4.5 kilobase pairs (Kb) but not to plasmids of less than 3.2 Kb or 6.5 Kb. Eleven of 16 strains of N meningitidis and two of six strains of N lactamica carried plasmids that showed strong hybridisation with the 4.2 Kb gonococcal plasmid. Hybridisation of plasmids from non-gonococcal species of neisseria with the gonococcal cryptic plasmid indicates that caution should be taken when using the cryptic plasmid as a diagnostic probe for gonorrhoea.

  6. IncHI2 Plasmids Are Predominant in Antibiotic-Resistant Salmonella Isolates

    PubMed Central

    Chen, Wenyao; Fang, Tingzi; Zhou, Xiujuan; Zhang, Daofeng; Shi, Xianming; Shi, Chunlei

    2016-01-01

    The wide usage of antibiotics contributes to the increase in the prevalence of antibiotic-resistant Salmonella. Plasmids play a critical role in horizontal transfer of antibiotic resistance markers in Salmonella. This study aimed to screen and characterize plasmid profiles responsible for antibiotic resistance in Salmonella and ultimately to clarify the molecular mechanism of transferable plasmid-mediated antibiotic resistance. A total of 226 Salmonella isolates were examined for antimicrobial susceptibility by a disk diffusion method. Thirty-two isolates (14.2%) were resistant to at least one antibiotic. The presence of plasmid-mediated quinolone resistance (PMQR) genes and β-lactamase genes were established by PCR amplification. PCR-based replicon typing revealed that these 32 isolates represented seven plasmid incompatibility groups (IncP, HI2, A/C, FIIs, FIA, FIB, and I1), and the IncHI2 (59.4%) was predominant. Antibiotic resistance markers located on plasmids were identified through plasmid curing. Fifteen phenotypic variants were obtained with the curing efficiency of 46.9% (15/32). The cured plasmids mainly belong to the HI2 incompatibility group. The elimination of IncHI2 plasmids correlated with the loss of β-lactamase genes (blaOXA-1 and blaTEM-1) and PMQR genes (qnrA and aac(6′)-Ib-cr). Both IncHI2 and IncI1 plasmids in a S. enterica serovar Indiana isolate SJTUF 10584 were lost by curing. The blaCMY -2-carrying plasmid pS10584 from SJTUF 10584 was fully sequenced. Sequence analysis revealed that it possessed a plasmid scaffold typical for IncI1 plasmids with the unique genetic arrangement of IS1294-ΔISEcp1-blaCMY -2-blc-sugE-ΔecnR inserted into the colicin gene cia. These data suggested that IncHI2 was the major plasmid lineage contributing to the dissemination of antibiotic resistance in Salmonella and the activity of multiple mobile genetic elements may contribute to antibiotic resistance evolution and dissemination between different plasmid

  7. Environmentally co-occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context-dependent fitness effects.

    PubMed

    Hall, James P J; Harrison, Ellie; Lilley, Andrew K; Paterson, Steve; Spiers, Andrew J; Brockhurst, Michael A

    2015-12-01

    Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co-occurring pQBR family environmental mercury resistance plasmids and measured their effects on competitive fitness of a Pseudomonas fluorescens SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid-borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus-encoding cluster and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolize sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity among co-occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity.

  8. Mobilization of the gonococcal 5.2 kb beta‐lactamase plasmid pSJ5.2 into Escherichia coli by cointegration with several Gram‐ conjugative plasmids

    PubMed Central

    Scharbaai‐Vázquez, R.; Candelas, T.; Torres‐Bauzá, L. J.

