Multimer Resolution Research Articles

Real-Time PCR Method for Assessment of ParA-Mediated Recombination Efficiency in Minicircle Production.

The in vivo intramolecular recombination of a parental plasmid allows excising prokaryotic backbone from the eukaryotic cassette of interest, leading to the formation of, respectively, a miniplasmid and a minicircle. Here we describe a real-time PCR protocol suitable for the determination of recombination efficiency of parental plasmids with multimer resolution sites (MRS). The protocol was successfully applied to purified DNA samples obtained from E. coli cultures, allowing a more reproducible determination of recombination efficiency than densitometry analysis of agarose gels.

Segregationally stabilised plasmids improve production of commodity chemicals in glucose-limited continuous fermentation

BackgroundThe production of chemicals via bio-based routes is held back by limited easy-to-use stabilisation systems. A wide range of plasmid stabilisation mechanisms can be found in the literature, however, how these mechanisms effect genetic stability and how host strains still revert to non-productive variants is poorly understood at the single-cell level. This phenomenon can generate difficulties in production-scale bioreactors as different populations of productive and non-productive cells can arise. To understand how to prevent non-productive strains from arising, it is vital to understand strain behaviour at a single-cell level.The persistence of genes located on plasmid vectors is dependent on numerous factors but can be broadly separated into structural stability and segregational stability. While structural stability refers to the capability of a cell to resist genetic mutations that bring about a loss of gene function in a production pathway, segregational stability refers to the capability of a cell to correctly distribute plasmids into daughter cells to maintain copy number. A lack of segregational stability can rapidly generate plasmid-free variants during replication, which compromises productivity.ResultsCitramalate synthase expression was linked in an operon to the expression of a fluorescent reporter to enable rapid screening of the retention of a model chemical synthesis pathway in a continuous fermentation of E. coli. Cells without additional plasmid stabilisation started to lose productivity immediately after entering the continuous phase. Inclusion of a multimer resolution site, cer, enabled a steady-state production period of 58 h before a drop in productivity was detected. Single-cell fluorescence measurements showed that plasmid-free variants arose rapidly without cer stabilisation and that this was likely due to unequal distribution of plasmid into daughter cells during cell division. The addition of cer increased total chemical yield by more than 50%.ConclusionsThis study shows the potential remains high for plasmids to be used as pathway vectors in industrial bio-based chemicals production, providing they are correctly stabilised. We demonstrate the need for accessible bacterial ‘toolkits’ to enable rapid production of known, stabilised bacterial production strains to enable continuous fermentation at scale for the chemicals industry.

Mobilization of pdif modules in Acinetobacter: A novel mechanism for antibiotic resistance gene shuffling?

XerCD-dif site-specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer-mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid-dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field.

Native nanodiscs formed by styrene maleic acid copolymer derivatives help recover infectious prion multimers bound to brain-derived lipids

Prions are lipidated proteins that interact with endogenous lipids and metal ions. They also assemble into multimers and propagate into the infectious scrapie form known as PrPSc The high-resolution structure of the infectious PrPSc state remains unknown, and its analysis largely relies on detergent-based preparations devoid of endogenous ligands. Here we designed polymers that allow isolation of endogenous membrane:protein assemblies in native nanodiscs without exposure to conventional detergents that destabilize protein structures and induce fibrillization. A set of styrene-maleic acid (SMA) polymers including a methylamine derivative facilitated gentle release of the infectious complexes for resolution of multimers, and a thiol-containing version promoted crystallization. Polymer extraction from brain homogenates from Syrian hamsters infected with Hyper prions and WT mice infected with Rocky Mountain Laboratories prions yielded infectious prion nanoparticles including oligomers and microfilaments bound to lipid vesicles. Lipid analysis revealed the brain phospholipids that associate with prion protofilaments, as well as those that are specifically enriched in prion assemblies captured by the methylamine-modified copolymer. A comparison of the infectivity of PrPSc attached to SMA lipid particles in mice and hamsters indicated that these amphipathic polymers offer a valuable tool for high-yield production of intact, detergent-free prions that retain in vivo activity. This native prion isolation method provides an avenue for producing relevant prion:lipid targets and potentially other proteins that form multimeric assemblies and fibrils on membranes.

