Plasmid pGA1 from Corynebacterium glutamicum codes for a gene product that positively influences plasmid copy number.

The complete nucleotide sequence (4,826 bp) of the cryptic plasmid pGA1 from Corynebacterium glutamicum was determined. DNA sequence analysis revealed four putative coding regions (open reading frame A [ORFA], ORFA2, ORFB, and ORFC). ORFC was identified as a rep gene coding for an initiator of plasmid replication (Rep) according to the high level of homology of its deduced amino acid sequence with the Rep proteins of plasmids pSR1 (from C. glutamicum) and pNG2 (from Corynebacterium diphtheriae). This function was confirmed by deletion mapping of the minimal replicon of pGA1 (1.7 kb) which contains only ORFC. Deletion derivatives of pGA1 devoid of ORFA exhibited significant decreases in the copy number in C. glutamicum cells and displayed segregational instability. Introduction of ORFA in trans into the cells harboring these deletion plasmids dramatically increased their copy number and segregational stability. The ORFA gene product thus positively influences plasmid copy number. This is the first report on such activity associated with a nonintegrating bacterial plasmid. The related plasmids pGA1, pSR1, and pNG2 lacking significant homology with any other plasmid seem to be representatives of a new group of plasmids replicating in the rolling-circle mode.     

R-loop-dependent rolling-circle replication and a new model for DNA concatemer resolution by mitochondrial plasmid mp1

The mitochondrial (mt) plasmid mp1 of Chenopodium album replicates by a rolling-circle (RC) mechanism initiated at two double-stranded replication origins (dso1 and dso2). Two-dimensional gel electrophoresis and electron microscopy of early mp1 replication intermediates revealed novel spots. Ribonucleotide (R)-loops were identified at dso1, which function as a precursor for the RCs in vivo and in vitro. Bacteriophage T4-like networks of highly branched mp1 concatemers with up to 20 monomer units were mapped and shown to be mainly formed by replicating, invading, recombining and resolving molecules. A new model is proposed in which concatemers were separated into single units by a "snap-back" mechanism and homologous recombination. dso1 is a recombination hotspot, with sequence homology to bacterial Xer recombination cores. mp1 is a unique eukaryotic plasmid that expresses features of phages like T4 and could serve as a model system for replication and maintenance of DNA concatemers.     

Characterization of the cryptic plasmid pCC1 from Corynebacterium callunae and its use for vector construction

The complete nucleotide sequence of the cryptic plasmid pCC1 from Corynebacterium callunae (4109 bp) was determined. DNA sequence analysis revealed five open reading frames longer than 200 bp. One of the deduced polypeptides showed homology with the Rep proteins encoded by plasmids of the pIJ101/pJV1 family of plasmids replicating by the rolling-circle (RC) mechanism. Within this plasmid family, the Rep protein of pCC1 showed the highest degree of similarity to the Rep proteins of corynebacterial plasmids pAG3 and pBL1. These data suggest that the plasmid pCC1 replicates by the RC mechanism. The Escherichia coli/Corynebacterium glutamicum shuttle cloning vector pSCCD1, carrying the pCC1 rep gene on the 2.1-kb DNA fragment and the streptomycin/spectinomycin resistance determinant, was constructed. This vector is stably maintained in population of C. glutamicum cells grown in the absence of selection pressure and it is compatible with plasmid vectors based on corynebacterial plasmids pBL1 and pSR1.     

Characterization of pGA1, a new plasmid from Corynebacterium glutamicum LP-6.

A new plasmid, pGA1, has been isolated from Corynebacterium glutamicum LP-6, and its detailed restriction map has been prepared. The 4.9-kb plasmid has a G + C content of 57%. It replicates in C. glutamicum ATCC13032 and is compatible with the three other plasmids, pCC1, pBL1 and pHM1519, commonly used for vector construction for amino acid-producing corynebacteria. Fusions of pGA1 with different Escherichia coli replicons (transferred from E. coli to Corynebacterium via transformation of spheroplasts or by filter mating experiments with intact cells) are shown to be suitable as shuttle plasmids; some of them are highly stable in C. glutamicum, even when propagated without any selection pressure.     

