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. Received: 12 October 1998 / Accepted: 28 February 1999     

The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding

Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB_V superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn²⁺ or Mg²⁺ cations in a dosage-dependent manner. However, whereas Mn²⁺ strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg²⁺. Furthermore, MobMN199 exhibited a high affinity binding for Mn²⁺ but not for Mg²⁺. We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26 nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein–DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn²⁺ during pMV158 transfer.     

Features of the plasmid pMV158-encoded MobM, a protein involved in its mobilization

The streptococcal promiscuous plasmid pMV158 can be mobilized between a number of bacterial species by means of three elements: (i) the plasmid-encoded nicking-closing protein MobM, involved in the initiation and termination of the conjugative transfer; (ii) the DNA sequence where the MobM-mediated nick takes place (the oriT(pMV158)); and (iii) the function(s) provided by auxiliary plasmids. MobM belongs to the Pre/Mob family of plasmid-encoded DNA-relaxing proteins (relaxases). Purified MobM protein has been used to assay cleavage conditions on plasmid supercoiled DNA. Some structural features of MobM have been addressed by analytical ultracentrifugation, circular dichroism, thermal denaturation, and fluorescence emission. The protein behaved as a dimer of identical subunits with an ellipsoidal shape. MobM showed a high (about 60%) alpha-helical content and a midpoint denaturation of about 40 degrees C. Cell fractionation assays showed that MobM was associated to the cell membrane. This association was abolished when a great alteration was introduced within a putative coiled-coil located at the C-terminal region of the protein. Emission fluorescence suggested that the three Trp residues of MobM are located within a hydrophobic environment. A molecular model of MobM on the known structure of colicin Ia has been built.     

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     

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.     

Relaxase DNA Binding and Cleavage Are Two Distinguishable Steps in Conjugative DNA Processing That Involve Different Sequence Elements of the nic Site

TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR₂). Mutation analysis in the nic region indicated that recognition of the IR₂ proximal arm and the nucleotides located between IR₂ and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR₂ cruciform and recognition of the distal IR₂ arm and loop were not necessary for these reactions to take place. On the other hand, IR₂ was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR₂-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.     

Lagging-Strand Replication from the ssoA Origin of Plasmid pMV158 in Streptococcus pneumoniae: In Vivo and In Vitro Influences of Mutations in Two Conserved ssoA Regions

The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RS_B] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication in Streptococcus pneumoniae. Cells containing plasmids with mutations in the RS_B accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RS_B region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.     

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.     

Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin.

The 4.2-kb cryptic plasmid pVA380-1 has been used as a vector for the cloning of antibiotic resistance genes directly in streptococci, and in the construction of Escherichia coli/Streptococcus shuttle vectors. The results of subcloning experiments located the basic replicon of pVA380-1 within a 2.5-kb region. The nucleotide base sequence of this region was determined and contained a single complete open reading frame (ORF) encoding a 237-amino-acid peptide with a predicted size of 29 kDa. This peptide and a region of the DNA molecule 5' to the ORF encoding it shared homology with the replication protein and plus origin, respectively, of the Staphylococcus aureus plasmid pUB110. Data from Tn5 mutagenesis and complementation studies indicated that the protein product of the ORF was required for pVA380-1 replication in streptococci. Deletion of a region of the basic replicon distal to the plus origin and ORF produced an unstable derivative, and resulted in the accumulation of single-stranded replicative intermediates, consistent with the loss of a minus origin. All of these results suggest that pVA380-1 replicates by a rolling circle mode, and is most closely related to the pC194 family of single-stranded DNA plasmids.     

The Bacteroides fragilis BtgA Mobilization Protein Binds to the oriT Region of pBFTM10

The Bacteroides fragilis conjugal plasmid pBFTM10 contains two genes, btgA and btgB, and a putative oriT region necessary for transfer in Bacteroides fragilis and Escherichia coli. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of btgA revealed three sets of inverted repeats, potentially locating the oriT region. A 304-bp DNA fragment comprising this putative oriT region was cloned and confirmed to be the functional pBFTM10 oriT by bacterial conjugation experiments using E. coli and B. fragilis. btgA was cloned and overexpressed in E. coli, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognate oriT. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to the oriT-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.     

