The repABC plasmid family

repABC plasmids are widely distributed among alpha-proteobacteria. They are especially common in Rhizobiales. Some strains of this bacterial order can contain multiple repABC replicons indicating that this plasmid family includes several incompatibility groups. The replication and stable maintenance of these replicons depend on the presence of a repABC operon. The repABC operons sequenced to date share some general characteristics. All of them contain at least three protein-encoding genes: repA, repB and repC. The first two genes encode proteins involved in plasmid segregation, whereas repC encodes a protein crucial for replication. The origin of replication maps within the repC gene. In contrast, the centromere-like sequence (parS) can be located at various positions in the operon. In this review we will summarize current knowledge about this plasmid family, with special emphasis on their structural diversity and their complex genetic regulation. Finally, we will examine some ideas about their evolutionary origin and trends.     

The expression of a novel antisense gene mediates incompatibility within the large repABC family of alpha-proteobacterial plasmids

Large extrachromosomal replicons in many members of the alpha-proteobacteria encode genes that are required for plant or animal pathogenesis or symbiosis. Most of these replicons encode repABC genes that control their replication and faithful segregation during cell division. In addition to its chromosome, the plant endosymbiont Sinorhizobium meliloti also maintains the 1.4 Mb pSymA and 1.7 Mb pSymB symbiotic megaplasmids both of which are repABC-type replicons. In all repABC loci that have been characterized, an apparently untranslated intergenic region between the repB and repC genes encodes a strong incompatibility determinant (referred to as incalpha). Here we report the isolation of mutations within the incalpha regions of pSymA and pSymB that eliminate incompatibility. These mutations map to and inactivate a promoter in the intergenic region that drives the expression of an approximately 56 nucleotide untranslated RNA molecule that mediates incompatibility. This gene, that we have named incA, is transcribed antisense to the repABC genes. Our analysis suggests that the incA gene is conserved in repABC loci from a diverse spectrum of bacteria.     

Diversity and evolution of repABC type plasmids in Rhodobacterales

The repABC operon is the prevalent replication unit of alphaproteobacterial plasmids. Their semi-autonomy is ensured by the essential replicase gene repC as well as the repAB partitioning cassette. While conserved repAB modules are widespread among bacterial plasmids and homologues are even responsible for chromosome partitioning, repC genes are exclusively present in Alphaproteobacteria . RepABC operons contain two strong incompatibility regions, namely a small regulative antisense RNA gene ( incα ) and a palindromic centromere region ( incβ ), which were previously used to classify these replicons. The present survey pursued a complementary strategy essentially following the rationale that all plasmids identified from a single bacterium are per se compatible. We established a novel classification scheme for plasmids based on comprehensive phylogenetic analyses of repC , repA and repB genes. Our case study is focused on the Roseobacter clade ( Rhodobacterales ), one of the most successful lineages of the marine bacterioplankton. Its global significance was shown in several studies and the interest in these organisms is reflected by more than 40 upcoming genome projects. Based on phylogenetic RepC analyses we identified nine compatibility groups that are expected to stably coexist within the same cell. This prediction is supported by RepA and RepB phylogenies, moreover independent evidence is delivered by the group specificity of highly conserved palindromes ( incβ ).     

Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis.

We have prepared and analyzed two types of gene fusion between the replication initiator gene, repC, and the reporter gene, blaZ, in order to investigate the relationship between pT181 plasmid copy number and RepC initiator protein production. A series of pT181 copy mutant plasmids, with copy numbers ranging from 70 to 800 copies per cell, were analyzed. In one type of gene fusion used in this study, blaZ was translationally coupled to the C-terminal end of the repC coding sequence such that native forms of both proteins were produced. This gene fusion arrangement, which permitted monitoring of RepC production (as BlaZ activity) by plasmids using the protein for their own replication, demonstrated a linear relationship, with one exception, between RepC production and plasmid copy number over a 20-fold range. In the second type of fusion, blaZ was translationally fused to the C-terminal end of repC. As the translational fusion did not produce active RepC protein, the fusion-containing pT181 derivatives were maintained in a strain which provided RepC in trans, and were thus analyzed at constant copy number. In contrast to previous analyses of this type, our translational fusion constructs expressed repC at levels proportional to the copy numbers of the plasmids from which the fusions were prepared. Using these data, we have calculated a minimum figure for the number of RepC molecules synthesized per replication event.     

Structural elements required for replication and incompatibility of the Rhizobium etli symbiotic plasmid

The symbiotic plasmid of Rhizobium etli CE3 belongs to the RepABC family of plasmid replicons. This family is characterized by the presence of three conserved genes, repA, repB, and repC, encoded by the same DNA strand. A long intergenic sequence (igs) between repB and repC is also conserved in all members of the plasmid family. In this paper we demonstrate that (i) the repABC genes are organized in an operon; (ii) the RepC product is essential for replication; (iii) RepA and RepB products participate in plasmid segregation and in the regulation of plasmid copy number; (iv) there are two cis-acting incompatibility regions, one located in the igs (incalpha) and the other downstream of repC (incbeta) (the former is essential for replication); and (v) RepA is a trans-acting incompatibility factor. We suggest that incalpha is a cis-acting site required for plasmid partitioning and that the origin of replication lies within incbeta.     

