Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014.

Summary The DNA sequences of a 2.4 kb plasmid (p353-2) from Lactobacillus pentosus MD353 and a 1.9 kb plasmid (p8014-2) from Lactobacillus plantarum ATCC 8014 show 81.5% overall similarity. Both plasmids carry elements (replication protein gene, plus-origin and minus-origin of replication), which are typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR). Direct evidence for an RCR mechanism was obtained by showing the accumulation of single-stranded plasmid intermediates in the presence of rifampicin. A minus-origin of replication was defined for plasmids p353-2 and p8014-2 based on DNA sequence analysis and on its ability to convert single-stranded into double-stranded plasmid DNA. Plasmids pLPE323, pLPE350 and pLPC37 that are derived from the p353-2 or p8014-2 replicon are structurally and segregationally stable in L. pentosus MD353, L. plantarum ATCC 8014 and in Lactobacillus casei ATCC 393. The presence of Escherichia coli or DNA fragments in vectors derived from p353-2 or p8014-2 does not affect the structural stability but results in segregational instability of the vectors. The instability increases with increasing size of the inserted DNA fragment. Since vectors based on these replicons can be efficiently propagated in a wide variety of Lactobacillus species, they are highly suitable for cloning and expression of foreign DNA in Lactobacillus, provided that selective pressure is applied.This paper is dedicated with great appreciation to Dr. Frits Berends on the occasion of his retirement as Head of the Biochemistry Department of the TNO Medical Biological Laboratory     

Complete DNA sequence and analysis of an emerging cryptic plasmid isolated from Yersinia pestis

A 6-kb cryptic plasmid (pYC; 5919 bp) has been recovered from Yersinia pestis isolates originating from regions of Yunnan province in China. The sequence of pYC was determined, and analysis of the sequence has revealed that two of the plasmid DNA regions (ORFs 10 and 11) are similar to the DinJ1 and DinJ2 gene products encoded by Escherichia coli chromosomal DNA. This plasmid is increasingly harbored by Y. pestis isolates recovered from a domestic rodent cycle in the southern regions of the province. Further studies will determine the origin and function of pYC.     

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.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

Sequence analysis and characterization of two cryptic plasmids derived from Lactobacillus buchneri CD034

Lactobacillus buchneri is probably the most beneficial microorganism for efficient preservation of animal feed silages made from grass, maize and other plant material against aerobic spoilage. Its obligatory heterofermentative nature, acid resistance and robustness have drawn attention to this species for applications as silage starter culture as well as for genetic engineering. For the first time, two cryptic plasmids present in the same L. buchneri strain, L. buchneri CD034, were isolated, sequenced and characterized. The larger plasmid, designated pCD034-1 was found to be 3424 bp in length with a G + C content of 38.36%. The smaller plasmid, designated pCD034-2 was found to be 2707 bp in length with a G + C content of 38.60%. On both plasmids we predicted three open reading frames. On pCD034-1, ORF 1 encodes a putative replication protein which shares 99% identity with the RepA protein of a Lactobacillus plantarum derived pC194/pUB110-family plasmid. ORF 2 encodes a putative protein of unknown function. ORF 1 and ORF 2 of pCD034-2 correspond to RepA and RepB proteins similar to those of plasmid pLB4 from L. plantarum. ORF 3 of both plasmids encodes a putative mobilization protein similar to that of the pediococcal plasmid pF8801. Double strand origins, putative single strand origins and typical mobilization start signals were identified. Both plasmids were shown to be maintained at relatively high plasmid copy numbers. Two shuttle vectors carrying the origins of replication of pCD034-1 and pCD034-2 were constructed and used to successfully transform two other species isolated from the same environment. Hence, we consider the two novel L. buchneri plasmids a valuable resource for the generation of shuttle and expression vectors for LAB.     

Complete nucleotide sequence of plasmid pST-III from Lactobacillus plantarum ST-III

The complete nucleotide sequence of the 53,560-bp plasmid pST-III from Lactobacillus plantarum ST-III has been determined. The plasmid contains 42 predicted protein-coding sequences, and the functions of 34 coding sequences could be assigned. Homology analysis for the replication protein and the typical features of the origin of replication suggested that pST-III replicates via the theta-type mechanism. Among the predicted genes, we identified a kdp gene cluster (a high-affinity K(+)-transport system) for the first time in the Lactobacillus genus and a system for osmolyte transport. Analysis of the plasmid-encoded functions and the plasmid-cured experiment showed that the genes of pST-III could serve for the niche adaptations of L. plantarum ST-III and make significant contributions to its viability under hyperosmotic conditions. Furthermore, the relative copy number of pST-III was determined to be 6.79±1.55 copies per cell.     