IN SILICO АНАЛИЗ СТРУКТУРНЫХ МОДИФИКАЦИЙ В КОНСЕРВАТИВНЫХ САЙТАХ MRS У РОДСТВЕННЫХ ПЛАЗМИД

In this work, a comparative structural in silico analysis of two related ColE1-type plasmids, pCS36-4CPA and pRHBSTW-00138_7, was provided. At the nucleotide sequence level, their identity was about 82.2%. In the plasmid pRHBSTW-00138_7, modules of replication, propagation, stability, as well as a cargo module were identified. The plasmid was identified as a member of the ColE1 family. The stability module was provided by the plasmid DNA multimer resolution site (mrs). At the nucleotide level, the mrs of both plasmids had a 61.3% identity. In the structure of mrs pRHBSTW-00138_7, despite the high identity of pCS36-4CPA, significant structural modifications were found. Among them, the change in the secondary structure of the RNA transcript Rcd. The results obtained indicate that, although the mrs of both plasmids have a fundamentally the same structure, they probably could have been obtained by them independently of different ancestral plasmids.

The Tcp plasmids of Clostridium perfringens require the resP gene to ensure stable inheritance

Many of the disease-causing toxins of the pathogenic bacterium Clostridium perfringens are harboured on large, highly stable, conjugative plasmids. Previous work has established the requirement of a ParMRC-like partitioning system for plasmid maintenance, but little is known about other mechanisms used to ensure stable plasmid inheritance. The archetypal 47 kb Tcp plasmid, pCW3, encodes a gene, resP, whose putative product has sequence similarity to members of the serine recombinase family of site-specific recombinases. ResP is therefore likely to function to resolve plasmid multimers. Sequence analysis identified that resP genes are present on all C. perfringens plasmid families, suggesting a conserved function in these plasmids. To assess the requirement of resP for the stability of pCW3, deletion mutants were constructed. Deletion of resP from pCW3 resulted in a marked instability phenotype that was rescued upon complementation with the wild-type resP gene. Complementation with resP genes from two different C. perfringens plasmids demonstrated that only closely related resP genes can complement the mutation on pCW3. The function of ResP in vivo was examined using an Escherichia coli model system, which determined that two directly repeated res sites were required for the resolution of DNA and that ResP could resolve multimeric plasmid forms into monomeric units. Based on these findings we concluded that ResP could catalyse the resolution of plasmid multimers and was required for the maintenance of Tcp plasmids within C. perfringens. Overall, the results of this study have significant implications for our understanding of the maintenance of toxin-encoding plasmids within C. perfringens.

Plasmid stability analysis based on a new theoretical model employing stochastic simulations.

Here, we present a simple theoretical model to study plasmid stability, based on one input parameter which is the copy number of plasmids present in a host cell. The Monte Carlo approach was used to analyze random fluctuations affecting plasmid replication and segregation leading to gradual reduction in the plasmid population within the host cell. This model was employed to investigate maintenance of pEC156 derivatives, a high-copy number ColE1-type Escherichia coli plasmid that carries an EcoVIII restriction-modification system. Plasmid stability was examined in selected Escherichia coli strains (MG1655, wild-type; MG1655 pcnB, and hyper-recombinogenic JC8679 sbcA). We have compared the experimental data concerning plasmid maintenance with the simulations and found that the theoretical stability patterns exhibited an excellent agreement with those empirically tested. In our simulations, we have investigated the influence of replication fails (α parameter) and uneven partition as a consequence of multimer resolution fails (δ parameter), and the post-segregation killing factor (β parameter). All of these factors act at the same time and affect plasmid inheritance at different levels. In case of pEC156-derivatives we concluded that multimerization is a major determinant of plasmid stability. Our data indicate that even small changes in the fidelity of segregation can have serious effects on plasmid stability. Use of the proposed mathematical model can provide a valuable description of plasmid maintenance, as well as enable prediction of the probability of the plasmid loss.