Prediction and functional analysis of the replication origin of the linear plasmid pSCL2 in Streptomyces clavuligerus

pSCL2 (120 kb), one of the linear plasmids found in Streptomyces clavuligerus NRRL3585, was isolated and partially sequenced. Computational analysis of the central region of pSCL2 revealed the presence of two open reading frames that appear to encode proteins highly homologous to RepL1 and RepL2, replication proteins from pSLA2-L, the large linear plasmid in Streptomyces rochei. The S. clavuligerus open reading frames were designated repC1 and repC2, encoding the proteins RepC1 (150 amino acids) and RepC2 (102 amino acids), respectively. The RepC and RepL proteins have identical translation features and very similar predicted secondary and tertiary structures. Functional analysis confirmed that RepC1 is essential for replication initiation of pSCL2, whereas RepC2 is dispensable but may play a role in copy number control. The RepC and RepL proteins do not show similarity to any other bacterial plasmid replication proteins. Three regions of DNA sequence, Box 1 (1050-850 bp), Box 2 (723-606 bp), and Box 3 (224-168 bp), located upstream of repC1, were also shown to be essential or very important for replication of pSCL2.     

The PcrA3 mutant binds DNA and interacts with the RepC initiator protein of plasmid pT181 but is defective in its DNA helicase and unwinding activities

Plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin (dso) by the initiator protein. The RepC initiator protein binds to the plasmid pT181 dso in a sequence-specific manner and recruits the PcrA helicase through a protein-protein interaction. Subsequently, PcrA unwinds DNA at the nick site followed by replication by DNA polymerase III. The pcrA3 mutant of Staphylococcus aureus has previously been shown to be defective in plasmid pT181 replication. Suppressor mutations in the repC initiator gene have been isolated that allow pT181 replication in the pcrA3 mutant. One such suppressor mutant contains a D57Y change in the RepC protein. To identify the nature of the defect in PcrA3, we have purified this mutant protein and studied its biochemical activities. Our results show that while PcrA3 retains its DNA binding activity, it is defective in its helicase and RepC-dependent pT181 DNA unwinding activities. We have also purified the RepC D57Y mutant and shown that it is similar in its biochemical activities to wild-type RepC. RepC D57Y supported plasmid pT181 replication in cell-free extracts made from wild-type S. aureus but not from the pcrA3 mutant. We also demonstrate that both wild-type RepC and its D57Y mutant are capable of a direct physical interaction with both wild-type PcrA and the PcrA3 mutant. Our results suggest that the inability of PcrA3 to support pT181 replication is unlikely to be due to its inability to interact with RepC. Rather, it is likely that a defect in its helicase activity is responsible for its inability to replicate the pT181 plasmid.     

Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein

pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.     

Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14

Summary The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. The rep product is trans-active and essential for plasmid replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB110 and protein A of pC194 (all these found in staphylococci) and the protein of the R6K plasmid of Escherichia coli. The predicted rep protein has highly homologous amino acid sequences with protein A of pC194 and RepC of pUB110 throughout the protein molecule, but not with RepC of pT181, of R6K or protein RepH encoded by and iniating the replication of pC194.     

Complete nucleotide sequence and structural organization of pPB1, a small Lactobacillus plantarum cryptic plasmid that originated by modular exchange

A small cryptic plasmid designated pPB1 was isolated from Lactobacillus plantarum BIFI-38 and its complete 2899 bp nucleotide sequence was determined. Sequence analysis revealed four putative open reading frames. Based on sequence analysis two modules could be identified. First, the replication module consisted of a sequence coding for a replication protein (RepB) and its corresponding target site, and two putative repressor proteins (RepA and RepC). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate RepB production. A putative lagging-strand initiation site was also found, suggesting that pPB1 replicates via a rolling circle mechanism. The second module of pPB1 consisted of a sequence coding for a putative mobilization protein and its corresponding oriT site. Since the nucleotide sequence of the replication module showed 94.5% identity to the similar region on the Leuconostoc lactis plasmid pCI411, and the nucleotide sequence of the mobilization module had 97.5% identity to L. plantarum plasmid pLB4, it is concluded that pPB1 originated by modular exchange between two such plasmids by homologous recombination. Putative recombination sites where crossover might have taken place were also identified.     

Identification of the partitioning site within the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1

The replicator region of composite plasmid pTAV1 of Paracoccus versutus (included in mini-replicon pTAV320) belongs to the family of repABC replicons commonly found in plasmids harbored by Agrobacterium and Rhizobium spp. The repABC replicons encode three genes clustered in an operon, which are involved in partitioning (repA and repB) and replication (repC). In order to localize the partitioning site of pTAV320, the two identified incompatibility determinants of this mini-replicon (inc1, located in the intergenic sequence between repB and repC; and inc2, situated downstream of the repC gene) were PCR amplified and used together with purified RepB fusion protein (homologous to the type B partitioning proteins binding to the partitioning sites) in an electrophoretic mobility shift assay. The protein bound only inc2, forming two complexes in a protein concentration-dependent manner. The inc2 region contains two long (14-bp) repeated sequences (R1 and R2). Disruption of these sequences completely eliminates RepB binding ability. R1 and R2 have sequence similarities with analogous repeats of another repABC replicon of plasmid pPAN1 of Paracoccus pantotrophus DSM 82.5 and with centromeric sequences of the Bacillus subtilis chromosome. Excess RepB protein resulted in destabilization of the inc2-containing plasmid in Escherichia coli. On the other hand, the inc2 region could stabilize another unstable replicon in P. versutus when RepA and RepB were delivered in trans, proving that this region has centromere-like activity. Thus, it was demonstrated that repA, repB, and inc2 constitute a functional system for active partitioning of pTAV320.