Lagging-Strand DNA Replication Origins Are Required for Conjugal Transfer of the Promiscuous Plasmid pMV158

The promiscuous streptococcal plasmid pMV158 is mobilizable by auxiliary plasmids and replicates by the rolling-circle mechanism in a variety of bacterial hosts. The plasmid has two lagging-strand origins, ssoA and ssoU, involved in the conversion of single-stranded DNA intermediates into double-stranded plasmid DNA during vegetative replication. Transfer of the plasmid also would involve conversion of single-stranded DNA molecules into double-stranded plasmid forms in the recipient cells by conjugative replication. To test whether lagging-strand origins played a role in horizontal transfer, pMV158 derivatives defective in one or in both sso''s were constructed and tested for their ability to colonize new hosts by means of intra- and interspecies mobilization. Whereas either sso supported transfer between strains of Streptococcus pneumoniae, only plasmids that had an intact ssoU could be efficiently mobilized from S. pneumoniae to Enterococcus faecalis. Thus, it appears that ssoU is a critical factor for pMV158 promiscuity and that the presence of a functional sso plays an essential role in plasmid transfer.Conjugation of bacterial plasmids is, together with transposition, the most important source of horizontal gene transfer among bacteria of the same or of different species (42). Conjugation implies the unidirectional transfer of one plasmid DNA strand from a donor to a recipient cell. This is initiated by the activity of a plasmid-encoded protein generically termed relaxase, in a process that resembles replication by the rolling-circle mechanism (13, 29). In the case of numerous, small plasmids (<10 kb) isolated primarily from gram-positive bacteria, two pioneer findings led to the discovery of the rolling-circle mechanism of replication (RCR) plasmids (reviewed in references 20 and 21). First, the strand-specific single-stranded DNA (ssDNA) molecules which act as replication intermediates were identified (41) and, second, the relaxing activity on the supercoiled DNA via the recognition of a specific sequence (the double-strand origin) of the Rep initiator proteins were described (22). Most RCR plasmids are not self-transmissible; instead, they encode not only the Rep topoisomerase-like initiator but also a Mob protein with relaxase activity involved in mobilization mediated by auxiliary plasmids. Such is the case of the promiscuous plasmid pMV158, which can be mobilized between various bacterial species by the pMV158-encoded MobM protein and by helper conjugative plasmids belonging to the Inc18 plasmid family, such as pAMβ1 (15), or even by IncP plasmids, such as RP4 (11). The relaxing activity of MobM on supercoiled DNA of pMV158 and the site of cleavage were first demonstrated in vitro (5, 17), and later the same activity of the MobA protein of the Staphylococcus aureus RCR plasmid pC221 was demonstrated (3, 39).Initiation of transfer, like initiation of RCR, involves cleavage of the phosphodiester bond of a specific dinucleotide on one of the plasmid strands. Cleavage is mediated either by the plasmid-encoded Mob protein at the origin of transfer (oriT) during conjugation or by the plasmid-encoded Rep protein at the dso during replication. In both processes, this initial stage is followed by displacement of the cleaved strand in a unidirectional manner (8, 21, 29, 36). Thus, RCR and conjugal transfer are equivalent processes in the sense that they generate strand-specific ssDNA plasmid intermediates that correspond only to the cleaved strand (9, 16, 41). The ssDNA intermediates are generated in the plasmid host by the activity of the Rep initiator protein (replication) or generated and transferred to the recipient cell (T-DNA) and closed by the Mob relaxase (conjugation), where they are converted into double-stranded plasmid DNA (dsDNA) molecules by lagging-strand synthesis. Replication of the lagging strand is initiated at the single-strand origins (sso) by the host RNA polymerase (RNAP), upon recognition of a specific site on ssDNA and synthesis of a short RNA primer (pRNA). The pRNA is used by DNA polymerase I for limited extension synthesis, followed by replication of the lagging strand by DNA polymerase III (27). Features of the sso include the potential to generate stem-loop structures on ssDNA (9, 16, 41) that can conform a ssDNA promoter, which is inactive in the dsDNA configuration. This kind of promoter was described in the coliphage N4 (18), as recognized by the virion RNAP (4, 14). A different kind of ssDNA promoter, F_rpo, was reported for the Escherichia coli plasmid F and was demonstrated to be used for gene expression and appeared to play a role during plasmid conjugation (34). The presence of ssDNA promoters has also been demonstrated in plasmids pMV158 (27) and ColI-P9 (1, 35). The organization of this kind of promoters showed that they are placed on the DNA strand that is partially complementary to the template strand.The first sso was described in the staphylococcal RCR plasmid pT181, in which a deletion located out of the replicon led to instability, reduction in copy number, and accumulation of ssDNA intermediates (16). Plasmid pMV158 exhibits two sso''s, ssoA and ssoU (23). Two conserved regions were found in the ssoA of pLS1 plasmid (a nonmobilizable pMV158 derivative lacking ssoU): a short region termed recombination site B, RS_B, supposedly involved in plasmid cointegration (16, 38), and a 6-nucleotide (nt) consensus sequence (5′-TAGCGT-3′, termed CS-6). Determination of the roles of these two conserved sites showed that, whereas RS_B was the primary site of RNAP binding (located at the stem of the hairpin), CS-6 was the termination site for the synthesis of a 20-nt pRNA in the loop of the hairpin (27). The predicted intrastrand pairings in the pMV158-ssoA showed the presence of an ssDNA promoter in the vicinity of the RS_B, which would have a consensus −35 region (5′-TTGACA-3′) but a weak −10 region (5′-TAcgcT-3′). With this situation, RNA synthesis should start and proceed in the direction toward the binding site of RNAP, being thus opposite to RNA synthesis from classic promoters (27) (see Fig. ​Fig.1A).1A). Sites homologous to RS_B and CS-6 were later observed in pMV158-ssoU (24).Open in a separate windowFIG. 1.Features of pMV158 and its two lagging-strand origins. (A) Schematic map of the plasmid transfer module indicating relevant restriction sites and the relative positions (shadowed) of the two lagging-strand origins of replication (ssoA and ssoU). Plasmid-encoded MobM protein (arrow below the map) and the position of the oriT are depicted. Direction of DNA transfer is indicated. The EcoRI fragment deleted to construct the pLS1 derivative (28) and the positions of primers used are also shown. A representation of the secondary structures of ssoA (left) and of ssoU (right) indicates the positions of the CS-6 and the RS_B regions (boxed). The locations of the G3 and G7 mutations in the ssoA and of the restriction sites used to generate deletions in the ssoU are shown. The start point and direction for the RNA primer (pRNA) synthesis, downstream to CS-6 sequence, is indicated by a wavy arrow. (B) Relevant sequence features of the two pMV158 sso. The RS_B and CS-6 sequences are shown in boxes. The restriction sites of BsaI and DraI used to generate ssoU-ΔBD mutant are also indicated, as well as the nucleotides changed (boldface) to construct the ssoA-G3G7 mutant (sequence indicated beneath).In the present study we have addressed the question of whether and, eventually, which of the two pMV158-ssos plays a role in conjugal transfer. With this objective, we constructed pMV158-derivatives defective in one or both sso''s and tested their role on intra- and interspecies mobilization. Whereas either sso supported transfer between strains of Streptococcus pneumoniae with the same efficiency as the parental pMV158, only the ssoU could do so when conjugal transfer was assayed between S. pneumoniae and Enterococcus faecalis. Our findings show that the functionality of ssoU is a critical factor in the colonization of a broad range of gram-positive bacteria for the pMV158 promiscuous plasmid and demonstrate that efficient transfer and replication in enterococci depend upon a functional ssoU. We suggest that sso''s lacking functionality for vegetative replication in a specific host should not be efficient in conjugative transfer and vice versa, since both events are mechanistically identical. As far as we know, this is the first report that shows the effect of sso functionality on horizontal gene transfer by plasmid conjugation, as well as the efficiency of the ssoA and ssoU in E. faecalis.     