Characterization and molecular cloning of cryptic plasmids isolated from Lactobacillus casei.

Four small cryptic plasmids were isolated from Lactobacillus casei strains, and restriction endonuclease maps of these plasmids were constructed. Three of the small plasmids (pLZ18C, pLZ19E, and pLZ19F1; 6.4, 4.9, and 4.8 kilobase pairs, respectively) were cloned into Escherichia coli K-12 by using pBR322, pACYC184, and pUC8 as vectors. Two of the plasmids, pLZ18C and pLZ19E, were also cloned into Streptococcus sanguis by using pVA1 as the vector. Hybridization by using nick-translated cloned 32P-labeled L. casei plasmid DNA as the probe revealed that none of the cryptic plasmids had appreciable DNA-DNA homology with the large lactose plasmids found in the L. casei strains, with chromosomal DNAs isolated from these strains. Partial homology was detected among several plasmids isolated from different strains, but not among cryptic plasmids isolated from the same strain.     

The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum

The complete nucleotide sequence of a cryptic plasmid isolated from a Lactobacillus plantarum strain has been determined. The plasmid, designated pC30i1, has a molecular size of 2140 bp and a GC content of 37%. The sequence contains one major open reading frame (ORF R) of 951 bp, encoding a basic polypeptide of 317 amino acids, and a molecular weight of 36,956. ORF R shows extensive sequence similarity with genes coding for replication-associated proteins in a group of gram-positive plasmids known to replicate via single-stranded intermediates (ssDNA plasmids), and a stretch of 9 amino acids in the translation of ORF R closely matches a conserved region in these proteins, as well as the active site of the phi X174 Rep protein. Sequences similar to the ssDNA plasmid origins of replication are also present in the pC30i1 sequence, strengthening the hypothesis that pC30i1 belongs to the ssDNA plasmid family. The other main feature of the pC30i1 sequence is a noncoding region consisting of 14 direct, imperfect repeats of a 17-bp sequence, which may have an incompatibility function.     

Complete genome sequence of the probiotic Lactobacillus plantarum ST-III

Lactobacillus plantarum strain ST-III, a probiotic strain with several functions, was isolated from kimchi. Here we report the complete genome sequence of ST-III and compared it with two published L. plantarum genomes.     

Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations

J.L. RUIZ-BARBA, J.C. PIARD AND R. JIMENEZ-DIAZ. 1991. Plasmid profiles of 35 Lactobacillus plantarum strains isolated from different green olive fermentors were obtained. A large number of plasmids in the CCC form (from 5 to 16) were present in all the tested strains as confirmed by a second dimension electrophoresis of DNA. These plasmids, all of which remain cryptic, ranged from 2.0 to 68 kb in size. Novobiocin, sodium dodecyl sulphate and ethidium bromide were used as plasmid-curing agents but only novobiocin induced loss of extrachromosomal DNA at a high frequency in these strains.     

Nucleotide sequence analysis of pRS2 and pRS3, two small cryptic plasmids from Oenococcus oeni

Nucleotide sequence analysis of two cryptic plasmids, pRS2 (2544 bp) and pRS3 (3948 bp), from Oenococcus oeni revealed the presence in both of three major open reading frames with significant similarity to other small cryptic plasmids from O. oeni. The results suggest that those plasmids could be separated into two subfamilies, one represented by pLo13 and pRS3, the other represented by pOg32, pRS1, and pRS2.     

Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations.

Plasmid profiles of 35 Lactobacillus plantarum strains isolated from different green olive fermentors were obtained. A large number of plasmids in the CCC form (from 5 to 16) were present in all the tested strains as confirmed by a second dimension electrophoresis of DNA. These plasmids, all of which remain cryptic, ranged from 2.0 to 68 kb in size. Novobiocin, sodium dodecyl sulphate and ethidium bromide were used as plasmid-curing agents but only novobiocin induced loss of extrachromosomal DNA at a high frequency in these strains.     

