Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.     

溶藻弧菌质粒pVAE259全序列测定与分子生物学特征分析

【目的】获得溶藻弧菌环状质粒pVAE259全序列,分析其分子生物学特征并探索该质粒可能具备的功能。【方法】使用酶切、克隆测序的方法获得pVAE259的全序列,利用软件分析DNA序列和可能的编码蛋白,推测质粒的生物学信息。【结果】pVAE259为闭合环状质粒,全长6,075 bp,GC含量为42.16%。在NCBI中比对发现pVAE259与Vibriosp.41隐蔽性质粒pPS41具有较高的相似性。我们在序列中找到一个oriT位点,另外全序列的4118-5494 bp推测为质粒复制区域。pVAE259中存在7个氨基酸序列长度大于100的开放式阅读框(ORF):ORF1-ORF7。其中ORF1编码蛋白属于释放酶超级家族(Relaxase Super-family)蛋白,在NCBI数据库中它与大肠杆菌(Escherichia coli)的MobA-like蛋白最相似;ORF2编码蛋白属于复制酶超级家族(Replicase Super-family),它与嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)的复制蛋白RepA最相似;ORF5与伸长盐单胞菌(Halomonas elongata)质粒pHE1的转移蛋白MobC相似。【结论】根据上述结果及相关文献分析,pVAE259可能是具有转移能力的质粒,该质粒是否影响宿主菌的表型性状还不清楚。     

Two replication regions in the pJM1 virulence plasmid of the marine pathogen Vibrio anguillarum

Vibrio anguillarum is a fish pathogen that causes vibriosis, a serious hemorrhagic septicemia, in wild and cultured fish. Many serotype O1 strains of this bacterium harbor the 65kb plasmid pJM1 carrying the majority of genes encoding the siderophore anguibactin iron transport system that is one of the most important virulence factors of this bacterium. We previously identified a replication region of the pJM1 plasmid named ori1. In this work we determined that ori1 can replicate in Escherichia coli and that the chromosome-encoded proteins DnaB, DnaC and DnaG are essential for its replication whereas PolI, IHF and DnaA are not required. The copy number of the pJM1 plasmid is 1-2, albeit cloned smaller fragments of the ori1 region replicate with higher copy numbers in V. anguillarum while in E. coli we did not observe an obvious difference of the copy numbers of these constructs which were all high. Furthermore, we were able to delete the ori1 region from the pJM1 plasmid and identified a second replication region in pJM1 that we named ori2. This second replication region is located on ORF25 that is within the trans-acting factor (TAFr) region, and showed that it can only replicate in V. anguillarum.     

小单孢菌40027菌株质粒pJTU112复制区的克隆与序列特性

福堤霉素A产生菌——小单孢菌40027菌株含有两个质粒pJTU101和pJTU 112.[目的]对质粒pJTU 112复制区进行克隆,并对质粒pJTU 112复制区序列进行测定和分析.[方法]克隆质粒pJTU 112的不同DNA片段导入消除质粒的小单孢菌40027菌株,通过复制功能的测定,确定质粒pJTU 112的复制区,并进行测序和生物信息学分析.[结果]质粒pJTU 112的复制区定位在约4.7 kb的SacI-KpnI DNA片段上,测序和生物信息学分析表明:4.7 kb的SacI-KpnI DNA片段包含5个ORFs(open reading frames),其中pJTU 112.1和pJTU 112.2与质粒接合转移有关,pJTU112.3、pJTU112.4和pJTU112.5与质粒复制有关.[结论] 质粒pJTU112的复制区定位在约4.7 kb的SacI-KpnI DNA片段上.     

Significance of Na+ in the fish pathogen, Vibrio anguillarum, under energy depleted condition

Vibrio anguillarum kills various kinds of fish over salinities ranging from seawater to freshwater. In this study, we investigated the role of Na(+) in V. anguillarum, especially under energy-depleted conditions such as in natural seawater. V. angustum S14, which is a typical marine vibrio, was used for comparison. V. anguillarum only required Na(+) for starvation-survival, but in contrast, V. angustum S14 always required Na(+) for both growth and starvation-survival. In marine vibrios, Na(+) is used in the Na(+)-dependent respiratory chain that produces the sodium motive force (SMF) across the cell membrane. It has been considered that marine vibrios always need a SMF produced by Na(+), however in the case of V. anguillarum, the SMF is not required for growth, but becomes more important for starvation-survival.     

Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775.

Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.     

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.     

RpoN gene,RAPD profile,antimicrobial resistance and plasmids of Vibrio anguillarum isolates from vibriosis infected Penaeus monodon

Aim: To evaluate the diversity of Vibrio anguillarum isolates from vibriosis‐infected Penaeus monodon collected from east coast of India. Methods and Results: Thirty‐six V. anguillarum were cultured from specific V. anguillarum medium, further identified using biochemical tests and confirmed by PCR detection of rpoN gene. Randomly amplified polymorphic DNA analysis revealed that in each location, the selected V. anguillarum isolates produced a unique band pattern, indicating that the members of this species are genetically heterogeneous. Antibiotic sensitivity results showed that 85%, 72%, 70%, 58%, 45% and 34% of the isolates were resistant to erythromycin, furazolidone, chloramphenicol, oxolinic acid, ciprofloxacin and nitrofurantoin, respectively. Plasmids were found in 70% of the isolates, and nine different plasmid profiles were observed. Conclusions: Wide ranges of diversity were noted in V. anguillarum isolates collected from P. monodon at different locations of east coast of India. Significance and Impact of the Study: Molecular typing, antibiotic resistance and plasmid profiles of V. anguillarum isolates from shrimp in India enables the prediction of possible risk for diseases in shrimp culture environment and the application of alternative management plans to prevent further spread of antibiotic resistance.     

