A first global analysis of plasmid encoded proteins in the ACLAME database

Many plasmids are mobile genetic elements (MGEs) and, as other members of that group of DNA entities, their genomes display a mosaic and combinatorial structure, making their classification extremely difficult. As other MGEs, plasmids play a major role in horizontal transfer of genetic materials and genome reorganization. Yet, the full impact of such phenomenon on major properties of the host cell, such as pathogenicity, the ability to use new carbon sources or resistance to antibiotics, remains to be fully assessed. More and more complete plasmid genome sequences are available. However, in the absence of standards for storing plasmid sequence data and annotating genes and gene products on sequenced plasmid genomes, the resulting information remains rather limited. Using 503 sequenced plasmids organized in the ACLAME database, we discuss how, by structuring information on the genomes, their host and the proteins they code for, one can gain access to either global or more detailed analysis of the plasmid sequence information, as illustrated by a network representation of the relationships between plasmids.     

Replication and Control of Circular Bacterial Plasmids

An essential feature of bacterial plasmids is their ability to replicate as autonomous genetic elements in a controlled way within the host. Therefore, they can be used to explore the mechanisms involved in DNA replication and to analyze the different strategies that couple DNA replication to other critical events in the cell cycle. In this review, we focus on replication and its control in circular plasmids. Plasmid replication can be conveniently divided into three stages: initiation, elongation, and termination. The inability of DNA polymerases to initiate de novo replication makes necessary the independent generation of a primer. This is solved, in circular plasmids, by two main strategies: (i) opening of the strands followed by RNA priming (theta and strand displacement replication) or (ii) cleavage of one of the DNA strands to generate a 3′-OH end (rolling-circle replication). Initiation is catalyzed most frequently by one or a few plasmid-encoded initiation proteins that recognize plasmid-specific DNA sequences and determine the point from which replication starts (the origin of replication). In some cases, these proteins also participate directly in the generation of the primer. These initiators can also play the role of pilot proteins that guide the assembly of the host replisome at the plasmid origin. Elongation of plasmid replication is carried out basically by DNA polymerase III holoenzyme (and, in some cases, by DNA polymerase I at an early stage), with the participation of other host proteins that form the replisome. Termination of replication has specific requirements and implications for reinitiation, studies of which have started. The initiation stage plays an additional role: it is the stage at which mechanisms controlling replication operate. The objective of this control is to maintain a fixed concentration of plasmid molecules in a growing bacterial population (duplication of the plasmid pool paced with duplication of the bacterial population). The molecules involved directly in this control can be (i) RNA (antisense RNA), (ii) DNA sequences (iterons), or (iii) antisense RNA and proteins acting in concert. The control elements maintain an average frequency of one plasmid replication per plasmid copy per cell cycle and can “sense” and correct deviations from this average. Most of the current knowledge on plasmid replication and its control is based on the results of analyses performed with pure cultures under steady-state growth conditions. This knowledge sets important parameters needed to understand the maintenance of these genetic elements in mixed populations and under environmental conditions.     

Behavior of autonomous plasmids and plasmids integrated into the chromosome during the sporulation of Bacillus subtilis cells

The behaviour of plasmids in free and integrated states was studied upon sporulation of Bacillus subtilis cells. Autonomous plasmids pBD12 and pGG10 were shown to be either transmitted into spores in small copy numbers or completely eliminated from the sporulating cell. However, insertion of the autonomous plasmid into the host chromosome may occur with a certain degree of probability (about 10(-3)) during sporulation. When in the integrated state, pBD12 plasmid may either excise from the host chromosome or amplify within the genome with the probability 1.8-2.10(-3) in the course of sporulation. The pGG102 plasmid carrying the fragment of wheat DNA and integrated by this fragment into the chromosome was shown to enter spores without whichever intragenome rearrangements.     