Molecular analysis of pDL10 from Acidianus ambivalens reveals a family of related plasmids from extremely thermophilic and acidophilic archaea.

The 7598-bp plasmid pDL10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic Archaeon Acidianus ambivalens was sequenced. It contains 10 open reading frames (ORFs) organized in five putative operons. The deduced amino acid sequence of the largest ORF (909 aa) showed similarity to bacterial Rep proteins known from phages and plasmids with rolling-circle (RC) replication. From the comparison of the amino acid sequences, a novel family of RC Rep proteins was defined. The pDL10 Rep protein shared 45-80% identical residues with homologous protein genes encoded by the Sulfolobus islandicus plasmids pRN1 and pRN2. Two DNA regions capable of forming extended stem-loop structures were also conserved in the three plasmids (48-69% sequence identity). In addition, a putative plasmid regulatory protein gene (plrA) was found, which was conserved among the three plasmids and the conjugative Sulfolobus plasmid pNOB8. A homolog of this gene was also found in the chromosome of S. solfataricus. Single-stranded DNA of both pDL10 strands was detected with a mung bean nuclease protection assay using PCR detection of protected fragments, giving additional evidence for an RC mechanism of replication.     

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.     

Unique features of the mitochondrial rolling circle-plasmid mp1 from the higher plant Chenopodium album (L.).

We analyzed the structure and replication of the mitochondrial (mt) circular DNA plasmid mp1 (1309 bp) from the higher plant Chenopodium album(L.). Two dimensional gel electrophoresis (2DE) revealed the existence of oligomers of up to a decamer in addition to the prevailing monomeric form. The migration behavior of cut replication intermediates during 2DE was consistent with a rolling circle (RC) type of replication. We detected entirely single-stranded (ss) plasmid copies hybridizing only with one of the two DNA strands. This result indicates the occurence of an asymmetric RC replication mechanism. mp1 has, with respect to its replication, some unique features compared with bacterial RC plasmids. We identified and localized a strand-specific nicking site (origin of RC replication) on the plasmid by primer extension studies. Nicks in the plasmid were found to occur at any one of six nucleotides (TAAG/GG) around position 735 of the leading strand. This sequence shows no homology to origin motifs from known bacterial RC replicons. mp1 is the first described RC plasmid in a higher plant.     

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...     

Characterization of putative rolling-circle plasmids from the Gram-negative bacterium Xylella fastidiosa and their use as shuttle vectors

This study was initiated to characterize a small Xylella fastidiosa (X. fastidiosa) plasmid and attempt to create a X. fastidosa/Escherichia coli shuttle vector that was stable in planta. Restriction enzyme analysis of a 1.3kb plasmid DNA from a grape-infecting strain of X. fastidiosa (UCLA) revealed the presence of three similar, but genetically distinct, plasmids, pUCLAs. Evidence that suggests the pUCLA plasmids replicate via a rolling-circle (RC) mechanism include: (i) the presence of ssDNA in X. fastidiosa cells; (ii) the presence of conserved motifs in the predicted ORF1 that are typical of initiator (Rep) proteins associated with RC replication; (iii) high amino acid identity between the putative Rep proteins of pUCLAs and Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa that replicates by a RC mechanism; and (iv) the presence of a putative origin of replication upstream of ORF1 that has the potential to form secondary hairpin structures. One DNA motif present in pUCLA shared sequence similarity to known nicking sites in the origins of replication of other RC plasmids and phages. The shuttle vector, pXF001, successfully transformed grape X. fastidiosa strains and was found to be present as autonomous, structurally unchanged DNA molecules in X. fastidiosa. However, pXF001 was not stably maintained in X. fastidiosa without antibiotic selection.     