Characterisation of an in vivo system for nicking at the origin of conjugal DNA transfer of the sex factor F

Transfer of the F plasmid between conjugating Escherichia coli cells has been assumed to require endonucleolytic cleavage at a specific site (oriT) on a specific strand of the F molecule. Using a lambda transducing phage which contains oriT we have detected this nicking process in vivo. Nicking of DNA occurred in the strand that included the “transferred” F strand and at a location within the transducing segment consistent with all previous genetic and restriction enzyme cleavage data on the position of oriT in F. Genetic study of the nicking process using Flac tra^? point and deletion mutants, and also λtra phages which carried various parts of the transfer region, indicated that the products of two transfer operon genes, traY and the previously unidentified gene traZ, were directly involved in nicking at oriT. The product of traJ was also required for nicking, but the possibility that this was solely due to the regulatory function of the traJ product could not be excluded. The plasmid specificities of oriT, traY and traZ between F and the related F-like plasmids R1-19 and R100-1 were investigated using the λoriT nicking system, and shown to be consistent with those determined in genetic complementation tests. The differences in specificity observed imply that the oriT sequence of F differs from those of R1-19 and R100-1.The products of the traM and traI genes are known to be required for the initiation of DNA transfer; their possible roles in modulating the activity of the traY Z endonuclease are discussed.     

Streptococcal R plasmid pIP501: endonuclease site map, resistance determinant location, and construction of novel derivatives

The streptococcal resistance plasmid pIP501 (30 kilobase pairs [kb]) encodes resistance to chloramphenicol (Cmr) and erythromycin (Emr) and is capable of conjugative transfer among numerous streptococcal species. By using a streptococcal host-vector recombinant DNA system, the Cmr and Emr determinants of pIP501 were localized to 6.3-kb HindIII and 2.1-kb HindIII-AvaI fragments, respectively. pIP501 was lost at a frequency of 22% in Streptococcus sanguis cells grown at 42 degrees C but was stable in cells grown at 37 degrees C (less than 1% frequency of loss). Sequences from a cryptic multicopy plasmid, pVA380-1, were substituted for the pIP501 Emr determinant in vitro, and the resulting recombinant plasmid, designated pVA797, was recovered in transformed S. sanguis cells. The replication of pVA797 was governed by the pVA380-1 sequences based on temperature-stable replication and incompatibility with pVA380-1-derived replicons. The self-ligation of partially cleaved HindIII pIP501 DNA fragments allowed the localization of a pIP501 region involved in autonomous plasmid replication. A small pIP501 derivative (pVA798) obtained from this experiment had a greatly increased copy number but was unstably inherited. Our data indicate that the sequences encoding the resistance determinants and some of the plasmid replication machinery are relatively clustered on the pIP501 molecule. The properties of pVA797 and pVA798 indicate that these molecules will enhance current streptococcal genetic systems from the standpoint of conjugative mobilization (pVA797) and gene amplification (pVA798).     

Helper plasmid cloning in Streptococcus sanguis: cloning of a tetracycline resistance determinant from the Streptococcus mutans chromosome

A model system for testing the helper plasmid cloning system of Gryczan et al. (Mol. Gen. Genet. 177:459-467, 1980) was devised for the Streptococcus sanguis (Challis) host-vector system. In this system, linearized pVA736 plasmid efficiently transformed an S. sanguis (Challis) host containing a homologous plasmid, pVA380-1, but did not transform a plasmidless host or a host containing a nonhomologous plasmid, pVA380. In addition, whereas monomeric circular pVA736 transformed a plasmidless host with two-hit kinetics, it transformed a pVA380-1-containing host with one-hit kinetics. This helper plasmid cloning system was used to isolate two HindIII fragments (5.0 megadaltons [Mdal] and 1.9 Mdal in size) from the chromosome of Streptococcus mutans V825 which conferred high-level tetracycline resistance. One tetracycline-resistant clone was examined and found to contain three plasmids which were sized and designated pVA868 (9.0 Mdal), pVA869 (9.5 Mdal), and pVA870 (9.8 Mdal). Results of Southern blot hybridization and restriction endonuclease digestion confirmed that all three chimeras were composed of two HindIII fragments of the S. mutans V825 chromosome, as well as a large portion, varying in size for each chimera, of the 2.8 Mdal cloning vector, pVA380-1. Incompatibility observed between pVA380-1 and each of the chimeras indicated that replication of the chimeras was governed by the pVA380-1 replicative origin. Southern blotting experiments revealed that the chimeras hybridized to Tn916, providing the first evidence that transposon-related genes of enteric streptococcal origin are disseminated among oral streptococci.     

