Functional Analysis of Three Plasmids from Lactobacillus plantarum

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.     

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin (dso) and a region with palindromic sequences that could function as single strand origin (sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55-58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.     

Complete nucleotide sequence of plasmid plca36 isolated from Lactobacillus casei Zhang

The complete 36,487 bp sequence of plasmid plca36 from Lactobacillus casei Zhang was determined. Plca36 contains 44 predicted coding regions, and to 23 of them functions could be assigned. For the first time, we identified a relBE toxin-antitoxin (TA) locus in a Lactobacillus genus, perhaps indicating a potential role for plca36 in host survival under extreme nutritional stress. A region encoding a cluster of conjugation genes (tra) was also identified. The cluster showed high similarity and co-linearity with tra regions of pWCFS103 and pMRC01 from Lactobacillus plantarum and Lactococcus lactis, respectively. Comparative gene analysis revealed that plasmids from the genus Lactobacillus may have contributed to the environmental adaptation mainly by providing carbohydrate and amino acid transporters. In addition, two chromosome-encoded relBE systems in Lactobacillus johnsonii and Lactobacillus gasseri were identified.     

植物乳杆菌KLDS1.0728质粒p141的分子分析

对分离自植物乳杆菌KLDS1.0728的质粒p141进行了全序列测定,结果显示,该质粒全长为3597bp,平均G+Cmol%值为38%,并利用DNAMAN6.0软件得到该质粒限制性内切酶图谱。经NCBI网站ORFFinder软件分析确定其编码序列即ORF为15个,通过与公共数据库比对,发现可以识别功能的ORF有2个,其中包括质粒复制所必需的rep基因,p141的rep基因与已知序列的植物乳杆菌WCFS1内源质粒pWCFS101以及植物乳杆菌内源质粒pM4的复制蛋白基因相似性高达91%。根据rep基因的相似性比较,判断p141的复制模式归属于RCR模式的GroupIII组,即pC194家族。另外,还发现质粒p141中存在mob基因,表明该质粒具有水平转移能力。但没有发现Tn4430转座子以及转座酶基因topl和topA,所以可以判断该质粒的基因比较稳定。此外,还发现该质粒序列中存在一定的与质粒复制和转移调控以及蛋白质表达等有关的重复序列,其对调控质粒的拷贝数有一定意义。     

Characterization of a phage resistance plasmid, pLKS, of silage-making Lactobacillus plantarum NGRI0101

Phage contamination has resulted in abnormal fermentation in silage. We isolated a phage-resistant strain, Lactobacillus plantarum NGRI0101 from silage. The strain carried two plasmids, pLKL (6.8 kb) and pLKS (2.0 kb). By curing and retransformation of the plasmids, we clarified that pLKS has phage resistant activity, characterized as no adsorption inhibition. pLKS has 2,025 bp and three orfs, orfl23, orf132, and orf918. The predicted amino acid sequence of the orf918 product showed high similarity to those of Rep proteins of Pediococcus halophilus plasmid pUCL287 and Lactobacillus acidophilus plasmid pLA103. The replication origin (ori) was upstream from orf918. There was no gene similar to typical phage resistant genes encoded by known plasmids. The phage resistance of L. plantarum NGRI0101 may possibly be due to a plasmid-encoded abortive infection.     

Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.     

Characterization of endogenous plasmids from Lactobacillus salivarius UCC118

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.     

Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4

A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.     

Genetic analysis of a novel plasmid pZMX101 from Halorubrum saccharovorum: determination of the minimal replicon and comparison with the related haloarchaeal plasmid pSCM201

The DNA sequence of a novel haloarchaeal plasmid pZMX101 (3918 bp) from Halorubrum saccharovorum was determined and six ORFs were predicted. The largest ORF encodes a putative replication initiation protein RepA, which shares 40% sequence similarity with the Rep201 of a theta-replication plasmid pSCM201 recently isolated from Haloarcula, suggesting that pZMX101 might replicate via a theta-type mechanism. Using pZMX101 as the only haloarchaeal replicon, a shuttle vector pZMX108 was constructed and successfully transformed into Haloferax volcanii DS70. Based on this in vivo system, the minimal replicon (1978 bp) of pZMX101 was determined. It is composed of the repA gene plus c. 400-bp upstream and 300-bp downstream sequences. Significantly, the putative replication origin of pZMX101 and that of pSCM201 contain different types of sequence motifs, and these two plasmids exhibit distinct host preference for Haloferax and Haloarcula, respectively.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.     

