Journal articles: 'RP4 plasmide conjugatif'

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Samuels, A. Lacey, Erich Lanka, and Julian E. Davies. "Conjugative Junctions in RP4-Mediated Mating ofEscherichia coli." Journal of Bacteriology 182, no. 10 (May 15, 2000): 2709–15. http://dx.doi.org/10.1128/jb.182.10.2709-2715.2000.

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ABSTRACT The physical association of bacteria during conjugation mediated by the IncPα plasmid RP4 was investigated. Escherichia colimating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (trbC), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.

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Cairns, Johannes, Matti Jalasvuori, Ville Ojala, Michael Brockhurst, and Teppo Hiltunen. "Conjugation is necessary for a bacterial plasmid to survive under protozoan predation." Biology Letters 12, no. 2 (February 2016): 20150953. http://dx.doi.org/10.1098/rsbl.2015.0953.

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Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens . A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.

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Backert, Steffen, Terry Kwok, and Wolfgang König. "Conjugative plasmid DNA transfer in Helicobacter pylori mediated by chromosomally encoded relaxase and TraG-like proteins." Microbiology 151, no. 11 (November 1, 2005): 3493–503. http://dx.doi.org/10.1099/mic.0.28250-0.

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One of the striking characteristics of Helicobacter pylori is the extensive genetic diversity among clinical isolates. This diversity has been attributed to an elevated mutation rate, impaired DNA repair, DNA transfer and frequent recombination events. Plasmids have also been identified in H. pylori but it remained unknown whether conjugation can contribute to DNA transfer between clinical isolates. To examine whether H. pylori possesses intrinsic capability for conjugative plasmid transfer, shuttle vectors were introduced into H. pylori containing an oriT sequence of the conjugative IncPα plasmid RP4 but no mobilization (mob) genes. It was shown that these vectors could stably replicate and be mobilized among clinical H. pylori strains. It was also demonstrated that traG and relaxase (rlx) homologues carried on the H. pylori chromosome were important for plasmid transfer. Primer extension studies and mutagenesis further confirmed that the relaxase homologue rlx1 in H. pylori encodes a functional enzyme capable of acting on the RP4 oriT. Furthermore, the findings of this study indicate that traG and rlx1 act independently of the previously described type IV secretion systems, including that encoded by the cag pathogenicity island and the comB transformation apparatus, in mediating conjugative plasmid DNA transfer between H. pylori strains.

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Bates, Steven, Annette M. Cashmore, and Brian M. Wilkins. "IncP Plasmids Are Unusually Effective in Mediating Conjugation of Escherichia coli and Saccharomyces cerevisiae: Involvement of the Tra2 Mating System." Journal of Bacteriology 180, no. 24 (December 15, 1998): 6538–43. http://dx.doi.org/10.1128/jb.180.24.6538-6543.1998.

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ABSTRACT Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Pα system caused detectable mobilization to yeast, giving peak values of 5 × 10−5 transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage oforiT in cis and the provision intrans of the Pα Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Pα conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Pα system in mediating gene transfer from bacteria to eukaryotes.

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Hamilton, Claire M., Hyewon Lee, Pei-Li Li, David M. Cook, Kevin R. Piper, Susanne Beck von Bodman, Erich Lanka, Walt Ream, and Stephen K. Farrand. "TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58." Journal of Bacteriology 182, no. 6 (March 15, 2000): 1541–48. http://dx.doi.org/10.1128/jb.182.6.1541-1548.2000.

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ABSTRACT Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraGRP4) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraGRP4. A. tumefaciensdonors transferred a chimeric plasmid that contains theoriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraGRP4 was expressed in the donors. Mutations intraG RP4 with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, thetra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraGRP4 nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG RP4 mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraGRP4-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraGRP4 and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.

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Dimitriu, Tatiana, Andrew C. Matthews, and Angus Buckling. "Increased copy number couples the evolution of plasmid horizontal transmission and plasmid-encoded antibiotic resistance." Proceedings of the National Academy of Sciences 118, no. 31 (July 29, 2021): e2107818118. http://dx.doi.org/10.1073/pnas.2107818118.

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Conjugative plasmids are mobile elements that spread horizontally between bacterial hosts and often confer adaptive phenotypes, including antimicrobial resistance (AMR). Theory suggests that opportunities for horizontal transmission favor plasmids with higher transfer rates, whereas selection for plasmid carriage favors less-mobile plasmids. However, little is known about the mechanisms leading to variation in transmission rates in natural plasmids or the resultant effects on their bacterial host. We investigated the evolution of AMR plasmids confronted with different immigration rates of susceptible hosts. Plasmid RP4 did not evolve in response to the manipulations, but plasmid R1 rapidly evolved up to 1,000-fold increased transfer rates in the presence of susceptible hosts. Most evolved plasmids also conferred on their hosts the ability to grow at high concentrations of antibiotics. This was because plasmids evolved greater copy numbers as a function of mutations in the copA gene controlling plasmid replication, causing both higher transfer rates and AMR. Reciprocally, plasmids with increased conjugation rates also evolved when selecting for high levels of AMR, despite the absence of susceptible hosts. Such correlated selection between plasmid transfer and AMR could increase the spread of AMR within populations and communities.

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Jalasvuori, Matti, Ville-Petri Friman, Anne Nieminen, Jaana K. H. Bamford, and Angus Buckling. "Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids." Biology Letters 7, no. 6 (June 2011): 902–5. http://dx.doi.org/10.1098/rsbl.2011.0384.

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Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.

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Rabel, Christian, A. Marika Grahn, Rudi Lurz, and Erich Lanka. "The VirB4 Family of Proposed Traffic Nucleoside Triphosphatases: Common Motifs in Plasmid RP4 TrbE Are Essential for Conjugation and Phage Adsorption." Journal of Bacteriology 185, no. 3 (February 1, 2003): 1045–58. http://dx.doi.org/10.1128/jb.185.3.1045-1058.2003.