Plasmid R6K Contains Two FunctionaloriTswhich can Assemble Simultaneously in Relaxosomesin vivo

Plasmid R6K contains two functional origins or transfer (oriT), in contrast to previously characterized conjugative plasmids. TheoriTsare formed by 98 bp palindromic sequences invertedly orientated with respect to each other and located in the immediate vicinity of the α and β origins of replication. The gene for R6KoriT-nickase,taxC, was identified by transposon mutagenesis and sequenced, revealing that TaxC belongs to the VirD2 nickase family. The protein was overproduced and purified. It catalysed a cleaving-joining reaction on single-stranded DNA containing its target sequence. Identification of thenicsites suggested that the R6KoriTsbelong to the RP4/VirD2oriTfamily. Cleavage was highly specific and did not occur with oligonucleotides cleaved by related nickases like TraI of RP4 or VirD2 of the Ti plasmid.niccleavage ofin vivopre- assembled relaxation complexes was induced by incubation of plasmid cleared lysates with ethidium bromide. Nicked molecules obtained in this way were treated with snake venom phosphodiesterase to produce double strand cleavages at thenicsites. 35% of the molecules were cleaved simultaneously at bothnicsites, both in the case of R6K and of R6Kdrd1, a derepressed mutant whose frequency of transfer is 1000-fold higher. This figure represents the minimum percentage of individual R6K molecules containing two pre-assembled relaxation complexes.     

Characterization of Multiple Regions Involved in Replication and Mobilization of Plasmid pNZ4000 Coding for Exopolysaccharide Production in Lactococcus lactis

We characterized the regions involved in replication and mobilization of the 40-kb plasmid pNZ4000, encoding exopolysaccharide (EPS) production in Lactococcus lactis NIZO B40. The plasmid contains four highly conserved replication regions with homologous rep genes (repB1, repB2, repB3, and repB4) that belong to the lactococcal theta replicon family. Subcloning of each replicon individually showed that all are functional and compatible in L. lactis. Plasmid pNZ4000 and genetically labeled derivatives could be transferred to different L. lactis strains by conjugation, and pNZ4000 was shown to be a mobilization plasmid. Two regions involved in mobilization were identified near two of the replicons; both included an oriT sequence rich in inverted repeats. Conjugative mobilization of the nonmobilizable plasmid pNZ124 was promoted by either one of these oriT sequences, demonstrating their functionality. One oriT sequence was followed by a mobA gene, coding for a trans-acting protein, which increased the frequency of conjugative transfer 100-fold. The predicted MobA protein and the oriT sequences show protein and nucleotide similarity, respectively, with the relaxase and with the inverted repeat and nic site of the oriT from the Escherichia coli plasmid R64. The presence on pNZ4000 of four functional replicons, two oriT sequences, and several insertion sequence-like elements strongly suggests that this EPS plasmid is a naturally occurring cointegrate.     

植物乳杆菌PC518质粒pLP224的发现以及pMV158家族质粒的保守性和多样性分析

【背景】植物乳杆菌含有丰富的天然质粒,分析这些质粒的序列特征有利于分析质粒所携带的遗传信息。【目的】分析从植物乳杆菌PC518分离的新质粒pLP224,聚类分析其所属家族质粒的保守性与多样性。【方法】提取植物乳杆菌PC518的质粒,酶切后构建质粒DNA文库,测序和BLAST鉴定文库中的新序列;通过反向PCR完成质粒全序列测定,注释新质粒;使用进化树软件MEGA X构建质粒的Rep蛋白进化树,并分析结合序列的变化。【结果】从植物乳杆菌PC518分离出一个质粒pLP224,大小为1766bp,其中(G+C)mol%含量为41.39%,与已知质粒的最大序列相似性为86.85%。推定其复制方式为滚环复制,属于pMV158家族成员。17个pMV158家族质粒的Rep蛋白分析表明:pMV158家族质粒的Rep蛋白进化距离越近,其dso位点的结合序列相似性越高,进化距离越远则其序列相似性越低。【结论】pLP224是pMV158家族的新成员。pMV158家族质粒在dso位点的切开序列上保守,在结合序列上多样。其Rep蛋白随结合序列变化而不同。这种差异有利于pMV158家族不同成员在同一宿主的共存,是家...     

Region of the streptococcal plasmid pMV158 required for conjugative mobilization.

The nonconjugative streptococcal plasmid pMV158 can be mobilized by the conjugative streptococcal plasmid pIP501. We determined the sequence of the 1.1-kilobase EcoRI fragment of pMV158 to complete the DNA sequence of the plasmid. We showed that an open reading frame, mob (able to encode a polypeptide of 58,020 daltons), is required for mobilization of pMV158. An intergenic region present in the EcoRI fragment contains four lengthy palindromes that are found also in one or more of the staphylococcal plasmids pT181, pE194, and pUB110. One palindromic sequence, palD, which is common to all four plasmids, also appeared to be necessary for mobilization. Circumstantial evidence indicates that this sequence contains both an oriT site and the mob promoter. The Mob protein is homologous in its amino-terminal half to Pre proteins encoded by pT181 and pE194 that were shown by others to be essential for site-specific cointegrative plasmid recombination; their main biological function may be plasmid mobilization.