Littérature scientifique sur le sujet « RP4 conjugative plasmid »

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

L’étude de la dynamique de conjugaison des plasmides conjugatifs chez les bactéries Gram-négatif est la thématique centrale de recherche de notre laboratoire et autour de laquelle s’est articulé mon projet de thèse. Mes travaux de recherche ont eu pour but d’apporter de véritables connaissances sur l’étendue et l’impact de la conjugaison dans les communautés bactériennes. Le biofilm est largement considéré par la communauté scientifique comme un hotspot favorisant le transfert de gènes principalement en raison des contacts cellulaires propices qui existent dans sa structure. Or, les seules études qui ont essayé de démontrer expérimentalement que le biofilm permet d’augmenter les transferts par conjugaison n’apportent aucunes données claires sur la dynamique de ces transferts qui ont lieu dans le biofilm et comment celui-ci impacte ces transferts. L’approche que nous avons utilisée pour étudier la dynamique de conjugaison dans le biofilm repose sur un projet collaboratif entre notre laboratoire et celui du Dr Knut Drescher, basé au Biozentrum de Bâle en Suisse. Cette collaboration a permis de déployer des techniques de microscopie à fluorescence innovantes développées par nos deux laboratoires et jusqu’ici jamais utilisées dans le contexte de l’étude de la conjugaison dans le biofilm.Nous nous sommes centrés sur le plasmide RP4 qui est un plasmide conjugatif de type IncP. Retrouvé au sein de nombreux environnements naturels, il a été le modèle plasmidique principal des études qui se sont intéressées à la conjugaison dans le biofilm, et il a été largement exploité comme outil génétique par la communauté scientifique. Malgré le fait qu’il ait été très utilisé, les mécanismes de transferts du plasmide RP4 sont très peu décrits. Le plasmide RP4 s’est donc révélé comme un modèle d’étude de la conjugaison très pertinent que nous avons utilisé à la fois dans un aspect biotechnologique pour élargir le spectre d’hôte des systèmes antibactériens TAPs et à la fois dans un aspect fondamental pour étudier sa dynamique de conjugaison, que ce soit au sein d’une population E. coli cultivées en 2D et au sein d’une population E. coli structurées en biofilm 3D.Durant mes travaux de thèse, j’ai donc exploité le plasmide RP4 pour véhiculer des systèmes CRISPR antibactériens chez diverses espèces bactériennes phylogénétiquement éloignées. J’ai apporté les premières images en temps réel du transfert du plasmide RP4 en 2D et de nouvelles données très intéressantes sur la chronologie de conversion de l’ADN en double brin dans la receveuse. Enfin, une approche totalement innovante a permis d’étudier la dynamique de conjugaison du plasmide RP4 dans le biofilm. Ces résultats constituent finalement la première étude qui décrit réellement comment la conjugaison a lieu dans le biofilm et qui va au-delà en termes de compréhension sur cette dynamique grâce à l’approche 2D que nous avions mis en place. Nous démontrons que biofilm n’est pas un hotspot pour le transfert du plasmide RP4 et que les facteurs de la matrice EPS qui compose sa structure n’empêchent pas la dissémination du plasmide. Mais qu’il s’agit plutôt du stade de développement du biofilm qui va rendre possible l’accessibilité des donneurs à l’attachement aux zones de contact avec la surface, à proximité des cellules receveuses
The study of conjugation dynamics of conjugative plasmids in Gram-negative bacteria is the central research theme of our laboratory and around which my thesis project was built. The aim of my research was to provide real knowledge on the extent and impact of conjugation in bacterial communities. The biofilm is widely considered by the scientific community as a hotspot for gene transfer mainly because of the favorable cell contacts that exist in its structure. However, the only studies that have attempted to demonstrate experimentally that biofilms increase gene transfer by conjugation do not provide clear data on the dynamics of these transfers that take place in the biofilm and how the biofilm impacts these transfers. The approach we used to study the dynamics of conjugation in biofilm is based on a collaborative project between our laboratory and that of Dr. Knut Drescher, based at the Biozentrum in Basel, Switzerland. This collaboration allowed us to deploy innovative fluorescence microscopy techniques developed by our two laboratories and never used before in the context of the study of conjugation in biofilm.We focused on the RP4 plasmid which is an IncP conjugative plasmid. Found within many natural environments, it has been the primary plasmid model for studies that have focused on conjugation in the biofilm, and has been widely exploited as a genetic tool by the scientific community. Despite the fact that it has been widely used, the transfer mechanisms of the RP4 plasmid are very poorly described. The RP4 plasmid has thus proven to be a very relevant model for studying conjugation that we have used both in a biotechnological aspect to broaden the host spectrum of antibacterial TAPs systems and in a fundamental aspect to study its conjugation dynamics, both within a 2D cultured E. coli population and within a 3D biofilm structured E. coli population.During my thesis work, I therefore exploited the RP4 plasmid to carry antibacterial CRISPR systems in various phylogenetically distant bacterial species. I provided the first real-time images of the RP4 plasmid transfer in 2D and very interesting new data on the timing of DNA double-strand conversion in the recipient. Finally, a totally innovative approach allowed to study the conjugation dynamics of the RP4 plasmid in the biofilm. These results finally constitute the first study that really describes how conjugation takes place in the biofilm and that goes beyond in terms of understanding this dynamic thanks to the 2D approach that we had set up. We demonstrate that biofilm is not a hotspot for the transfer of the RP4 plasmid and that the factors of the EPS matrix that compose its structure do not prevent the dissemination of the plasmid. Rather, it is the stage of biofilm development that makes it possible for the donors to attach to the surface contact areas near the recipient cells