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

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

Complete nucleotide sequence and characterization of a cryptic plasmid from Lactobacillus helveticus subsp. jugurti

A small cryptic plasmid, pLJ1, was isolated from Lactobacillus helveticus subsp. jugurti and was cloned into Escherichia coli HB101 by using pBR329 as a vector. Plasmid pLJ1 was 3,292 base pairs long and had single restriction endonuclease sites for PvuII, KpnI, AvaII, Acci, HindIII, and EcoRI. In a maxicell system, pLJ1 produced a protein of about 41 kilodaltons.     

Complete sequence analysis of two methanotroph-specific repABC-containing plasmids from Methylocystis sp. strain SC2

The complete nucleotide sequences of two large, low-copy-number plasmids of 229.6 kb (pBSC2-1) and 143.5 kb (pBSC2-2) were determined during assembly of the whole-genome shotgun sequences of the methane-oxidizing bacterium Methylocystis sp. strain SC2. The physical existence of the two plasmids in strain SC2 was confirmed by pulsed-field gel electrophoresis followed by Southern hybridization. Both plasmids have a conserved replication module of the repABC system and carry genes involved in their faithful maintenance and conjugation. In addition, they contain genes that might be involved in essential metabolic processes. These include several heavy metal resistance genes and copper transport genes in pBSC2-1 and a complete nitrous oxide reductase operon and a pmoC singleton in pBSC2-2, the latter encoding the PmoC subunit of particulate methane monooxygenase.     

两株母乳源植物乳杆菌的全基因组测序分析

【背景】母乳是一个重要的益生菌筛选库,其中植物乳杆菌是一种用途广泛、适应性强的益生菌。然而不同菌株具有不同的功能,现有的生理生化方法对其潜在益生特性研究十分有限,有必要采用高通量的方法寻找具有种群特异性的优质益生菌。【目的】结合菌株生化特征在全基因组的测序与分析的基础上对两株植物乳杆菌的潜在功能进行预测,并重点找寻与肠液耐受性及细菌素的合成相关的基因,即在基因组的结构上对菌株的表型进行探究。【方法】分离筛选出两株母乳源植物乳杆菌MP55、MP37,并利用Illumina genome analyzer对菌株的全基因组进行测序,采用Prokka软件对细菌基因组进行注释,采用Carbohydrate-active enzymes (CAZy)、Koyto encyclopedia of genes and genomes(KEGG)和Clusters of orthologous genes(COG)数据库对基因组进行功能注释;同时采用Prodigal、RNAmmer等工具对编码序列、核糖体RNA进行预测,并用CGView软件绘制菌株的基因组环形图谱。【结果】通过基因组装得到了两株植物乳杆菌的全基因组信息,植物乳杆菌MP37、MP55基因组大小分别为3 204 421 bp和3 299 180 bp;(G+C)mol%含量分别为44.36%和44.46%;分别包含3 012个和3 101个DNA编码序列,结合菌株生化特征在基因组上找到4个与肠液耐受相关的基因及一段细菌素合成相关基因簇。基因组序列原始数据和拼接结果已提交至"gcMeta"平台。【结论】通过高通量测序分析在基因组水平上揭示了植物乳杆菌MP55、MP37在肠道存活性与抑制病原菌相关的可能机理。植物乳杆菌MP55、MP37是两株潜在的益生菌候选菌株,实验结果为进一步阐明其益生菌特性的功能机制提供了遗传学基础。     

一个植物乳杆菌新质粒的序列分析与归类

[目的]分离鉴定植物乳杆菌PC518的质粒并分析滚环复制p C194家族复制起点特征。[方法]从植物乳杆菌PC518中提取质粒,HindⅢ单酶切后克隆测序,然后用反向PCR方法验证质粒序列的完整性。使用DNAMAN V6. 0软件和MEGA X软件对43个p C194家族质粒的复制起点序列和复制蛋白进行比对分析。[结果]分离得到一个3 325 bp的新质粒p LP325。43个p C194家族质粒复制起点中:24个在nick上、下游均有反向重复序列,12个只在nick上游有反向重复序列,4个只在下游有反向重复序列。复制蛋白的聚类与复制起点中反向重复序列的位置是对应的。[结论]p LP325的复制方式推定为滚环复制,属于p C194家族。p C194家族复制起点的bind以反向重复序列为特征,位于nick上游或下游。