Molecular characterization of the pA3J1 plasmid from the psychrotolerant Antarctic bacterium Pseudomonas sp. ANT_J3

The knowledge on plasmids of cold-active bacteria is highly limited. In this study, the molecular characterization of the pA3J1 plasmid of Antarctic psychrotolerant bacterium Pseudomonas sp. ANT_J3 was performed. Within this plasmid, thirteen putative open reading frames were identified. Nine of them encoded proteins involved in replication, partitioning, postsegregational elimination of plasmid-less cells (via a toxin-antitoxin system activity), multimer resolution and mobilization by conjugal transfer. These genes constitute the plasmid backbone. The functional analysis of the pA3J1 maintenance region revealed that it is a narrow host range replicon, stably maintained in the host cells by the combined activities of the partitioning and relBE-type toxin-antitoxin systems. It was also suggested that the replication system of the pA3J1 plasmid may be temperature-sensitive. Comparative analyses revealed the presence of 16 Pseudomonas plasmids encoding homologous replication proteins and 5 plasmids carrying mobA genes homologous to the corresponding gene of pA3J1. The relaxase (MobA) of the pA3J1 plasmid was classified into MOBQ family, and the phylogenetic analysis suggested that this may be a representative of a novel group (or subgroup) within this family. The structural and comparative analyses revealed that the arrangement of genetic modules in the pA3J1 plasmid is unique.

Analysis of pCERC7, a small antibiotic resistance plasmid from a commensal ST131 Escherichia coli, defines a diverse group of plasmids that include various segments adjacent to a multimer resolution site and encode the same NikA relaxase accessory protein enabling mobilisation

The ampicillin resistance plasmid pCERC7, carrying transposon Tn2 with an IS4 insertion, was detected in the draft genome of a commensal Escherichia coli isolate. The genome data also revealed that this isolate belongs to ST131, clade B. pCERC7 is 9712bp comprised of a 3319bp backbone, Tn2::IS4 (6388bp) and 5bp of target site duplication, and was present at a copy number of 40. pCERC7 is related to several plasmids composed of only the backbone, or the backbone with the Tn2 insertion in the same position. These plasmids have been found previously in Escherichia coli or Salmonella enterica recovered in several different countries from as early as the 1970s. This group was named the NTP16 group after the best studied example. pCERC7 was annotated using available information about plasmids in this group and additional analyses. The backbone includes genes for RNA I and RNA II to initiate replication and the Tn2 interrupts a gene found here to encode a protein 66% identical to the Rom regulatory protein of ColE1. NTP16 family plasmids include a gene, previously designated mobA, that was found to encode a homologue (53% identical) of the NikA relaxase accessory protein of the conjugative IncI1 plasmid R64, which is known to bind to the R64 oriT. However, a nikB relaxase gene is not present, indicating that a relaxase must be supplied in trans for mobilisation by R64 to occur, as demonstrated previously for NTP16. Hence, MobA of NTP16 and relatives was renamed NikA. Upstream of nikA, we found a region closely related to the oriT of R64. pCERC7 and all members of the NTP16 family also include a multimer resolution site, nmr, similar to the cer site of ColE1. The backbone of the NTP16 family also includes genes for a demonstrated toxin-antitoxin system, LsoAB. Several more distantly related groups of plasmids that include a very closely related nmr-nikA-oriT segment (99.4–93.7% DNA identity) were identified in the GenBank non-redundant DNA database. All use an RNA I/RNA II-Rom system for replication initiation, but each contains a unique fragment adjacent to the nmr site. The segment of the NTP16/pCERC7 group that encodes the LsoAB toxin-antitoxin system is replaced by a different segment in other family groups. The point at which the sequences diverge is between the XerC and XerD sites of the dif site at one end of nmr, suggesting that the evolution of this broad group of plasmids involves XerC/XerD recombination.