一株动性球菌ZOYM的初步鉴定及其内源性质粒pPCZ1和pPCZ2的分析

从来自中国深海的样品中分离到一株菌株ZOYM, PCR获得其16S rRNA基因序列, 经比对, 与已发表的动性球菌属(Planococcus)的不同种高度相似。提取质粒DNA, 在琼脂糖凝胶上检测到多条DNA带, 对其中2个小的环型质粒pPCZ1和pPCZ2进行了克隆和测序。 pPCZ1和pPCZ2的全长分别为4738 bp和7935 bp, 编码3个和8个蛋白。预测的pPCZ1和pPCZ2的复制蛋白Rep分别与链球菌(Staphylococcus)质粒pSCFS1和芽孢杆菌(Bacillus) 质粒pBM19的Rep有很高的相似性。此外, pPCZ1和pPCZ2的复制蛋白Rep之间也有一定的同源性。质粒pPCZ1和pPCZ2上均未找到滚环复制质粒保守的dso和sso序列, 而在复制基因的上游均发现有多个重复序列和富含AT的序列, 因此, 推测它们可能不是以滚环方式进行复制, 而是theta方式。这是首次报道动性球菌属两个质粒的完整序列。     

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.     

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.     

Characterization of two small cryptic plasmids from Pseudomonas sp. strain S-47

Two small cryptic plasmids, p47L and p47S, identified in Pseudomonas sp. S-47 were characterized by determination of DNA sequences and physical and functional maps. They are 3084 and 1782 bp in length, respectively, with GC contents of 63.55 and 65.21%. The detection of single-strand DNAs of both plasmids indicates that they replicate by a rolling-circle mechanism. The deduced polypeptide encoded by the rep gene of p47L is homologous with Rep proteins of plasmids belonging to the pIJ101/pJV1 family, which are known to replicate by the rolling-circle mechanism. Despite containing a homologous signature with Rep proteins of rolling-circle replicating (RCR) plasmids in the pT181 family, the Rep of p47S lacks significant homology with Rep proteins of this family and is missing a region similar to the family's replication origin (dso). Based on the rep sequence comparisons, p47L falls into a previously defined plasmid family whereas p47S defines a new family of RCR plasmid.     

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.     

Expression of Kanamycin resistance introduced byAgrobacteriutn binary vector intoNicotiana tabacum andAtropa belladonna

Kanamycin resistance gene was introduced into tobacco and Atropa belladonna cells by binary vectors, based on Agrobacterium, by means of inoculation of seedlings. The plasmid pGA472, which carries chimaeric kanamycin resistance gene expressed in plants was introduced by transformation into A. tumefaciens Bo542, harbouring pTiBo542 plasmid and A. rhizogenes 8196, carrying pRi8196 plasmids and the resulting two strains were used as binary vectors. Tobacco tumors induced by A. tumefaciens Bo542(pGA472) grew as undifferentiated, kanamycin resistant tissues. Those induced by A. rhizogenes 8196(pGA472) differentiated into transformed plants. When cultivated in vitro on 200 μg ml^-1 kanamycin medium, they showed yellow green sectoring, which was not selected out during vegetative propagation. Atropa belladonna tissues transformed by both A. tumefaciens Bo542(pGA472) and A. rhizogenes 8196(pGA472) differentiated plants which grew well on 200 μg ml^-1 kanamycin as green, non-sectoring plants; sensitive cells obviously did not divide at all. Selection of Atropa belladonna transformed tissues on kanamycin medium is much more efficient than selection of transformed tobacco tissues with introduced kanamycin resistance gene.     

Improvement of DNA vaccine immunogenicity by a dual antigen expression system

This study examined whether increased antigen expression resulted in enhanced antigen-specific immune responses in the context of DNA vaccines. To increase antigen expression, two copies of antigen expression cassettes were arranged in a plasmid pDX. BALB/c mice were intramuscularly immunized with various constructs that express influenza antigens and analysed for DNA-raised immunity. The plasmid pDX that expresses two copies of the antigen gene induced stronger antigen-specific immune responses than the plasmid pGA which expresses single antigen gene. To explore the in vivo transgene expression by pDX and pGA, luciferase activity was measured in the muscles transduced with luciferase expression plasmids. The pDX expressing two copies of luciferase induced the highest luciferase activity, which corresponded to the results from vaccination. We concluded that increasing the number of antigen expression cassettes in a vaccine construct improved antigen expression in the transduced tissue, which induced stronger DNA-raised immune responses.