Fitness of the pMV158 replicon in Streptococcus pneumoniae

Promiscuous, rolling-circle replication plasmid pMV158 determines tetracycline resistance to its host and can be mobilized by conjugation. Plasmid pLS1 is a deletion derivative of pMV158 that has lost its conjugative mobilization ability. Both plasmids replicate efficiently and are stably inherited in Streptococcus pneumoniae. We have analyzed the effect of pMV158 and pLS1 carriage on the bacterial growth rate. Whereas the parental plasmid does not significantly modify the cell doubling time, pLS1 slows down the growth of the bacterial host by 8-9%. The bases of the differential burden caused by pMV158 and pLS1 carriage are not yet understood. The negligible cost of the pMV158 parental natural plasmid on the host might explain the prevalence of small, multicopy, rolling-circle replication plasmids, even though they lack any selectable trait.     

Direction of transcription affects the replication mode of lambda in an in vitro system

Summary pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably mantained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30° C. The optimal concentration of template DNA was 40 g/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its serivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.     

In vivo definition of the functional origin of leading strand replication on the lactococcal plasmid pFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose prototype is the streptococcal plasmid pMV158, that replicates by the rolling circle mechanism. Determination of the nucleotide sequence of the repX gene of pFX2 allowed us to make some minor corrections in the published sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia coli and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 and the pMV158 derivative pLS1 exhibit a moderate degree of incompatibility in S. pneumoniae. Cloning of the double strand origin (dso) of pFX2 into a high-copy-number plasmid that is compatible with the pMV158 replicon led to an increase in incompatibility toward pLS1. Plasmids pFX2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the observed incompatibility indicates that cross-recognition of Rep proteins and dso takes place. Received: 25 May 1998 / Accepted: 8 July 1998     

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RS_R.     

Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids.

Staphylococcus aureus plasmid pE194 manifests a natural thermosensitivity for replication and can be established in several species, both gram positive and gram negative, thus making it attractive for use as a delivery vector. Like most characterized plasmids of gram-positive bacteria, pE194 generates single-stranded DNA. The direction of pE194 replication is clockwise, as determined by the strandedness of free single-stranded DNA. Significant homology exists between a 50-base-pair sequence in the origin of pE194 and sequences present in plasmids pMV158 (Streptococcus agalactiae), pADB201 (Mycoplasma mycoides), and pSH71 (Lactococcus lactis). We used an initiation-termination reaction, in which pE194 initiates replication at its own origin and is induced to terminate at the related pMV158 sequence, to demonstrate that pE194 replicates by a rolling-circle mechanism; the initiation nick site was localized to an 8-base-pair sequence.     

Localization of the start sites of lagging-strand replication of rolling-circle plasmids from Gram-positive bacteria

A number of small, multicopy plasmids from Gram-positive bacteria replicate by an asymmetric rolling-circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence, SSO_A, that acts as the single-strand origin (SSO) for the replication of the lagging-strand DNA. Although not all the SSO_A sequences share ONA sequence homology, they are structurally very similar. We have used an in vitro system to study the lagging-strand replication of several plasmids from Gram-positive bacteria using the SSO_A sequences of pT181, pE194 and pSN2 as representative of three different groups of Staphylococcus aureus plasmids. In addition, we have investigated the lagging-strand replication of the pUB110 plasmid that contains an alternative single-strand origin, sso_u. Our results confirm that RNA polymerase is involved in lagging-strand synthesis from both SSO_A and ssou-type lagging-strand origins. Interestingly, while initiation of lagging-strand DNA synthesis of pUB110 occurred predominantly at a single position within sso_u, replication of pT181, pSN2 and pE194 plasmids initiated at multiple positions from SSO_A.     

Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae

A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413bp) has been isolated from the multidrug-resistant clone Poland(23F)-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene (orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.     