Isolation and sequence analysis of pCS36-4CPA, a small plasmid from Citrobacter sp. 36-4CPA.

A small plasmid designated pCS36-4CPA with a size of 5217 base pairs and G-C content of 50.74% was isolated from Citrobacter sp. 36-4CPA. The origin of replication (ori) of the plasmid was identified as a region of about 800 bp in length with an identity of 67.1% to the ColE1 plasmid at the nucleotide level. The replication region contained typical elements of ColE1-like plasmids: RNA I and RNA II with their corresponding −10 and −35 boxes, a single-strand initiation site (ssi), and a lagging-strand termination site (terH). As seen in other ColE1-like plasmids, pCS36-4CPA carried mobilisation machinery that include mobABCD genes but it did not possess the rom gene. Analysis of the multimer resolution site (mrs) was performed and XerC and XerD binding sites were identified. Also, the 70-nt transcript Rcd of pCS36-4CPA was predicted and similarity of the transcript’s secondary structure with those of the ColE1-family was shown. The cargo module of pCS36-4CPA contained three open reading frames (ORFs). Two of them (ORF5 and ORF6) showed no significant homology to any known gene sequences but contained putative THAP DNA-binding (DBD) and type II restriction endonuclease EcoO109I domains. The seventh open reading frame (ORF7) encodes YhdJ-like DNA modification methylase. The region highly homologous to pCS36-4CPA was found in the Salmonella phage SE2 genome.

Plasmid pEC156, a Naturally Occurring Escherichia coli Genetic Element That Carries Genes of the EcoVIII Restriction-Modification System, Is Mobilizable among Enterobacteria.

Type II restriction-modification systems are ubiquitous in prokaryotes. Some of them are present in naturally occurring plasmids, which may facilitate the spread of these systems in bacterial populations by horizontal gene transfer. However, little is known about the routes of their dissemination. As a model to study this, we have chosen an Escherichia coli natural plasmid pEC156 that carries the EcoVIII restriction modification system. The presence of this system as well as the cis-acting cer site involved in resolution of plasmid multimers determines the stable maintenance of pEC156 not only in Escherichia coli but also in other enterobacteria. We have shown that due to the presence of oriT-type F and oriT-type R64 loci it is possible to mobilize pEC156 by conjugative plasmids (F and R64, respectively). The highest mobilization frequency was observed when pEC156-derivatives were transferred between Escherichia coli strains, Enterobacter cloacae and Citrobacter freundii representing coliform bacteria. We found that a pEC156-derivative with a functional EcoVIII restriction-modification system was mobilized in enterobacteria at a frequency lower than a plasmid lacking this system. In addition, we found that bacteria that possess the EcoVIII restriction-modification system can efficiently release plasmid content to the environment. We have shown that E. coli cells can be naturally transformed with pEC156-derivatives, however, with low efficiency. The transformation protocol employed neither involved chemical agents (e.g. CaCl2) nor temperature shift which could induce plasmid DNA uptake.

The Legacy of Nat Sternberg: The Genesis of Cre-lox Technology

Cre-lox of bacteriophage P1 has become one of the most widely used tools for genetic engineering in eukaryotes. The origins of this tool date to more than 30 years ago when Nat L. Sternberg discovered the recombinase, Cre, and its specific locus of crossover, lox, while studying the maintenance of bacteriophage P1 as a stable plasmid. Recombinations mediated by Cre assist in cyclization of the DNA of infecting phage and in resolution of prophage multimers created by generalized recombination. Early in vitro work demonstrated that, although it shares similarities with the well-characterized bacteriophage λ integration, Cre-lox is in many ways far simpler in its requirements for carrying out recombination. These features would prove critical for its development as a powerful and versatile tool in genetic engineering. We review the history of the discovery and characterization of Cre-lox and touch upon the present direction of Cre-lox research.