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ABSTRACT Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems. The central feature of VirB4 proteins is a nucleotide binding site. In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides. A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities. We defined four common motifs and one conserved domain. One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis. Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli. Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.

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Lian, Zheng Jie, Minh-Duy Phan, Steven J. Hancock, Nguyen Thi Khanh Nhu, David L. Paterson, and Mark A. Schembri. "Genetic basis of I-complex plasmid stability and conjugation." PLOS Genetics 19, no. 6 (June 22, 2023): e1010773. http://dx.doi.org/10.1371/journal.pgen.1010773.

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Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination.

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Picardeau, Mathieu. "Conjugative Transfer between Escherichia coli and Leptospira spp. as a New Genetic Tool." Applied and Environmental Microbiology 74, no. 1 (November 9, 2007): 319–22. http://dx.doi.org/10.1128/aem.02172-07.

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ABSTRACT Our understanding of leptospiral pathogenesis, which remains poorly understood, depends on reliable genetic tools for functional analysis of genes in pathogenic strains. In this study, we report the first demonstration of conjugation between Escherichia coli and Leptospira spp. by using RP4 derivative conjugative plasmids. The DNA transfer described here was due to authentic conjugation, as shown by the requirement for cell-to-cell contact and the resistance of DNA transfers to the addition of DNase I. Transposition via conjugation of a plasmid delivering Himar1 yielded frequencies ranging from 1 × 10−6 to 8.5 × 10−8 transconjugants/recipient cell in the saprophyte L. biflexa and the pathogen L. interrogans, respectively. Analysis of mutants indicated that transposition occurs randomly, and at single sites in the genome of these strains, allowing the utilization of this system to generate libraries of transposon mutants.

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Elsas, J. D. Van, J. T. Trevors, L. S. Van Overbeek, and M. E. Starodub. "Survival of Pseudomonas fluorescens containing plasmids RP4 or pRK2501 and plasmid stability after introduction into two soils of different texture." Canadian Journal of Microbiology 35, no. 10 (October 1, 1989): 951–59. http://dx.doi.org/10.1139/m89-157.

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Survival of Pseudomonas fluorescens R2f containing either the conjugative plasmid RP4 or the nonconjugative plasmid pRK2501, and stability of the plasmids were studied in two soils, Ede loamy sand and Guelph loam, and in extracts prepared from these soils. In sterile soils, the introduced bacterial populations initially increased and then remained stable over a 47-day period. The presence of wheat roots did not significantly influence bacterial numbers in Guelph loam, whereas a slight increase occurred in Ede loamy sand. In Guelph loam, both plasmids were stably maintained in the introduced populations, but in Ede loamy sand substantial plasmid loss was observed. The presence of added phosphate in Ede loamy sand enhanced plasmid maintenance in the introduced R2f population. In nonsterile Guelph loam, a slow decline in the introduced populations was noted, regardless of plasmid type, whereas in Ede loamy sand the decline was more rapid. There was no detectable effect of plasmid type on host survival. Both plasmids RP4 and pRK2501 remained present in the R2f populations in these soils. The results obtained with sterile soil extracts substantiated the data on plasmid loss in both soils; both plasmids were rather unstable during starvation in minimal medium. The results indicated the absence of an effect of plasmid type on host survival. Soil type significantly affected host survival and plasmid maintenance, and higher survival and stability were observed in the heavier-textured Guelph loam.Key words: survival, plasmid stability, Pseudomonas spp., soil, microcosms.

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Sher, Azam A., Mia E. VanAllen, Husnain Ahmed, Charles Whitehead-Tillery, Sonia Rafique, Julia A. Bell, Lixin Zhang, and Linda S. Mansfield. "Conjugative RP4 Plasmid-Mediated Transfer of Antibiotic Resistance Genes to Commensal and Multidrug-Resistant Enteric Bacteria In Vitro." Microorganisms 11, no. 1 (January 12, 2023): 193. http://dx.doi.org/10.3390/microorganisms11010193.

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Many antibiotic-resistant bacteria carry resistance genes on conjugative plasmids that are transferable to commensals and pathogens. We determined the ability of multiple enteric bacteria to acquire and retransfer a broad-host-range plasmid RP4. We used human-derived commensal Escherichia coli LM715-1 carrying a chromosomal red fluorescent protein gene and green fluorescent protein (GFP)-labeled broad-host-range RP4 plasmid with ampR, tetR, and kanR in in vitro matings to rifampicin-resistant recipients, including Escherichia coli MG1655, Dec5α, Vibrio cholerae, Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter rodentium, and Salmonella Typhimurium. Transconjugants were quantified on selective media and confirmed using fluorescence microscopy and PCR for the GFP gene. The plasmid was transferred from E. coli LM715-1 to all tested recipients except P. aeruginosa. Transfer frequencies differed between specific donor–recipient pairings (10−2 to 10−8). Secondary retransfer of plasmid from transconjugants to E. coli LM715-1 occurred at frequencies from 10−2 to 10−7. A serial passage plasmid persistence assay showed plasmid loss over time in the absence of antibiotics, indicating that the plasmid imposed a fitness cost to its host, although some plasmid-bearing cells persisted for at least ten transfers. Thus, the RP4 plasmid can transfer to multiple clinically relevant bacterial species without antibiotic selection pressure.

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Dahlberg, Cecilia, and Lin Chao. "Amelioration of the Cost of Conjugative Plasmid Carriage in Eschericha coli K12." Genetics 165, no. 4 (December 1, 2003): 1641–49. http://dx.doi.org/10.1093/genetics/165.4.1641.

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Abstract 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.

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Grahn, A. Marika, Jana Haase, Dennis H. Bamford, and Erich Lanka. "Components of the RP4 Conjugative Transfer Apparatus Form an Envelope Structure Bridging Inner and Outer Membranes of Donor Cells: Implications for Related Macromolecule Transport Systems." Journal of Bacteriology 182, no. 6 (March 15, 2000): 1564–74. http://dx.doi.org/10.1128/jb.182.6.1564-1574.2000.