Characterization of putative circular plasmids in sponge‐associated bacterial communities using a selective multiply‐primed rolling circle amplification

Plasmid transfers among bacterial populations can directly influence the ecological adaptation of these populations and their interactions with host species and environment. In this study, we developed a selective multiply‐primed rolling circle amplification (smRCA) approach to enrich and characterize circular plasmid DNA from sponge microbial symbionts via high‐throughput sequencing (HTS). DNA (plasmid and total community DNA) obtained from sponge (Cinachyrella sp.) samples and a bacterial symbiont (Vibrio sp. CyArs1) isolated from the same sponge species (carrying unknown plasmids) were used to develop and validate our methodology. The smRCA was performed during 16 hr with 141 plasmid‐specific primers covering all known circular plasmid groups. The amplified products were purified and subjected to a reamplification with random hexamer primers (2 hr) and then sequenced using Illumina MiSeq. The developed method resulted in the successful amplification and characterization of the sponge plasmidome and allowed us to detect plasmids associated with the bacterial symbiont Vibrio sp. CyArs1 in the sponge host. In addition to this, a large number of small (<2 kbp) and cryptic plasmids were also amplified in sponge samples. Functional analysis identified proteins involved in the control of plasmid partitioning, maintenance and replication. However, most plasmids contained unknown genes, which could potentially serve as a resource of unknown genetic information and novel replication systems. Overall, our results indicate that the smRCA‐HTS approach developed here was able to selectively enrich and characterize plasmids from bacterial isolates and sponge host microbial communities, including plasmids larger than 20 kbp.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.     

Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population

A soil population of 16 Rhizobium leguminosarum bv. trifolii isolates was characterized by using three Sym (for symbiotic) plasmid-specific DNA hybridization probes: (i) an R. leguminosarum bv. trifolii-specific, repeated-sequence probe; (ii) a nifHDK gene probe, and (iii) a nod gene probe. A predominant Sym plasmid family was identified among the isolates. Three other unrelated Sym plasmid families were also identified. The isolates were also classified either by using a chromosomal DNA hybridization probe or by serological relatedness to 25 different R. leguminosarum bv. trifolii antisera. With either method, it was possible to group the 16 soil isolates into identical or related families. However, the correlation between the two techniques was not high. Irrespective of the means used to classify the bacterial host strain, it was possible to identify the same Sym plasmids in unrelated strains, as well as unrelated Sym plasmids in identical host strains. These data indicate that, within this soil population, there has been genetic exchange of Sym plasmids, and in one instance the hybridization pattern indicates that in vivo recombination of two different Sym plasmids may have occurred. Symbiotic effectiveness tests on red, strawberry, and subterranean clovers clearly differentiated the isolates. In general, the pattern of response was similar within groupings on the basis of Sym plasmid and chromosomal profiles but different between such groups.     

Copy number control and compatibility of nuclear plasmids in Dictyostelium discoideum

Copy number of the endogenous nuclear plasmids of Dictyostelium discoideum is a plasmid-specific trait. Copy number is stable over time, is constant relative to ploidy level, is independent of host cell genetic background, and is independent of the presence of a second unrelated plasmid in the same nucleus. Unrelated plasmids are compatible with one another within a single nucleus. Pairwise combinations of Ddp1, Ddp2, and Ddp5 were stably maintained over many generations in the absence of selection. In contrast, one of the D. discoideum plasmids (Ddp2) was incompatible with a recombinant plasmid derived from it (p7d2). In the absence of selection for retention of p7d2, transformants contain either one or the other but not both plasmids. The plasmids are stably maintained in host cells with differing genetic backgrounds, although plasmid-free colonies were detected at a frequency of about 1-2% in populations of some strains after 50 generations growth following a previous cloning.     

Effect of repair deficiency and R plasmids on spontaneous and radiation-induced mutability in Pseudomonas aeruginosa.