Sequence Analysis of a Small Cryptic Plasmid Isolated from Streptococcus suis Serotype 2

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism. Received: 26 June 1999 / Accepted: 10 August 1999     

The roles of mutS, sbcCD and recA in the propagation of TGG repeats in Escherichia coli

A 24 triplet TGG·CCA repeat array shows length- and orientation-dependent propagation when present in the plasmid pUC18. When TGG₂₄ is present as template for leading-strand synthesis, plasmid recovery is normal in all strains tested. However, when it acts as template for lagging-strand synthesis, plasmid propagation is seriously compromised. Plasmids carrying deletions in the 5′ side of this sequence can be isolated and products carrying 15 TGG triplets do not significantly interfere with plasmid propagation. Mutations in sbcCD, mutS and recA significantly improve the recovery of plasmids with TGG₂₄ on the lagging-strand template. These findings suggest that TGG₂₄ can fold into a structure that can interfere with DNA replication in vivo but that TGG₁₅ cannot. Furthermore, since the presence of the MutS and SbcCD proteins are required for propagation interference, it is likely that stabilisation of mismatched base pairs and secondary structure cleavage are implicated. In contrast, there is no correlation of triplet repeat expansion and deletion instability with predicted DNA folding. These results argue for a dissociation of the factors affecting DNA fragility from those affecting trinucleotide repeat expansion–contraction instability.     

A functional lagging strand origin does not stabilize plasmid pMV158 inheritance in Escherichia coli

Plasmid rolling circle replication generates single-stranded DNA intermediates. The intracellular amount of these molecules depends upon the efficiency of the conversion of single-stranded into double-stranded plasmid forms, that is, the functionality of the lagging strand origin (sso). The broad-host-range streptococcal plasmid pMV158 harbors two different ssos, both of which function efficiently in Streptococcus pneumoniae but poorly in Escherichia coli. Plasmid pMV158 is stably inherited in the pneumococcal host, but it is unstable in E. coli. A pMV158 derivative lacking its two ssos is unstable in both strains. We have cloned into this derivative the coliphage f1 lagging strand origin. Whereas the f1 sso was fully functional in E. coli, it did not show any activity in S. pneumoniae, a bacteria closely related to the pMV158 natural host. The presence of the f1 sso did not stabilize pMV158 inheritance in either the gram-positive or the gram-negative host.     

Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1

The plasmid pJB01 (GenBank Accession No. AY425961) isolated from the pathogenic bacterium, Enterococcus faecium JC1, is 2235 base pairs in length and consists of a putative double-strand origin (dso), a single-strand origin, a counter-transcribed RNA, and three open reading frames. A comparison of a few replication factors and motifs, bind and nic regions, for replication initiation on the nucleotide sequence level revealed that it belongs to the pMV158 family among RC-replicating plasmids. A runoff DNA synthesis assay demonstrated that nicking occurred between G525 and A526, which is located on the internal loop of a putative secondary structure in the dso. Unlike all the other plasmids of the pMV158 family having two or three direct repeats, pJB01 has three non-tandem direct repeats of 5'-CAACAAA-3' separated by four nucleotides, as the RepB-binding site in the dso. Moreover, the nick site on the internal loop is located at 77 nucleotides upstream from the RepB-binding region. Irrespective of the structural difference of direct repeats from other members of the pMV158 family, we think, it is still a new member of this plasmid family. The introduction of mutations in conserved regions of RepB confirmed that RepB N-moiety is important for nicking/nick-closing activity. Within N-moiety, especially all of the motif R-III, the Y100 in R-IV and Y116 in R-V residues, played particularly critical roles in this activity, however, for its binding, both of the N- and C-moieties of RepB were needed.     

Rolling-circle replication of bacterial plasmids.

Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication.     

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro.     

Small cryptic plasmids of Streptococcus pneumoniae belong to the pC194/pUB110 family of rolling circle plasmids

The DNA sequences of two related plasmids pPR1 and pPR3 described previously in Streptococcus pneumoniae isolates from Germany and Spain were now determined. Both plasmids belong to a family of rolling circle (RC) plasmids found in a variety of bacteria. Their GC content with 32% is lower than that of the S. pneumoniae chromosomal DNA. The plasmid pPR3 has a molecular size of 3160 bp with four putative open reading frames, whereas pPR1 contained a deletion of 313 bp that included the 5′-part of ORF2 and upstream regions and differed by three bp from pPR3. The predicted protein of ORF1 showed high similarity to replication proteins of RC plasmids with 74% identical amino acids to RepA of Streptococcus thermophilus plasmids. Sequences similar to the plus origin of replication of ssDNA plasmids were present in both plasmids. They also contained a 152-bp region with over 83% identity to the minus origin of replication of the Streptococcus agalacticae plasmid pMV158.