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ABSTRACT During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPα plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the twoE. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.

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Froehlich, Barbara, Erik Holtzapple, Timothy D. Read, and June R. Scott. "Horizontal Transfer of CS1 Pilin Genes of Enterotoxigenic Escherichia coli." Journal of Bacteriology 186, no. 10 (May 15, 2004): 3230–37. http://dx.doi.org/10.1128/jb.186.10.3230-3237.2004.

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ABSTRACT CS1 is one of a limited number of serologically distinct pili found in enterotoxigenic Escherichia coli (ETEC) strains associated with disease in people. The genes for the CS1 pilus are on a large plasmid, pCoo. We show that pCoo is not self-transmissible, although our sequence determination for part of pCoo shows regions almost identical to those in the conjugative drug resistance plasmid R64. When we introduced R64 into a strain containing pCoo, we found that pCoo was transferred to a recipient strain in mating. Most of the transconjugant pCoo plasmids result from recombination with R64, leading to acquisition of functional copies of all of the R64 transfer genes. Temporary coresidence of the drug resistance plasmid R64 with pCoo leads to a permanent change in pCoo so that it is now self-transmissible. We conclude that when R64-like plasmids are transmitted to an ETEC strain containing pCoo, their recombination may allow for spread of the pCoo plasmid to other enteric bacteria.

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Furuya, Nobuhisa, and Teruya Komano. "Initiation and Termination of DNA Transfer during Conjugation of IncI1 Plasmid R64: Roles of Two Sets of Inverted Repeat Sequences within oriT in Termination of R64 Transfer." Journal of Bacteriology 182, no. 11 (June 1, 2000): 3191–96. http://dx.doi.org/10.1128/jb.182.11.3191-3196.2000.

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ABSTRACT Intercellular transfer of plasmid DNA during bacterial conjugation initiates and terminates at a specific origin of transfer,oriT. We have investigated the oriT structure of conjugative plasmid R64 with regard to the initiation and termination of DNA transfer. Using recombinant plasmids containing two tandemly repeated R64 oriT sequences with or without mutations, the subregions required for initiation and termination were determined by examining conjugation-mediated deletion between the repeated oriTs. The oriT subregion required for initiation was found to be identical to the 44-bp oriT core sequence consisting of two units, the conserved nick region sequence and the 17-bp repeat A sequence, that are recognized by R64 relaxosome proteins NikB and NikA, respectively. In contrast, the nick region sequence and two sets of inverted repeat sequences within the 92-bp minimal oriT sequence were required for efficient termination. Mutant repeat A sequences lacking NikA-binding ability were found to be sufficient for termination, suggesting that the inverted repeat structures are involved in the termination process. A duplication of the DNA segment between the repeated oriTs was also found after mobilization of the plasmid carrying initiation-deficient but termination-proficient oriT and initiation-proficient but termination-deficient oriT, suggesting that the 3′ terminus of the transferred strand is elongated by rolling-circle-DNA synthesis.

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Zhang, Ning, Xiang Liu, Bing Li, Limei Han, Xuejiao Ma, Fanbin Meng, and Miao Li. "Modified U-Tube for Ruling out Naked DNA Transfer during Conjugation and Application in Antibiotic Resistance Genes Transfer Research." Water 10, no. 10 (September 22, 2018): 1313. http://dx.doi.org/10.3390/w10101313.

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Antibiotic resistance is currently a major global public health issue. In particular, the emergence and transfer of antibiotic resistance genes (ARGs) is a matter of primary concern. This study presented a method for ruling out the transfer of naked DNA (plasmid RP4 lysed from donor cells) during the cell-to-cell conjugation, using a modified “U-tube”. A series of gene transfer assays was conducted in both flask and modified U-tube, using Pseudomonas putida KT2440 (P. putida (RP4)) harboring the RP4 plasmid as the donor strain, Escherichia coli (E. coli, ATCC 25922) in pure culture as sole recipient, and bacteria from reclaimed water microcosms as multi-recipients. The verification experiments showed that the U-tube device could prevent direct contact of bacteria without affecting the exchange of free plasmid. In the experiments involving a sole recipient, the transconjugants were obtained in flask samples, but not in modified U-tube. Furthermore, in experiments involving multi-recipients, transfer of naked DNA in the modified U-tube accounted for 5.18% in the transfer frequency of the flask transfer experiment. The modified U-tube proved to be useful for monitoring the interference of naked DNA in the research of conjugative transfer and calculating the exact conjugative transfer rate. This device is identified as a promising candidate for distinguishing different gene transfers in practical application because of its convenient use and easy and simple manufacture.

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Fernandez-Lopez, Raul, Cristina Machón, Christopher M. Longshaw, Steve Martin, Soren Molin, Ellen L. Zechner, Manuel Espinosa, Erich Lanka, and Fernando de la Cruz. "Unsaturated fatty acids are inhibitors of bacterial conjugation." Microbiology 151, no. 11 (November 1, 2005): 3517–26. http://dx.doi.org/10.1099/mic.0.28216-0.

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This report describes a high-throughput assay to identify substances that reduce the frequency of conjugation in Gram-negative bacteria. Bacterial conjugation is largely responsible for the spread of multiple antibiotic resistances in human pathogens. Conjugation inhibitors may provide a means to control the spread of antibiotic resistance. An automated conjugation assay was developed that used plasmid R388 and a laboratory strain of Escherichia coli as a model system, and bioluminescence as a reporter for conjugation activity. Frequencies of conjugation could be measured continuously in real time by the amount of light produced, and thus the effects of inhibitory compounds could be determined quantitatively. A control assay, run in parallel, allowed elimination of compounds affecting cell growth, plasmid stability or gene expression. The automated conjugation assay was used to screen a database of more than 12 000 microbial extracts known to contain a wide variety of bioactive compounds (the NatChem library). The initial hit rate was 1·4 %. From these, 48 extracts containing active compounds and representing a variety of organisms and extraction conditions were subjected to fractionation (24 fractions per extract). The 52 most active fractions were subjected to a secondary analysis to determine the range of plasmid inhibition. Plasmids R388, R1 and RP4 were used as representatives of a variety of plasmid transfer systems. Only one fraction (of complex composition) affected transfer of all three plasmids, while four other fractions were active against two of them. Two separate compounds were identified from these fractions: linoleic acid and dehydrocrepenynic acid. Downstream analysis showed that the chemical class of unsaturated fatty acids act as true inhibitors of conjugation.