Genetic analysis of the inter-relationship between plasmid replication and incompatibility

Summary The relationship between replication control and plasmid incompatibility has been investigated using a composite replicon, pPM1, which consists of the pSC101 plasmid ligated to another small multicopy plasmid, RSF1050. Since pPM1 can utilise the replication system of either of the two functionally distinct components, propagation of the composite plasmid can occur in the presence of a mutation of one of its moieties. Such mutants are detected by their inability to rescue the composite plasmid under conditions not permissive for replication of the other moiety. Mutations in incompatibility functions can be detected by the failure of the composite replicon to exclude co-existing plasmids carrying a replication system identical to the one on pPM1.The inability of the composite plasmid to replicate at 42° in a host synthesizing temperature-sensitive DNA polymerase I, which is required by the RSF1050 replication system, was used to isolate pPM1 mutants defective in replication of the pSC101 component. Mutants defective in the incompatibility functions of pSC101 were obtained by selecting derivatives that allow the stable coexistence of a second pSC101 replicon in the same cell. Analysis of these two classes of mutants indicates that plasmids selected for defective pSC101 replication ability nevertheless retain pSC101 incompatibility. In contrast, plasmid mutants that have lost incompatibility functions were found always to be defective in replication ability.     

Sequence and analysis of pBM02, a novel RCR cryptic plasmid from Lactococcus lactis subsp cremoris P8-2-47

This paper reports the complete nucleotide sequence of the 3.85 kbp plasmid pBM02 from Lactococcus lactis subsp. cremoris P8-2-47. Analysis of the sequence predicted six ORFs larger than 25 amino acids. They all were transcribed from the same strand and organized in two functional cassettes: the replication region and a putative mobilization region. In the replication region, two ORFs specifying proteins homologous to others found in some classes of rolling circle-replicating plasmids were encountered (copG and repB). In fact, single-stranded DNA was detected as a replication intermediate of pBM02. copG and repB, together with some upstream sequences, formed part of the minimal replication unit of the plasmid. Interestingly, pBM02 shared a 212 bp stretch with plasmids of the pWV01 type, in which the whole single-strand origin of replication is included. In the mobilization region, an ORF coding for a mobilization-like protein was present, preceded by a putative oriT sequence homologous to that of plasmid pMV158. The replicon of pBM02 is of the wide-host range type, and functions in both Gram-positive and Gram-negative bacteria, including Lactobacillus casei, Lactobacillus plantarum, Bacillus subtilis, and Escherichia coli.     

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.     

Conjugal transfer of group B streptococcal plasmids and comobilization of Escherichia coli-Streptococcus shuttle plasmids to Lactobacillus plantarum.

The antibiotic resistance group B streptococcal plasmids, pIP501 and pVA797, were conjugally transferred from Streptococcus faecalis to Lactobacillus plantarum. The Escherichia coli-Streptococcus shuttle plasmids, pVA838 and pSA3, were mobilized from S. sanguis to L. plantarum by pVA797 via cointegrate formation. pVA838 readily resolved from pVA797 and was present in L. plantarum as deletion derivatives. The pVA797::pSA3 cointegrate failed to resolve in L. plantarum.     

Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the L. reuteri population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.     

Influence of Different Functional Elements of Plasmid pGT232 on Maintenance of Recombinant Plasmids in Lactobacillus reuteri Populations In Vitro and In Vivo

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the L. reuteri population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.     

植物乳杆菌编码复制起始蛋白天然质粒的系统进化分析

【目的】探究复制起始蛋白(Replication initiation protein,Rep)是否可以作为天然质粒系统进化关系研究的分子标记。【方法】以植物乳杆菌天然质粒编码的Rep为研究对象,通过构建Rep系统进化树详细分析和讨论这些质粒的系统进化关系。【结果】植物乳杆菌45个编码Rep天然质粒可以划分为5个进化关系紧密的家族和1个独立进化质粒p G6302,家族1-4质粒可以进一步划分为10个进化关系更近的亚家族类群,因此这些质粒可能起源于6个祖先质粒。【结论】Rep氨基酸序列显示了适度的保守性和变异性,是植物乳杆菌编码Rep质粒进化研究理想的分子标记,为植物乳杆菌天然质粒系统进化研究提供了一种简单、有效的分析方法和标准,并为植物乳杆菌或其他乳酸菌天然质粒系统进化研究提供了分子水平的佐证和依据。     

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.     

Lactobacillus plantarum and Lactobacillus buchneri as Expression Systems: Evaluation of Different Origins of Replication for the Design of Suitable Shuttle Vectors

The objectives of this study were to establish transformation protocols for Lactobacillus plantarum CD033 and Lactobacillus buchneri CD034, two industrial silage strains and to test the influence of selected origins of replication on plasmid copy number, plasmid stability, and plasmid incompatibility in these strains. Electro-transformation protocols were optimized by examination of the influence of different electroporation solutions and cell wall weakening agents on transformation efficiency. Using Lithium acetate as cell wall weakening agent, we could achieve transformation efficiencies of 8?×?10(4) transformants per 1?μg DNA for L. buchneri CD034 which is to our knowledge the highest described for this species up to now. In order to test feasibility of previously described origins of replication derived from Bacillus subtilis, L. plantarum, Lactococcus lactis, and two novel L. buchneri CD034 plasmids to drive replication in our two selected Lactobacillus strains, six shuttle vectors were constructed. Results indicate that, in terms of stable propagation and high gene copy numbers (up to 238 copies/chromosome), the most suitable origins of replication for the construction of expression vectors for the selected silage strains were the ones derived from the novel L. buchneri CD034 plasmids.     

Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure

The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.