Characterization of an F18+ enterotoxigenic Escherichia coli strain from post weaning diarrhoea of swine, and of its conjugative virulence plasmid pTC

The enterotoxigenic Escherichia coli (ETEC) strain Ec2173, causing post weaning diarrhoea in swine, harbours six plasmids ranging from 13 to 200 kb in size. The heat stable toxin genes sta, stb and a tetracycline resistance gene were located on a self conjugative 120-kb plasmid, called pTC. In the cloned ColE1 type origin of replication of pTC a deletion was detected compared to other ColE1 replicons affecting the replication modulator gene rom. Epidemiological studies on ETEC isolates showed that pTC-like plasmids are widely distributed among porcine ETEC strains; thus representing an example of co-evolution of antibacterial resistance and virulence in pathogenic E. coli.     

Complete nucleotide sequence of a virulence plasmid of Salmonella enterica serovar Dublin and its phylogenetic relationship to the virulence plasmids of serovars Choleraesuis, Enteritidis and Typhimurium

The complete nucleotide sequence of pOU1113 (pSDVu), one of the two types of virulence plasmids of Salmonella enterica serovar Dublin, was determined. It contained 80 156 bp with 53.8 mol% G+C content. Approximately 70 genes could be discerned. Compared with pSTV, the virulence plasmid of serovar Typhimurium, pOU1113 was shorter owing to a missing region amounting to c. 10 kb; furthermore, except for a unique 10 849-bp region, the nucleotide as well as deduced amino acid sequences of pOU1113 were nearly identical to the corresponding regions of three S. enterica virulence plasmids, namely pSCV (virulence plasmid of Choleraesuis), pSTV and pSEV (virulence plasmids of Enteritidis), confirming their close phylogenetic relationship. Comparative analysis indicated that these virulence plasmids appeared to have descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. From a biological and evolutionary point of view, if the decreasing sizes of pOU1113 and pSCV truly reflect a process in which the virulence plasmid has been shedding unnecessary genes during evolution, our data suggest that some genes in the missing region, such as the pef and tra operons, could have a minimal role in maintaining the survival of the bacteria in their environmental niche.     

Complete sequence and genetic organization of pDTG1, the 83 kilobase naphthalene degradation plasmid from Pseudomonas putida strain NCIB 9816-4

The complete 83,042 bp sequence of the circular naphthalene degradation plasmid pDTG1 from Pseudomonas putida strain NCIB 9816-4 was determined in order to examine the process by which the nah and sal operons may have been compiled and distributed in nature. Eighty-nine open reading frames were predicted using computer analyses, comprising 80.0% of the pDTG1 DNA sequence. The most distinctive feature of the plasmid is the upper and lower naphthalene degradation operons, which occupy 9.5 kb and 13.4 kb regions, respectively, bordered by numerous defective mobile genetic element fragments. Identified on this plasmid were homologues of genes required for large plasmid replication, maintenance, and conjugation, as well as transposases, resolvases, and integrases, suggesting an evolution that involved the lateral transfer of DNA between bacterial species. Also found were genes that contain a high degree of sequence similarity to other known degradation genes, as well as genes involved in chemotaxis. Although the incompatibility group designation of pDTG1 remains unresolved, striking sequence organization and homology exists between the plasmid backbones of pDTG1 and the IncP-9 toluene-degradation plasmid pWW0, which suggests a divergent evolution from a progenitor plasmid prior to degradative gene incorporation.     

Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775.

Insertions were created in three iron uptake genes in plasmid pJM1 of Vibrio anguillarum 775 to assess their in vivo effects on virulence in fish. Insertions that blocked p40, pOM2, and pAngR expression resulted in iron uptake-negative strains and in 4.2 x 10(5)-, 8.8 x 10(5)-, and 2.5 x 10(5)-fold attenuations in virulence, respectively. A strain with an insertion in the pAngR coding region still synthesized significant constitutive levels of the outer membrane protein pOM2 and persisted in fish for at least 14 days postinjection. The results demonstrate a direct relationship between virulence and three pJM1-encoded gene products and also the feasibility of constructing live attenuated strains of V. anguillarum that might be useful in future vaccines.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

Bacteria Shigella spp. are highly contagious, severely harmful and gram-negative facultative intracellular pathogens. They may cause shigellosis characterized by fever, dehydration and hematochezia in clinic, and shigellosis has been remaining a leading cause of infant mortality in the world. Shigella belongs to the family Enterobacteriaceae and the group Escherichiaeae, which are divided into four species and at least 47 serotypes: Shigella dysenteriae (13 serotypes), Shigella flexneri (15 …     

Complete nucleotide sequence of a cryptic plasmid from the marine bacterium Vibrio splendidus and identification of open reading frames

A 2.3-kb replication-proficient fragment was previously obtained from a cryptic plasmid (pPS41) isolated from a marine Vibrio splendidus isolate (P. A. Sobecky, T. J. Mincer, M. C. Chang, A. Toukdarian, and D. R. Helinski, 1998, Appl. Environ. Microbiol. 64, 2822-2830). Analysis of the complete nucleotide sequence of plasmid pPS41 revealed two additional open reading frames (ORFs). Analysis of ORF-1 revealed that its translated product has 125 amino acids with a predicted MW of 16,978 and ORF-2 encodes a putative protein of 151 amino acids with a predicted MW of 19,802. The ORF-2 encoded protein showed 31 to 35% sequence homology to proteins identified to have a role in plasmid mobilization. These proteins are encoded on plasmids found in Escherichia coli and Pasteurella multocida. Plasmid pPS41 could be mobilized by a conjugative plasmid at frequencies of 1 x 10(-2) to 2 x 10(-2).