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Wang, Jiamin, Qingtong Wu, Yifan Liu, and Mengying Shao. "Triadimefon Promoted Plasmid-mediated Conjugative Transfer of Multiple Antibiotic Resistance Genes within Bacterial Genus." BIO Web of Conferences 59 (2023): 01014. http://dx.doi.org/10.1051/bioconf/20235901014.

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The rapid spread of antibiotic resistance genes (ARGs) has been a worldwide threat to the public health, especially via horizontal gene transfer (HGT). Conjugation is one of the major pathways of HGT. Triadimefon (TF) is a broad-spectrum antifungal compound used to control rust and powdery mildew on crops. Nevertheless, it is not known whether TF could affect the conjugation of ARGs and potential molecular mechanisms. Here, the effects of TF on conjugative transfer of RP4 plasmid within Escherichia coli were systematically investigated. The results demonstrated that TF increased the transconjugant number by 1.41–2.21 folds and the frequency of conjugative transfer by 1.49–2.22 folds at concentrations of 0.1–10 mg/L. There was no obvious change in the number of donor and recipient strains in the mating system of intra-genus with TF. The main mechanisms include increasing intracellular reactive oxygen species and membrane permeability. Our findings highlight the promotion effect of TF on ARG conjugation, providing evidence of the risk of non-antibiotic agrochemical use in ARG dissemination.

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McLeod, Sarah M., Vincent Burrus, and Matthew K. Waldor. "Requirement for Vibrio cholerae Integration Host Factor in Conjugative DNA Transfer." Journal of Bacteriology 188, no. 16 (August 15, 2006): 5704–11. http://dx.doi.org/10.1128/jb.00564-06.

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ABSTRACT The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. In the absence of IHF, V. cholerae displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT integration into or excision from the V. cholerae chromosome, which requires an SXT-encoded integrase related to λ integrase, did not require IHF. Therefore, the defect in SXT transmission in the V. cholerae IHF mutant is probably not related to IHF's ability to promote DNA recombination. The V. cholerae IHF mutant was also highly impaired as a donor of RP4, a broad-host-range conjugative plasmid. Thus, the V. cholerae IHF mutant appears to have a general defect in conjugation. Escherichia coli IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a V. cholerae-specific conjugation factor.

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Furuya, Nobuhisa, and Teruya Komano. "NikAB- or NikB-Dependent Intracellular Recombination between Tandemly Repeated oriT Sequences of Plasmid R64 in Plasmid or Single-Stranded Phage Vectors." Journal of Bacteriology 185, no. 13 (July 1, 2003): 3871–77. http://dx.doi.org/10.1128/jb.185.13.3871-3877.2003.

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ABSTRACT The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.

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Wu, Qingtong, Mile Du, Yingzhen Zhang, and Mengying Shao. "Chlorpyrifos And Chlorpyrifos-methyl Can Promote Conjugative Transfer of Antibiotic Resistance Genes." BIO Web of Conferences 59 (2023): 01015. http://dx.doi.org/10.1051/bioconf/20235901015.

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Antibiotic misuse induces the production of antibiotic resistance genes (ARGs), leading to the global spread of antimicrobial resistance (AMR), which poses a major threat to human health. Conjugative transfer, as the main process of ARGs propagation, is sensitively influenced by coexisting contaminants. Chlorpyrifos and chlorpyrifos-methyl, as organophosphorus insecticides widely used in agriculture, have been shown to induce cytotoxicity such as elevated levels of reactive oxygen radicals (ROS) and lipid peroxidation. This is similar to the mechanism by which antibiotics promote the conjugative transfer of ARGs, based on which we hypothesized that chlorpyrifos and chlorpyrifos-methyl could promote conjugative transfer. However, the effect of chlorpyrifos and chlorpyrifos-methyl on conjugative transfer is unclear. Therefore, we constructed RP4 plasmid-mediated conjugation system and confirmed that chlorpyrifos and chlorpyrifos-methyl can promote conjugative transfer by inducing oxidative stress in donor and recipient bacteria. Our research reveals the risk of ARM spread in organophosphorus insecticides and ARGs co-contaminated environments.

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Aparicio, Tomás, Jillian Silbert, Sherezade Cepeda, and Víctor de Lorenzo. "Propagation of Recombinant Genes through Complex Microbiomes with Synthetic Mini-RP4 Plasmid Vectors." BioDesign Research 2022 (August 4, 2022): 1–15. http://dx.doi.org/10.34133/2022/9850305.

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The promiscuous conjugation machinery of the Gram-negative plasmid RP4 has been reassembled in a minimized, highly transmissible vector for propagating genetically encoded traits through diverse types of naturally occurring microbial communities. To this end, the whole of the RP4-encoded transfer determinants (tra, mob genes, and origin of transfer oriT) was excised from their natural context, minimized, and recreated in the form of a streamlined DNA segment borne by an autoselective replicon. The resulting constructs (the pMATING series) could be self-transferred through a variety of prokaryotic and eukaryotic recipients employing such a rationally designed conjugal delivery device. Insertion of GFP reporter into pMATING exposed the value of this genetic tool for delivering heterologous genes to both specific mating partners and complex consortia (e.g., plant/soil rhizosphere). The results accredited the effective and functional transfer of the recombinant plasmids to a diversity of hosts. Yet the inspection of factors that limit interspecies DNA transfer in such scenarios uncovered type VI secretion systems as one of the factual barriers that check otherwise high conjugal frequencies of tested RP4 derivatives. We argue that the hereby presented programming of hyperpromiscuous gene transfer can become a phenomenal asset for the propagation of beneficial traits through various scales of the environmental microbiome.