    2007-01-01

    We report the mobilization by cointegration of the gonococcal 5.2 kb beta‐lactamase plasmid pSJ5.2 in an Escherichia coli background. Transfer of pSJ5.2 was measured by filter mating assays with five different conjugative plasmids from Enterobacteriaceae and the gonococcal 41 kb tet(M). Plasmid pSJ5.2 was mobilized to E. coli at frequencies of 1.7 × 10−6, 9.3 × 10−8 and 2.7 × 10−5 by the tet(M), R64 drd‐33 and N3 conjugative plasmids, respectively. Mobilization of pSJ5.2 by the 41 kb tet(M) conjugative plasmid resulted in stable AmpR E. coli transconjugants consisting of pSJ5.2 plasmid with an insertion located in the 2.4 kb BamH1‐ BamH1 fragment. Mobilization of pSJ5.2 by R64drd‐33 and N3 conjugative plasmids involved stable cointegrates as detected by Southern Blot with a DIG‐labelled Pst1‐digested pSJ5.2 probe. Restriction analysis of the R64::pSJ5.2 and N3::pSJ5.2 cointegrates and Southern Blot with the pSJ5.2 probe showed that cointegrates formed by deletion of DNA regions within the 1.8 kb BamH1‐HindIII fragment of pSJ5.2. The plasmid thus appears to use multiple recombination mechanisms for cointegration with different conjugative plasmids. The complete nucleotide sequence of pSJ5.2 was determined, and will be a useful tool to further investigate the molecular mechanisms leading to its cointegrative transfer. PMID:17027960

  9. Mobilization of Bacillus thuringiensis plasmid pTX14-3.

    PubMed

    Andrup, L; Bendixen, H H; Jensen, G B

    1995-05-01

    The Bacillus thuringiensis subsp. israelensis (Bti) plasmid pTX14-3 has been reported to contain a gene, mob14-3, with considerable homology to genes encoding mobilization proteins from other gram-positive bacteria. We have used the aggregation-mediated conjugation system recently discovered in Bti to compare the mobilization kinetics of different derivatives of plasmid pTX14-3. Plasmid pTX14-3 has been found to replicate by the rolling-circle mechanism and to contain a locus suppressing the formation of high-molecular-weight DNA. We found that deleting a DNA fragment containing this locus increased the transfer frequency about twofold. The mobilization frequency of the plasmid containing the intact mob14-3 gene did not indicate a mobilization-enhancing activity of the encoded polypeptide. However, the presence of the mob14-3 gene seemed to increase the stability of the plasmid in exponential growth.

  10. Persistence of Antibiotic Resistance Plasmids in Biofilms

    DTIC Science & Technology

    2014-10-01

    useful in the care of patients with combat-related wound infections. 15. SUBJECT TERMS Antibiotic resistance, plasmid, biofilm, coevolution , bacteria...Antibiotic!resistance,!plasmid,!biofilm,! coevolution ,!bacteria,!wound!infections! ! ! ! 3! 3. OVERALL PROJECT SUMMARY: The! successful! persistence

  11. Genome Sequence of Listeria monocytogenes Plasmid pLM-C-273 Carrying Genes Related to Stress Resistance.

    PubMed

    Liang, Lindsay; Gnaneshan, Saravanamuttu; Garduño, Rafael A; Mallo, Gustavo V

    2016-10-13

    Mobile genetic elements in bacteria, such as plasmids, act as important vectors for the transfer of antibiotic resistance, virulence, and metal resistance genes. Here, we report the genome sequence of a new plasmid pLM-C-273, identified in a Listeria monocytogenes strain isolated from a clinical sample in Ontario, Canada.

  12. Genome Sequence of Listeria monocytogenes Plasmid pLM-C-273 Carrying Genes Related to Stress Resistance

    PubMed Central

    Liang, Lindsay; Gnaneshan, Saravanamuttu; Garduño, Rafael A.

    2016-01-01

    Mobile genetic elements in bacteria, such as plasmids, act as important vectors for the transfer of antibiotic resistance, virulence, and metal resistance genes. Here, we report the genome sequence of a new plasmid pLM-C-273, identified in a Listeria monocytogenes strain isolated from a clinical sample in Ontario, Canada. PMID:27738039