PKBuS13, a KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 833, carrying Tn4401b inserted into an Xer site-specific recombination locus.

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported.

Genetic analysis of maintenance of pEC156, a naturally occurring Escherichia coli plasmid that carries genes of the EcoVIII restriction–modification system

In the present study the role of the mechanisms responsible for maintenance of a natural plasmid pEC156, that carries genes of the EcoVIII restriction–modification system was investigated. Analysis of this plasmid's genetic content revealed the presence of genetic determinants suggesting two such mechanisms. The first of them relies on site specific recombination utilizing the Xer/cer molecular machinery, while the second involves a restriction–modification system as an addiction module. Our analysis indicated that three factors affect the maintenance of pEC156: (i) a cis-acting cer site involved in resolution of plasmid multimers, (ii) a gene coding for EcoVIII endonuclease, and (iii) plasmid copy number control. The lowest stability was observed with pEC156 derivatives deprived of the cer site. Decreased stability of pEC156 derivatives was also observed in E. coli strains deficient in genes coding for proteins involved in plasmid multimer resolution (XerC, XerD, ArgR and PepA). A similar effect, but to a much lesser extent was observed for the pEC156 derivative without a functional gene coding for EcoVIII endonuclease. Our results indicate that the presence of the cer site is more important for pEC156 stable maintenance than the presence of a functional gene coding for EcoVIII endonuclease. In our work we also tested maintenance of pEC156 possessing a ColE1-type replicon in bacteria belonging to Enterobacteriaceae family. We have found that pEC156 was most stably maintained in Enterobacter cloacae and Klebsiella oxytoca representing coli-type enterobacteria. We have found that in all enterobacteria tested pEC156 derivatives deficient in the cer site were significantly less stably maintained than cer+ variants.

Xer Site-Specific Recombination: Promoting Vertical and Horizontal Transmission of Genetic Information.

Two related tyrosine recombinases, XerC and XerD, are encoded in the genome of most bacteria where they serve to resolve dimers of circular chromosomes by the addition of a crossover at a specific site, dif. From a structural and biochemical point of view they belong to the Cre resolvase family of tyrosine recombinases. Correspondingly, they are exploited for the resolution of multimers of numerous plasmids. In addition, they are exploited by mobile DNA elements to integrate into the genome of their host. Exploitation of Xer is likely to be advantageous to mobile elements because the conservation of the Xer recombinases and of the sequence of their chromosomal target should permit a quite easy extension of their host range. However, it requires means to overcome the cellular mechanisms that normally restrict recombination to dif sites harbored by a chromosome dimer and, in the case of integrative mobile elements, to convert dedicated tyrosine resolvases into integrases.

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

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

The tyrosine recombinase MrpA and its target sequence: a mutational analysis of the recombination site mrpS resulting in a new left element/right element (LE/RE) deletion system

MrpA is the multimer resolution protein of the Streptomyces coelicolor A3(2) plasmid SCP2*. Previously, MrpA was found to be a site-specific tyrosine recombinase that acts with the 36-bp recombination site mrpS. The present report gives a comprehensive characterization of the composition as well as the position of the spacer and MrpA binding sites within mrpS. Experiments revealed a spacer consisting of 6 remarkably variable nucleotides in the middle of the mrpS-site. A reduction in the spacer to 5 nucleotides abolished recombination. Investigation of the MrpA binding sites showed the importance of its 15 nucleotides on an effective recombination. Among almost randomly exchangeable nucleotides, two nucleotides were identified as essential for MrpA binding. Alteration of either of these nucleotides led to a reduction in MrpA binding down to 2% or even to no binding. Based on these results, a new left element/right element (LE/RE) deletion system was developed. The constructed heteromeric mrpS-sites are efficiently resolved by MrpA. The resulting double mutated (LE/RE) site can no longer be used as a recombination site by MrpA. The system has been successfully applied for the generation of multiple-targeted deletions in the genome of E. coli.