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Ehlers, Laura J., and Edward J. Bouwer. "RP4 plasmid transfer among species of pseudomonas in a biofilm reactor." Water Science and Technology 39, no. 7 (April 1, 1999): 163–71. http://dx.doi.org/10.2166/wst.1999.0353.

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Conjugation, the most prevalent mechanism of horizontal gene transfer, has been extensively studied for its role in the dissemination of antibiotic resistance and catabolic genes among bacteria. Very little research on conjugation has been conducted in natural biofilm systems, although over 99% of bacteria in nature are attached to surfaces. Previous studies suggest that rates of conjugation on surfaces elevated compared to rates in liquid media. The goal of this research was to observe conjugation between bacteria growing in a biofilm reactor. Conjugation of the broad host range plasmid RP4 between two species of Pseudomonas occurred in the biofilm reactor at high frequencies. The most important environmental para-meter was the shear stress at the biofilm-liquid interface. Conjugation was only observed below a shear of 0.0851 N/m2, corresponding to a laminar flow regime. Increasing temperature from 15°C to 28°C increased conjugation frequencies 10,000-fold. Conjugation frequency was unchanged in experiments conducted with 3.5, 7, and 35 mg/l acetate, though total cell concentration in the biofilm increased as expected. These data suggest ways to manipulate environmental parameters to affect plasmid transfer rates among biofilm bacteria.

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Haines, Anthony S., Parveen Akhtar, Elton R. Stephens, Karen Jones, Christopher M. Thomas, Caroline D. Perkins, Jacqueline R. Williams, Martin J. Day, and John C. Fry. "Plasmids from freshwater environments capable of IncQ retrotransfer are diverse and include pQKH54, a new IncP-1 subgroup archetype." Microbiology 152, no. 9 (September 1, 2006): 2689–701. http://dx.doi.org/10.1099/mic.0.28941-0.

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Nine mercury-resistance plasmids isolated from river epilithon were assessed for their ability to retrotransfer the non-conjugative IncQ plasmid, R300B, derivatives of which have commercial uses that may result in accidental or deliberate release into the environment. Retrotransfer frequencies ranging from 2.1×10−4 to 1.75×10−5 were obtained for five of the nine plasmids – the remaining plasmids showed low or undetectable retrotransfer ability. The majority of the retrotransfer-proficient plasmids could not be classified by the tests used. Classical incompatibility testing with RP4 identified pQKH6, pQKH54 and pQM719 as IncP-1. Hybridization to replicon probes confirmed this for pQKH6 and pQM719 and added pQKH33. PCR with primers designed to amplify trfA and korA regions of IncP-1 plasmids did not identify any other plasmids. Plasmids pQKH6 and pQM719 but not pQKH54 produced similar SphI restriction profiles to the IncP-1β subgroup. The complete nucleotide sequence of pQKH54 was determined, revealing it to have a complete IncP-1 backbone but belonging to a new distinct subgroup which was designated IncP-1γ. The results emphasize the ubiquity and diversity of IncP-1 plasmids in the environment but demonstrate that plasmids of as yet unknown groups are also able to retrotransfer IncQ plasmids efficiently.

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Zienkiewicz, M., I. Kern-Zdanowicz, M. Gołȩbiewski, J. Żyliñska, P. Mieczkowski, M. Gniadkowski, J. Bardowski, and P. Cegłowski. "Mosaic Structure of p1658/97, a 125-Kilobase Plasmid Harboring an Active Amplicon with the Extended-Spectrum β-Lactamase Gene blaSHV-5." Antimicrobial Agents and Chemotherapy 51, no. 4 (April 2007): 1164–71. http://dx.doi.org/10.1128/aac.00772-06.

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ABSTRACT Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum β-lactamase (ESBL) of the SHV type. The β-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the bla SHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

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Xiao, Yawen, Yan Zhang, Fengjun Xie, Rikke Heidemann Olsen, Lei Shi, and Lili Li. "Inhibition of Plasmid Conjugation in Escherichia coli by Targeting rbsB Gene Using CRISPRi System." International Journal of Molecular Sciences 24, no. 13 (June 24, 2023): 10585. http://dx.doi.org/10.3390/ijms241310585.

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Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic-resistant genes (ARGs) among human pathogens. The spread of ARGs can be halted or diminished by interfering with the conjugation process. In this study, we explored the possibility of using an rbsB gene as a single target to inhibit plasmid-mediated horizontal gene transfer in Escherichia coli by CRISPR interference (CRISPRi) system. Three single-guide RNAs (sgRNAs) were designed to target the rbsB gene. The transcriptional levels of the rbsB gene, the conjugation-related genes, and the conjugation efficiency in the CRISPRi strain were tested. We further explored the effect of the repressed expression of the rbsB gene on the quorum sensing (QS) system and biofilm formation. The results showed that the constructed CRISPRi system was effective in repressing the transcriptional level of the rbsB gene at a rate of 66.4%. The repressed expression of the rbsB gene resulted in the reduced conjugation rate of RP4 plasmid by 88.7%, which significantly inhibited the expression of the conjugation-related genes (trbBp, trfAp, traF and traJ) and increased the global regulator genes (korA, korB and trbA). The repressed rbsB gene expression reduced the depletion of autoinducer 2 signals (AI-2) by 12.8% and biofilm formation by a rate of 68.2%. The results of this study indicated the rbsB gene could be used as a universal target for the inhibition of conjugation. The constructed conjugative CRISPRi system has the potential to be used in ARG high-risk areas.