  13. Distribution and diversity of mycoplasma plasmids: lessons from cryptic genetic elements

    PubMed Central

    2012-01-01

    Background The evolution of mycoplasmas from a common ancestor with Firmicutes has been characterized not only by genome down-sizing but also by horizontal gene transfer between mycoplasma species sharing a common host. The mechanisms of these gene transfers remain unclear because our knowledge of the mycoplasma mobile genetic elements is limited. In particular, only a few plasmids have been described within the Mycoplasma genus. Results We have shown that several species of ruminant mycoplasmas carry plasmids that are members of a large family of elements and replicate via a rolling-circle mechanism. All plasmids were isolated from species that either belonged or were closely related to the Mycoplasma mycoides cluster; none was from the Mycoplasma bovis-Mycoplasma agalactiae group. Twenty one plasmids were completely sequenced, named and compared with each other and with the five mycoplasma plasmids previously reported. All plasmids share similar size and genetic organization, and present a mosaic structure. A peculiar case is that of the plasmid pMyBK1 from M. yeatsii; it is larger in size and is predicted to be mobilizable. Its origin of replication and replication protein were identified. In addition, pMyBK1 derivatives were shown to replicate in various species of the M. mycoides cluster, and therefore hold considerable promise for developing gene vectors. The phylogenetic analysis of these plasmids confirms the uniqueness of pMyBK1 and indicates that the other mycoplasma plasmids cluster together, apart from the related replicons found in phytoplasmas and in species of the clade Firmicutes. Conclusions Our results unraveled a totally new picture of mycoplasma plasmids. Although they probably play a limited role in the gene exchanges that participate in mycoplasma evolution, they are abundant in some species. Evidence for the occurrence of frequent genetic recombination strongly suggests they are transmitted between species sharing a common host or niche. PMID

  14. Transformation of Shewanella baltica with ColE1-like and P1 plasmids and their maintenance during bacterial growth in cultures.

    PubMed

    Milewska, Klaudia; Węgrzyn, Grzegorz; Szalewska-Pałasz, Agnieszka

    2015-09-01

    The presence of natural plasmids has been reported for many Shewanella isolates. However, knowledge about plasmid replication origin and segregation mechanisms is not extensive for this genus. Shewanella baltica is an important species in the marine environment due to its denitrification ability in oxygen-deficient zones and the potential role in bioremediation processes. However, no information about possible use of plasmid vectors in this species has been reported to date. Here we report that plasmids with ColE1-type and plasmid P1 origin can transform S. baltica and replicate in this bacterium. Without the antibiotic selection pressure plasmid maintenance is less efficient than in Escherichia coli. Nevertheless, cultivation of S. baltica in the presence of appropriate antibiotics caused relatively stable maintenance of ColE1-like and P1-derived plasmids. This indicates that plasmid-based genetic manipulations and gene transfer in S. baltica are possible.

  15. Prevalence and significance of plasmid maintenance functions in the virulence plasmids of pathogenic bacteria.

    PubMed

    Sengupta, Manjistha; Austin, Stuart

    2011-07-01

    Virulence functions of pathogenic bacteria are often encoded on large extrachromosomal plasmids. These plasmids are maintained at low copy number to reduce the metabolic burden on their host. Low-copy-number plasmids risk loss during cell division. This is countered by plasmid-encoded systems that ensure that each cell receives at least one plasmid copy. Plasmid replication and recombination can produce plasmid multimers that hinder plasmid segregation. These are removed by multimer resolution systems. Equitable distribution of the resulting monomers to daughter cells is ensured by plasmid partition systems that actively segregate plasmid copies to daughter cells in a process akin to mitosis in higher organisms. Any plasmid-free cells that still arise due to occasional failures of replication, multimer resolution, or partition are eliminated by plasmid-encoded postsegregational killing systems. Here we argue that all of these three systems are essential for the stable maintenance of large low-copy-number plasmids. Thus, they should be found on all large virulence plasmids. Where available, well-annotated sequences of virulence plasmids confirm this. Indeed, virulence plasmids often appear to contain more than one example conforming to each of the three system classes. Since these systems are essential for virulence, they can be regarded as ubiquitous virulence factors. As such, they should be informative in the search for new antibacterial agents and drug targets.

  16. Requirements for mobilization of plasmids RSF1010 and ColE1 by the IncW plasmid R388: trwB and RP4 traG are interchangeable.

    PubMed Central

    Cabezón, E.; Lanka, E.; de la Cruz, F.

