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.     

Construction of the mobilizable plasmid pMV158GFP,a derivative of pMV158 that carries the gene encoding the green fluorescent protein

Plasmid pMV158 has been employed to construct cloning non-mobilizable vectors for various Gram-positive organisms. Here we report the construction of a mobilizable pMV158-based plasmid that harbors the gene encoding the green fluorescent protein under the control of a promoter inducible by maltose. The plasmid was mobilized between strains of Streptococcus pneumoniae as well as from S. pneumoniae to Lactococcus lactis or Enterococcus faecalis at the same frequency as its parental. Transconjugant that received the GFP-tagged plasmid could be detected by their fluorescence, which was especially high in E. faecalis cells.     

Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lactis subsp. lactis.

pMV158, a non-self-transmissible plasmid encoding tetracycline resistance, was conjugally transferred from Enterococcus faecalis JH203 to Lactococcus lactis subsp. lactis IL1403. This transfer appeared to be dependent on the cotransfer of the conjugative plasmids pAM beta 1 or pIP501. Intraspecies conjugal transfer of pMV158 also occurred in strain IL1403. In contrast to the transfer from E. faecalis, transfer in IL1403 did not require the presence of a conjugative plasmid in the donor strain but, rather, appeared to be dependent on putative chromosomal functions in strain IL1403. The transfer of pMV158 from strain IL1403 required the presence of an active pMV158-encoded protein, which showed homology to the Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from Staphylococcus aureus, such as pT181.     

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.     

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...     

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.     

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.     

Transfer of plasmids by conjugation in Streptococcus pneumonias

Transfer of resistance plasmids occurred by conjugation in Streptococcus pneumoniae (pneumococcus) similarly to the process in other streptococcal groups. The 20-megadalton plasmid pIP501 mediated its own DNase-resistant transfer by filter mating and mobilized the 3.6-megadalton non-self-transmissible pMV158. Pneumococcal strains acted as donors or as recipients for intraspecies transfers and for interspecific transfers with Streptococcus faecalis. Transconjugants contained the plasmids expected from their phenotypes and acted as donors for further transfers. Deficiency in an endonuclease essential for entry of transforming DNA did not affect the frequency of transfer. Transfer-deficient mutants of pIP501 have been found.     

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     

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.     

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.     

Similarity of minus origins of replication and flanking open reading frames of plasmids pUB110, pTB913 and pMV158.

Plasmids pMV158 and pTB913, originating from Streptococcus agalactiae and a thermophilic Bacillus respectively, were sequenced to completion. Both contained a BA3-type minus origin of replication and an RSA-site, believed to constitute a site-specific recombination site. These two regions were more than 99% homologous to the corresponding regions of the Staphylococcus aureus plasmid pUB110. Deleting the BA3-type minus origin resulted in the accumulation of a considerable amount of single-stranded DNA, both in L. lactis subsp. lactis and B. subtilis, indicating that this minus origin was functional in both bacterial species. Like pUB110, both plasmids contained an open reading frame encoding a putative plasmid recombination enzyme (Pre protein), which was located downstream of the RSA-site. On the basis of sequence comparisons between pUB110, pMV158, pTB913, pT181, pE194, pNE131 and pT48 two distinct families of RSA-sites and Pre proteins could be distinguished.     

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.     

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     

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.     

Exogenous isolation of conjugative plasmids from pesticide contaminated soil

Exogenous plasmid isolation method was used to assess conjugative plasmids conferring pesticide tolerance/multiple metal and antibiotic resistance from contaminated soil using bacteria detached from soil samples as a donor and rifampicin resistant E. coli HMS as a recipient strain on mineral salt agar medium supplemented with γ-HCH, and antibiotics ampicillin, tetracycline, chloramphenicol and kanamycin. Transconjugants were obtained on ampicillin (10?μg/ml) and tetracycline (20?μg/ml) amended MSA plates and frequency of ampicillin and tetracycline resistance gene transfer was 7.2?×?10(-6) and 9.2?×?10(-4) transconjugants/recipient, respectively. PCR typing methods were used to assess the presence of plasmids of the incompatibility groups IncP, IncN, IncW, IncQ and rolling circle plasmids of pMV158 type in DNA derived from transconjugants. All transconjugants were PCR amplified for the detection of Inc group plasmids and rolling circle plasmids of pMV158 family in which TM2, 3, 4, 11 and 12 (tet) transconjugants gave PCR products with the IncP-specific primers for both replication and transfer functions (trfA2 (IncP) and oriT (IncP)), while TM 14 (amp) gave an IncP specific PCR product for the replication gene trfA2 (IncP) only. TM15, 16, 18 and 21 (amp) gave a PCR product for the IncW-specific oriT (IncW). Out of 24 transconjugants, only TM 5 (tet) gave a PCR product with the pMV158 specific primer pair for oriT (RC). Our findings indicate that Inc group plasmids and rolling circle plasmids of pMV158 type may be responsible for transferring multiple antibiotic resistance genes among the bacterial soil community.     

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.