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Lumjiaktase, Putthapoom, Claudio Aguilar, Tom Battin, Kathrin Riedel, and Leo Eberl. "Construction of Self-Transmissible Green Fluorescent Protein-Based Biosensor Plasmids and Their Use for Identification of N-Acyl Homoserine-Producing Bacteria in Lake Sediments." Applied and Environmental Microbiology 76, no. 18 (July 30, 2010): 6119–27. http://dx.doi.org/10.1128/aem.00677-10.

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ABSTRACT Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

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van Kranenburg, Richard, and Willem M. de Vos. "Characterization of Multiple Regions Involved in Replication and Mobilization of Plasmid pNZ4000 Coding for Exopolysaccharide Production in Lactococcus lactis." Journal of Bacteriology 180, no. 20 (October 15, 1998): 5285–90. http://dx.doi.org/10.1128/jb.180.20.5285-5290.1998.

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ABSTRACT We characterized the regions involved in replication and mobilization of the 40-kb plasmid pNZ4000, encoding exopolysaccharide (EPS) production in Lactococcus lactis NIZO B40. The plasmid contains four highly conserved replication regions with homologous rep genes (repB1, repB2,repB3, and repB4) that belong to the lactococcal theta replicon family. Subcloning of each replicon individually showed that all are functional and compatible in L. lactis. Plasmid pNZ4000 and genetically labeled derivatives could be transferred to different L. lactis strains by conjugation, and pNZ4000 was shown to be a mobilization plasmid. Two regions involved in mobilization were identified near two of the replicons; both included an oriT sequence rich in inverted repeats. Conjugative mobilization of the nonmobilizable plasmid pNZ124 was promoted by either one of these oriT sequences, demonstrating their functionality. One oriT sequence was followed by a mobA gene, coding for atrans-acting protein, which increased the frequency of conjugative transfer 100-fold. The predicted MobA protein and theoriT sequences show protein and nucleotide similarity, respectively, with the relaxase and with the inverted repeat andnic site of the oriT from the Escherichia coli plasmid R64. The presence on pNZ4000 of four functional replicons, two oriT sequences, and several insertion sequence-like elements strongly suggests that this EPS plasmid is a naturally occurring cointegrate.

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Ferrières, Lionel, Gaëlle Hémery, Toan Nham, Anne-Marie Guérout, Didier Mazel, Christophe Beloin, and Jean-Marc Ghigo. "Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery." Journal of Bacteriology 192, no. 24 (October 8, 2010): 6418–27. http://dx.doi.org/10.1128/jb.00621-10.

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ABSTRACT Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.

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Merryweather, A., P. T. Barth, and B. M. Wilkins. "Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation." Journal of Bacteriology 167, no. 1 (1986): 12–17. http://dx.doi.org/10.1128/jb.167.1.12-17.1986.

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MACDONALD, J., B. SMETS, and B. RITTMANN. "The effects of energy availability on the conjugative-transfer kinetics of plasmid RP4." Water Research 26, no. 4 (April 1992): 461–68. http://dx.doi.org/10.1016/0043-1354(92)90046-7.

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Balzer, Dietmar, Güunter Ziegelin, Werner Pansegrau, Volker Kruft, and Erich Lanka. "KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer." Nucleic Acids Research 20, no. 8 (1992): 1851–58. http://dx.doi.org/10.1093/nar/20.8.1851.

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Riccobono, Eleonora, Vincenzo Di Pilato, Tiziana Di Maggio, Carmen Revollo, Alessandro Bartoloni, Lucia Pallecchi, and Gian Maria Rossolini. "Characterization of IncI1 Sequence Type 71 Epidemic Plasmid Lineage Responsible for the Recent Dissemination of CTX-M-65 Extended-Spectrum β-Lactamase in the Bolivian Chaco Region." Antimicrobial Agents and Chemotherapy 59, no. 9 (June 22, 2015): 5340–47. http://dx.doi.org/10.1128/aac.00589-15.

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ABSTRACTDuring the last decade, a significant diffusion of CTX-M-type extended-spectrum β-lactamases (ESBLs) was observed in commensalEscherichia colifrom healthy children in the Bolivian Chaco region, with initial dissemination of CTX-M-2, which was then replaced by CTX-M-15 and CTX-M-65. In this work, we demonstrate that the widespread dissemination of CTX-M-65 observed in this context was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid lineage. The structure of the epidemic plasmid population was characterized by complete sequencing of four representatives and PCR mapping of the remainder (n= 16). Sequence analysis showed identical plasmid backbones (similar to that of the reference IncI1 plasmid, R64) and a multiresistance region (MRR), which underwent local microevolution. The MRR harbored genes responsible for resistance to β-lactams, aminoglycosides, florfenicol, and fosfomycin (with microevolution mainly consisting of deletion events of resistance modules). TheblaCTX-M-65module harbored by the IncI1 ST71 epidemic plasmid was apparently derived from IncN-type plasmids, likely via IS26-mediated mobilization. The plasmid could be transferred by conjugation to several different enterobacterial species (Escherichia coli,Cronobacter sakazakii,Enterobacter cloacae,Klebsiella oxytoca,Klebsiella pneumoniae, andSalmonella enterica) and was stably maintained without selective pressure in these species, with the exception ofK. oxytocaandS. enterica. Fitness assays performed inE. colirecipients demonstrated that the presence of the epidemic plasmid was apparently not associated with a significant biological cost.

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Le Roux, Frï¿½dï¿½rique, Johan Binesse, Denis Saulnier, and Didier Mazel. "Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector." Applied and Environmental Microbiology 73, no. 3 (November 22, 2006): 777–84. http://dx.doi.org/10.1128/aem.02147-06.

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ABSTRACT Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the P BAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.

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Kornstein, Laura B., Virginia L. Waters, and Robert C. Cooper. "A natural mutant of plasmid RP4 that confers phage resistance and reduced conjugative transfer." FEMS Microbiology Letters 91, no. 2 (March 1992): 97–100. http://dx.doi.org/10.1111/j.1574-6968.1992.tb05191.x.