    1994-01-01

    Mobilization of plasmid RSF1010 by the IncW plasmid R388 requires the genes involved in W pilus synthesis plus trwB. traG of the IncP plasmid RP4 can substitute for trwB in RSF1010 mobilization by R388 but not in self-transfer of R388. This result suggests a dual specificity of TrwB-like proteins in conjugation. The same genetic requirements were found for R388 to mobilize the unrelated plasmid ColE1. PMID:8021231

  17. Separate F-Type Plasmids Have Shaped the Evolution of the H30 Subclone of Escherichia coli Sequence Type 131

    PubMed Central

    Danzeisen, Jessica L.; Youmans, Bonnie; Case, Kyle; Llop, Katharine; Munoz-Aguayo, Jeannette; Flores-Figueroa, Cristian; Aziz, Maliha; Sokurenko, Evgeni; Price, Lance B.; Johnson, James R.

    2016-01-01

    ABSTRACT The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B− plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B− plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring blaCTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical

  18. Effect of lipopolysaccharide mutations on recipient ability of Salmonella typhimurium for incompatibility group H plasmids.

    PubMed Central

    Sherburne, C; Taylor, D E

    1997-01-01

    Previous investigations of the incompatibility group F, P, and I plasmid systems revealed the important role of the outer membrane components in the conjugal transfer of these plasmids. We have observed variability in transfer frequency of three incompatibility group H plasmids (IncHI1 plasmid R27, IncHI2 plasmid R478, and a Tn7 derivative of R27, pDT2454) upon transfer into various Salmonella typhimurium lipopolysaccharide (LPS) mutants derived from a common parental strain, SL1027. Recipients with truncated outer core via the rfaF LPS mutation increased the transfer frequency of the IncH plasmids by up to a factor of 10(3). Mutations which resulted in the truncation of the residues following 3-deoxy-D-manno-octulosonic acid, such as the rfaE and rfaD mutations, decreased the transfer frequency to undetectable levels. Addition of phosphorylethanolamine, a component of wild-type LPS, to the media decreased the frequency of transfer of R27 into wild-type and rfaF LPS mutant recipients tested. Reversing the direction of transfer, by mating LPS mutant donors with wild-type recipients, did not affect the frequency of transfer compared to the standard matings of wild-type donor with LPS mutant recipient. These findings demonstrate that conjugation interactions affected by LPS mutation are not specific for the recipient cell. Our results suggest that LPS mutation does not affect conjugation via altered pilus binding but affects some later steps in the conjugative process, and alteration of transfer frequency by O-phosphorylethanolamine and LPS truncation is due to charge-related interactions between the donor and recipient cell. PMID:9006054

  19. Characterization and comparative analysis of antibiotic resistance plasmids isolated from a wastewater treatment plant.

    PubMed

    Rahube, Teddie O; Viana, Laia S; Koraimann, Günther; Yost, Christopher K

    2014-01-01

    A wastewater treatment plant (WWTP) is an environment high in nutrient concentration with diverse bacterial populations and can provide an ideal environment for the proliferation of mobile elements such as plasmids. WWTPs have also been identified as reservoirs for antibiotic resistance genes that are associated with human pathogens. The objectives of this study were to isolate and characterize self-transmissible or mobilizable resistance plasmids associated with effluent from WWTP. An enrichment culture approach designed to capture plasmids conferring resistance to high concentrations of erythromycin was used to capture plasmids from an urban WWTP servicing a population of ca. 210,000. DNA sequencing of the plasmids revealed diversity of plasmids represented by incompatibility groups IncU, col-E, IncFII and IncP-1β. Genes coding resistance to clinically relevant antibiotics (macrolide, tetracycline, beta-lactam, trimethoprim, chloramphenicol, sulphonamide), quaternary ammonium compounds and heavy metals were co-located on these plasmids, often within transposable and integrative mobile elements. Several of the plasmids were self-transmissible or mobilizable and could be maintained in the absence of antibiotic selection. The IncFII plasmid pEFC36a showed the highest degree of sequence identity to plasmid R1 which has been isolated in England more than 50 years ago from a patient suffering from a Salmonella infection. Functional conservation of key regulatory features of this F-like conjugation module were demonstrated by the finding that the conjugation frequency of pEFC36a could be stimulated by the positive regulator of plasmid R1 DNA transfer genes, TraJ.

  20. Plasmid profiling of bacterial isolates from confined environments

    NASA Astrophysics Data System (ADS)

    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 psych