Instability of Escherichia coli R-factors in Salmonella enterica serovar Typhi involves formation of recombinant composite plasmid structures

In spite of a well-documented ability of Samonella enterica Typhi strains to receive R factors from Escherichia coli and other enterobacteria, epidemiological data show that Typhi is a rather poor host of antibiotic-resistance genes and in fact, of plasmids, suggesting that most of the plasmids naturally acquired by Typhi strains become unstable and eventually segregate. We have previously reported evidence that each of three plasmids conjugatively transferred to S. enterica Typhi experienced deletion-mediated loss of a resistance determinant before plasmid segregation occurred.We now report that in Typhi strains containing these unstable plasmids a superhelical DNA species of lower mobility is detected, probably representing plasmid dimer structures. Plasmid deletion is a RecA-dependent process since it is not detected in derivatives of a recA1 S. enterica Typhi strain containing the corresponding plasmids, and in such strains we were unable to detect either the low-mobility species. We propose that the deletable segments contain key information for plasmid stability in S. enterica Typhi, possibly a multimer resolution system.

Site-specific deletions in the tomato genome by the CinH-RS2 and ParA-MRS recombination systems

We have tested the CinH-RS2 and ParA-MRS site-specific deletion systems in tomato (Solanum lycopersicum L.). The ParA-MRS system is derived from the broad-host-range plasmid RK2, where the 222 aa ParA recombinase recognizes a 133 bp multimer resolution site (MRS). The CinH-RS2 system is derived from Acinetobacter plasmids pKLH2 and pKLH204, where the 188 amino acid CinH recombinase recognizes a 113-bp recombination site known as RS2. In this study, target lines containing a DNA segment flanked by recombination sites were crossed to recombinase-expressing lines producing CinH or ParA recombinase. CinH-mediated recombination of RS2 substrates was detected in 2 of 3 F1 plants that harbor both the target and recombinase loci. On the other hand, recombination mediated by ParA was not detected among F1 plants, but was found among 13 of 47 F2 plants. These data show that both systems can mediate site-specific DNA deletion in the tomato genome, and, upon further refinement, can provide additional molecular tools for tomato improvement through precise genome manipulation. As the target construct also contains additional recombination sites for site-specific integration by other recombination systems, these tomato lines could be used for future testing of gene stacking through site-specific integration.

Prevalence and Significance of Plasmid Maintenance Functions in the Virulence Plasmids of Pathogenic Bacteria

Virulence functions of pathogenic bacteria are often encoded on large extrachromosomal plasmids. These plasmids are maintained at low copy number to reduce the metabolic burden on their host. Low-copy-number plasmids risk loss during cell division. This is countered by plasmid-encoded systems that ensure that each cell receives at least one plasmid copy. Plasmid replication and recombination can produce plasmid multimers that hinder plasmid segregation. These are removed by multimer resolution systems. Equitable distribution of the resulting monomers to daughter cells is ensured by plasmid partition systems that actively segregate plasmid copies to daughter cells in a process akin to mitosis in higher organisms. Any plasmid-free cells that still arise due to occasional failures of replication, multimer resolution, or partition are eliminated by plasmid-encoded postsegregational killing systems. Here we argue that all of these three systems are essential for the stable maintenance of large low-copy-number plasmids. Thus, they should be found on all large virulence plasmids. Where available, well-annotated sequences of virulence plasmids confirm this. Indeed, virulence plasmids often appear to contain more than one example conforming to each of the three system classes. Since these systems are essential for virulence, they can be regarded as ubiquitous virulence factors. As such, they should be informative in the search for new antibacterial agents and drug targets.