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Wang, Qing, Daqing Mao, and Yi Luo. "Ionic Liquid Facilitates the Conjugative Transfer of Antibiotic Resistance Genes Mediated by Plasmid RP4." Environmental Science & Technology 49, no. 14 (July 8, 2015): 8731–40. http://dx.doi.org/10.1021/acs.est.5b01129.

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Gliesche, Christian G., and Peter Hirsch. "Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522." Canadian Journal of Microbiology 38, no. 11 (November 1, 1992): 1167–74. http://dx.doi.org/10.1139/m92-191.

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Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N′ -nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine–methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10−6 to 10−8 for each marker. Key words: methylotrophs, Hyphomicrobium, N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis, transposon mutagenesis, promiscuous plasmids, chromosome mobilization.

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Demarre, Gaëlle, Anne-Marie Guérout, Chiho Matsumoto-Mashimo, Dean A. Rowe-Magnus, Philippe Marlière, and Didier Mazel. "A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPα) conjugative machineries and their cognate Escherichia coli host strains." Research in Microbiology 156, no. 2 (March 2005): 245–55. http://dx.doi.org/10.1016/j.resmic.2004.09.007.

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Al-Doori, Zainab. "Heterospecific transcription of theEscherichia coli rpoB-3 allele in Gram-negative bacteria." Genetical Research 50, no. 2 (October 1987): 87–90. http://dx.doi.org/10.1017/s0016672300023478.

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SummaryrpoB is the structural gene for the β-subunit ofE. coliRNA polymerase. TherpoB-3 allele confers resistance to the antibiotic rifampicin and is unusual in being dominant to the wild-type allele. We used the plasmid pZD23, a derivative of the broad host range conjugative plasmid RP4, to introduce therpoB-3 allele into a range of bacterial species. Species belonging to the same family asE. coli(Enterobacter aerogenes, Citrobacter freundii, Hafnia alvei møller, Klebsiella pneumoniae, Salmonella typhimurium) expressedrpoB-3 to give a rifampicin resistant phenotype; this demonstrated heterospecific transcription. The transfer of pZD23 to the non-Enterobacteriaceae speciesAzotobacter vinelandiiandRhizobium leguminosarumdid not result in rifampicin resistance. In the former case this was due to non-expression of therpoB-3 resistance phenotype, in the latter case the dominant resistance phenotype had been lost from pZD23. Heterospecific transcription can be used as a criterion for the investigation of genetic relatedness between bacterial species.

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Balzer, D., W. Pansegrau, and E. Lanka. "Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4." Journal of Bacteriology 176, no. 14 (1994): 4285–95. http://dx.doi.org/10.1128/jb.176.14.4285-4295.1994.

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Ziegelin, G., W. Pansegrau, R. Lurz, and E. Lanka. "TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin." Journal of Biological Chemistry 267, no. 24 (October 1992): 17279–86. http://dx.doi.org/10.1016/s0021-9258(18)41923-0.

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Reimmann, Cornelia, and Dieter Haas. "Mode of Replicon Fusion Mediated by the Duplicated Insertion Sequence IS21 in Escherichia coli." Genetics 115, no. 4 (April 1, 1987): 619–25. http://dx.doi.org/10.1093/genetics/115.4.619.

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ABSTRACT The insertion sequence IS21 (2.1 kb) originating from the broad-host-range IncP plasmid R68 transposes infrequently; by contrast, the IS21 tandem repeat found on the derivative R68.45 is highly active in transpositional mobilization of other replicons in a variety of Gram-negative bacteria. The mobilized plasmids are joined to R68.45 by single IS21 copies in direct orientation.—The formation of IS21 tandem duplications was observed in cointegrates between R68.45 and pBR325::IS21 and also in an RP1::IS21 plasmid derivative in which a segment located between two directly repeated copies of IS21 was deleted spontaneously. We speculate that IS21 tandem repeats can arise when the termini of two IS21 elements are specifically joined in a transposition or deletion event.—A resistance gene flanked by two IS21 elements in direct orientation did not behave as a transposon. The Ω fragment carrying transcription and translation stop signals was inserted into various sites of the IS21 tandem repeat; in this way it could be shown that the left IS21 element (which is next to the kanamycin resistance gene in R68.45) was 100 times more active in cointegrate formation than was the righthand element.—Cointegrates between the conjugative plasmid R751 and pBR325 derivatives carrying IS21 and IS21::Ω in tandem contained a single IS21 at one replicon junction and a single IS21::Ω at the other. In the IS21 duplications the inner IS21 ends were preferentially recognized (presumably by IS21 transposase), whereas the outer termini were not required for cointegrate formation. Based on these findings a conservative (simple) pathway of transposition is proposed for R68.45 and other plasmids with an IS21 tandem repeat. In this model R68.45 is pictured as a large transposon whose ends are joined together to form a circular molecule which is capable of autonomous replication.

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Mally, Manuela, and Guy R. Cornelis. "Genetic Tools for Studying Capnocytophaga canimorsus." Applied and Environmental Microbiology 74, no. 20 (August 22, 2008): 6369–77. http://dx.doi.org/10.1128/aem.01218-08.

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ABSTRACT Capnocytophaga canimorsus, a commensal bacterium from canine oral flora, has been isolated throughout the world from severe human infections caused by dog bites. Due to the low level of evolutionary relationship to Proteobacteria, genetic methods suitable for the genus Capnocytophaga needed to be established. Here, we show that Tn4351, derived from Bacteroides fragilis, could be introduced by conjugation into C. canimorsus and conferred resistance to erythromycin. By mapping and sequencing a naturally occurring plasmid isolated from a clinical isolate of C. canimorsus, we identified a repA gene that allowed us to construct Escherichia coli-Capnocytophaga shuttle vectors. Most commonly used antibiotic markers were not functional in C. canimorsus, but cefoxitin (cfxA), tetracycline (tetQ), and erythromycin (ermF) resistances could be used as markers for plasmid maintenance in C. canimorsus and even in some other Capnocytophaga spp. Shuttle vectors were introduced into C. canimorsus either by conjugation using the origin of transfer (oriT) of RP4 or by electrotransformation. Taking advantage of the promoter of ermF, an expression vector was constructed. Finally, a method that allows site-directed mutagenesis is described. All these genetic tools pave the way, not only for molecular studies of the pathogenesis of C. canimorsus, but also for studies of other oral Capnocytophaga species.