The effect of R plasmids on spontaneous and radiation (ultraviolet and gamma)-induced mutability in Pseudomonas aeruginosa was studied in strains containing the radiation-sensitive markers polA3 or rec-2 and the revertable auxotrophic markers hisO27 and trpB1. In the absence of an R plasmid, the radiation-induced mutability was dependent on the recA+ genotype and independent of the polA+ genotype, whereas spontaneous mutability was similar in all genetic backgrounds. R plasmids pPL1, R2, and pMG15 increased the ultraviolet radiation survival and ultraviolet-induced mutability of wild-type and polA host cells but did not alter either effect in a recA mutant. These R plasmids also increased the gamma radiation survival and gamma-induced mutability of wild-type host cells bud pMG15 also enhanced the level of spontaneous mutagenesis in wild-type host cells but not in a polA or recA mutant. These data suggested that a common plasmid gene product(s) may participate in various recA-dependent, error-prone deoxyribonucleic acid repair pathways of P. aeruginosa. The properties of a mutant R plasmid, pPL2, originally selected because it lacked enhanced ultraviolet-induced mutability, supported this conclusion.     

DNA sequence and functional analysis of Lactobacillus delbrueckii subsp. lactis plasmids pN42 and pJBL2

The plasmids pN42 and pJBL2 were isolated from the Lactobacillus delbrueckii subsp. lactis strains NCC88 and JCL414. DNA sequence determination and bioinformatic analysis revealed a strikingly conserved genetic organization containing five major, highly conserved open reading frames (ORFs). Transformation studies indicated that ORF2 (consisting of a primase fused to a replicative DNA helicase), ori, and ORF3 constitute the minimal requirements for replication of pN42 in the heterologous host Lactococcus lactis. The ORF1's are predicted to encode type I restriction-modification (R-M) system HsdS subunits with different specificities on either plasmid, suggesting that these plasmids may be involved in host defense by expanding their host R-M system repertoire. These plasmids constitute the basis for the construction of novel L. delbrueckii vectors.     

Vesicle-mediated transfer of virulence genes from Escherichia coli O157:H7 to other enteric bacteria

Membrane vesicles are released from the surfaces of many gram-negative bacteria during growth. Vesicles consist of proteins, lipopolysaccharide, phospholipids, RNA, and DNA. Results of the present study demonstrate that membrane vesicles isolated from the food-borne pathogen Escherichia coli O157:H7 facilitate the transfer of genes, which are then expressed by recipient Salmonella enterica serovar Enteritidis or E. coli JM109. Electron micrographs of purified DNA from E. coli O157:H7 vesicles showed large rosette-like structures, linear DNA fragments, and small open-circle plasmids. PCR analysis of vesicle DNA demonstrated the presence of specific genes from host and recombinant plasmids (hly, L7095, mobA, and gfp), chromosomal DNA (uidA and eaeA), and phage DNA (stx1 and stx2). The results of PCR and the Vero cell assay demonstrate that genetic material, including virulence genes, is transferred to recipient bacteria and subsequently expressed. The cytotoxicity of the transformed enteric bacteria was sixfold higher than that of the parent isolate (E. coli JM109). Utilization of the nonhost plasmid (pGFP) permitted the evaluation of transformation efficiency (ca. 10(3) transformants microg of DNA(-1)) and demonstrated that vesicles can deliver antibiotic resistance. Transformed E. coli JM109 cells were resistant to ampicillin and fluoresced a brilliant green. The role vesicles play in genetic exchange between different species in the environment or host has yet to be defined.     

Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.     

Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids

The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.     

Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue

Plasmids have been described in almost all bacterial species analysed and have proven to be essential genetic tools. In many bacteria these extrachromosomal DNAs are cryptic with no known markers or function, which makes their characterization and genetic exploitation extremely difficult. Here we describe a system that will allow the rescue of any circular DNA (plasmid or phage) using an in vitro transposition system to deliver both a selectable marker (kanamycin) and an Escherichia coli plasmid origin of replication. In this study, we demonstrate the rescue of four cryptic plasmids from the opportunistic pathogen Mycobacterium avium. To evaluate the host range of the rescued plasmids, we have examined their ability to be propagated in Mycobacterium smegmatis and Mycobacterium bovis BCG, and their compatibility with other mycobacterial plasmids. In addition, we use a library of transposon insertions to sequence one plasmid, pVT2, and to begin a genetic analysis of plasmid genes. Using this approach, we identified a putative conjugative relaxase, suggesting this myco-bacterial plasmid is transferable, and three genes required for plasmid establishment and replication.     

农杆菌T-复合物的形成与转运研究进展

在简要介绍农杆菌T-DNA转运全过程的基础上,结合作者近年的工作,重点对T-复合物的形成和T-复合物在农杆菌细胞内的转运机理的最新进展进行归纳和评述．农杆菌能够将其Ti质粒上的一段DNA以单链DNA-蛋白质复合物(简称T-复合物)的形式,通过其细胞两端的四型分泌系统(typeⅣ secretion system,T4SS)转运到宿主植物中,并使宿主发生遗传转化,因而农杆菌介导的T-DNA转运技术已成为应用最广泛的植物转基因技术,同时,由于转运T-复合物的T4SS也是某些质粒接合转移和许多病源微生物分泌致病效应蛋白的通道,因此,农杆菌T-DNA转运机理的研究受到了广泛的重视和关注,使得这方面的研究进展非常迅速．     

Origin and evolution of plasmids

Studies on the origin and evolution of plasmids may provide valuable insights on the promiscuous nature of DNA. The first examples of the selfish nature of nucleic acids are exemplified by primordial oligoribonucleotides which evolved into primitive replicons. The propagation of these molecules were likely patterned after the current viral RNA ribozymes, which have been recently shown to possess RNA synthesizing and template mediated polymerizing capabilities in the absence of proteins. The parasitic nature of nucleic acids is depicted by satellite nucleic acid molecules associated with viruses. The satellites of adenovirus and tobacco ringspot virus serve as established examples: they contain no open reading frames. Comparative analysis of the replication origins of virions and plasmids show them to be conserved, originating from the simplest autocatalytic replicon to highly complex and evolved plasmids, replicating by a rolling circle mechanism. The eventual association of proteins with nucleic acids provided added efficiency and protective advantages for molecular perpetuation. The promiscuous and selfish nature of plasmids is demonstrated by their ability to genetically engineer their host so that the host cell is best able to cope and survive in hostile environments. Survival of the host ensures survival of the plasmid. Sequestering of genes by plasmids occurs when the environmental conditions negatively affect the host. The sequestering mechanism is fundamental and forms the outreach mechanisms to generate and propagate macromolecules of increasing size when necessary for survival. The level of sophistication of plasmids increases with the addition of new genes such as those that allow the host to occupy a specific environment normally inhospitable to the host cell. The vast range of plasmid types which have obtained genes interchangeably reflect the levels of sophistication achieved by these macromolecules. The Ti plasmid in Agrobacterium tumefaciens and the pSym and accessory plasmids in Rhizobium illustrate the level of complexity attained by replicons.     

Stress responses and replication of plasmids in bacterial cells

Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage lambda that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.     

Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.     

Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids

Chromosomal mutants were isolated in Escherichia coli that altered carotenoid production from transformed carotenoid biosynthesis genes on a pACYC-derived plasmid (pPCB15). The mutations were mapped by sequencing. One group of mutations appeared to affect the cell metabolism without changing the copy number of the carotenoid synthesis plasmid. The other group of mutations either increased or decreased the copy number of the pPCB15 plasmid as determined by real-time PCR. The copy number change in most mutants was likely specific for ColE1-type plasmids for which copy number is controlled by a small antisense RNA. This collection of host strains would be useful for fine tuning expression of proteins and adjusting production of desired molecules without recloning to different vectors.