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Antonenka, Uladzimir, Christina Nölting, Jürgen Heesemann, and Alexander Rakin. "Horizontal transfer of Yersinia high-pathogenicity island by the conjugative RP4 attB target-presenting shuttle plasmid." Molecular Microbiology 57, no. 3 (June 15, 2005): 727–34. http://dx.doi.org/10.1111/j.1365-2958.2005.04722.x.

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Zahrl, Doris, Maria Wagner, Karin Bischof, Michaela Bayer, Barbara Zavecz, Andreas Beranek, Christoph Ruckenstuhl, Gernot E. Zarfel, and Günther Koraimann. "Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems." Microbiology 151, no. 11 (November 1, 2005): 3455–67. http://dx.doi.org/10.1099/mic.0.28141-0.

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Specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of Gram-negative bacteria. Specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. This paper reports the analysis of selected members of the specialized lytic transglycosylase family from type III and type IV secretion systems. These proteins were analysed in vivo by assaying their ability to complement the DNA transfer defect of the conjugative F-like plasmid R1-16 lacking a functional P19 protein, the specialized lytic transglycosylase of this type IV secretion system. Heterologous complementation was accomplished using IpgF from the plasmid-encoded type III secretion system of Shigella sonnei and TrbN from the type IV secretion system of the conjugative plasmid RP4. In contrast, neither VirB1 proteins (Agrobacterium tumefaciens, Brucella suis) nor IagB (Salmonella enterica) could functionally replace P19. In vitro, IpgF, IagB, both VirB1 proteins, HP0523 (Helicobacter pylori) and P19 displayed peptidoglycanase activity in zymogram analyses. Using an established test system and a newly developed assay it was shown that IpgF degraded peptidoglycan in solution. IpgF was active only after removal of the chaperonin GroEL, which co-purified with IpgF and inhibited its enzymic activity. A mutant IpgF protein in which the predicted catalytic amino acid, Glu42, was replaced by Gln, was completely inactive. IpgF-catalysed peptidoglycan degradation was optimal at pH 6 and was inhibited by the lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A.

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Christie-Oleza, Joseph Alexander, Isabel Brunet-Galmés, Jorge Lalucat, Balbina Nogales, and Rafael Bosch. "MiniUIB, a Novel Minitransposon-Based System for Stable Insertion of Foreign DNA into the Genomes of Gram-Negative and Gram-Positive Bacteria." Applied and Environmental Microbiology 79, no. 5 (December 28, 2012): 1629–38. http://dx.doi.org/10.1128/aem.03214-12.

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ABSTRACTTransposition of the insertion sequence (IS) ISPpu12is actively induced after conjugative interaction. The transposase of this IS can act intranson structures flanked by inverted repeats similar to those of the transposon. Based on that fact, an ISPpu12-based minitransposon, miniUIB, has been constructed in order to biotechnologically exploit the self-regulation of ISPpu12and its increased activity after conjugative interaction. Mobilization of the miniUIB structure into the genome ofPseudomonas stutzeriAN10 after conjugative interaction was demonstrated. A single gene, i.e., the kanamycin resistance determinant, or large genetic structures of >12 kb, i.e.,alkBFGHJKLandalkSToperons ofPseudomonas putidaTF4-1L (GPo1), have been easily integrated inP. stutzeriAN10 by an RP4-based delivery system. Therefore, the integration of thealkdeterminants by use of the miniUIB system has extended the biodegradation capabilities of this strain. Plasmid pJOC100, containing the transposase and regulator genes of ISPpu12adjacent to the miniUIB structure, was constructed in order to extend the host range of this biotechnologically useful genetic tool to other model and real-world bacteria. The effectiveness of the system for random mutagenesis in a phylogenetic wide range of bacteria and for the insertion of novel functions has been demonstrated, even in successive steps.

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48

Narahara, Koji, Erkin Rahman, Nobuhisa Furuya, and Teruya Komano. "Requirement of a Limited Segment of thesogGene for Plasmid R64 Conjugation." Plasmid 38, no. 1 (July 1997): 1–11. http://dx.doi.org/10.1006/plas.1997.1297.

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Tsvetkova, Krassimira, Jean-Christophe Marvaud, and Thierry Lambert. "Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn1549, a Member of a New Family of Conjugative Elements." Journal of Bacteriology 192, no. 3 (December 4, 2009): 702–13. http://dx.doi.org/10.1128/jb.00680-09.

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ABSTRACT Conjugative transfer from Clostridium symbiosum to enterococci of Tn1549, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (oriT) in the element. Transcription analysis of Tn1549 indicated that orf29, orf28, orfz, and orf27 were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to orf29-orf30 of Tn1549 was mobilized in Escherichia coli recA::RP4::Δnic provided that orf28 and orf29 were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in trans, but a cis-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp oriT was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp oriT fragment. The minimal oriT contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOBp superfamily. The nick site was identified within oriT by an oligonucleotide cleavage assay. Closely related oriTs linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn1549 is a member of a MOBp clade. Interestingly, the Tn1549-derived constructs were mobilized by RP4 in E. coli, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn1549 to be mobilized by a heterologous transfer system.

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Voisard, Christophe, Manuela Rella, and Dieter Haas. "Conjugative transfer of plasmid RP1 to soil isolates ofPseudomonas fluorescensis facilitated by certain large RP1 deletions." FEMS Microbiology Letters 55, no. 1 (September 1988): 9–13. http://dx.doi.org/10.1111/j.1574-6968.1988.tb02790.x.

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Journal articles on the topic 'RP4 plasmide conjugatif'

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