Plasmid-mediated iron uptake and virulence in Vibrio anguillarum

The plasmid pJM1 of Vibrio anguillarum harbors genes encoding proteins that enable the bacterial cell to survive under iron limiting conditions. A subset of these proteins are involved in the biosynthesis of the siderophore anguibactin and in the internalization of the ferric-siderophore into the cell cytosol. We have identified several genes encoding non-ribosomal peptide synthetases that catalyze the synthesis of anguibactin, these genes are: angB/G, angM, angN, angR, and angT. In addition, the genes fatA, fatB, fatC, and fatD are involved in the transport of ferric-anguibactin complexes. These transport genes, together with the biosynthesis genes angR and angT, are included in the iron transport biosynthesis operon (ITBO). Both the biosynthesis and the transport genes are under tight positive as well as negative control. We have identified four regulators; two of them, a chromosomally encoded Fur and a plasmid-mediated antisense RNA, RNAbeta, act in a negative fashion, while positive regulation is facilitated by AngR and TAFr. We also have evidence that the siderophore itself plays a positive role in the regulatory mechanism of the expression of both transport and biosynthesis genes.     

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes (vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.     

Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum.

Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.     

Restriction fragment length polymorphism of the Vibrio anguillarum serovar O1 virulence plasmid

Seventy-eight strains of Vibrio anguillarum serovar O1, all harboring one 65- to 70-kilobase plasmid, were typed according to restriction fragment length polymorphism of the plasmid. Six types, three of which comprised 96% of the strains examined, were produced with the restriction endonuclease BamHI. The fragment length polymorphism type did not correlate to any of 12 different phenotypic properties tested.     

Serotyping of Vibrio anguillarum

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

利用抑制性消减杂交（SSH）技术研究不同毒力的鳗弧菌菌株的基因多样性

[目的和方法]鳗弧菌是一种嗜盐的革兰氏阴性细菌,也是鱼类弧菌病的主要病原.对斑马鱼的半数致死量研究结果表明,鳗弧菌菌株VIB72具有较高的毒力,而菌株CW1的毒力较低.本文利用抑制性消减杂交(SSH)技术对这两个菌株的遗传差异进行了研究.[结果]通过对差减文库筛选,分离到59个对菌株VIB72的阳性克隆,并对这些克隆的DNA序列进行了测定.17个基因片断与其它细菌的已知功能的基因有较高的同源性,其中包括可溶性溶胞壁质转糖基酶、转移蛋白MobA和MobC、转座子IS66、抑制相关蛋白(金属β-内酰胺酶和乙酰转移酶家族)、毒素蛋白(DT-201和alveicin A免疫蛋白)、与OLD家族相似的ATP依赖性核酸内切酶以及SocE和GTP结合蛋白HflX(有高频率的溶原化).这些基因片断有可能是鳗弧菌毒力岛的一部分.其他的基因片断与其它的已知基因没有明显的相关性.[结论]这些结果表明,SSH技术成功地鉴定了不同致病性的鳗弧菌菌株的基因差异及潜在的毒力基因.     

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.     

Restriction fragment length polymorphism of the Vibrio anguillarum serovar O1 virulence plasmid.

Seventy-eight strains of Vibrio anguillarum serovar O1, all harboring one 65- to 70-kilobase plasmid, were typed according to restriction fragment length polymorphism of the plasmid. Six types, three of which comprised 96% of the strains examined, were produced with the restriction endonuclease BamHI. The fragment length polymorphism type did not correlate to any of 12 different phenotypic properties tested.     

Multiflagellate variants of Vibrio anguillarum

An ultrastructural examination of six strains of Vibrio anguillarum of varying virulence for eels revealed an apparent correlation between pathogenicity and the possession of more than one flagellum. The relationship between V. anguillarum surface appendages and virulence is discussed.     

Serotyping of Vibrio anguillarum.

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.     

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.     

Characterization of ferric-anguibactin transport in Vibrio anguillarum

The fish pathogen Vibrio anguillarum is the causative agent of a fatal hemorrhagic septicemia in salmonid fish. Many serotype O1 strains harbors a 65 Kbp plasmid (pJM1 encoding an iron sequestering system essential for virulence. The genes involved in the biosynthesis of the indigenous siderophore anguibactin are encoded by both the pJM1 plasmid and the chromosome, while those involved in the transport of the ferric-siderophore complex, including the outer membrane receptor, are plasmid-encoded. This work describes the role of specific amino acid residues of the outer membrane receptor FatA in the mechanism of transport of ferric-anguibactin. FatA modeling indicated that this protein has a 22 stranded ß-barrel blocked by the plug domain, the latter being formed by residues 51–54. Deletion of the plug domain resulted in a receptor unable to act as an open channel for the transport of the ferric anguibactin complex.     

P. aeruginosa quorum-sensing systems and virulence

Quorum sensing is an important mechanism for the regulation of genes in many Gram-negative and Gram-positive bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, the absence of one or more components of the quorum-sensing system results in a significant reduction in virulence. Recent advances in the past year have demonstrated that the quorum-sensing signal molecule 3O-C(12)-HSL is also a potent stimulator of multiple eukaryotic cells and thus may alter the host response during P. aeruginosa infections. Therefore, via the regulation of multiple factors and the production of 3O-C(12)-HSL, quorum-sensing systems have a significant effect on the virulence of the bacteria and also on how the host responds to P. aeruginosa infections.     

Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid pJM1.

Vibrio anguillarum 775 harboring the virulence plasmid pJM1 synthesized an outer membrane protein of 86 kilodaltons, OM2, that was inducible under conditions of iron limitation. pJM1 DNA fragments obtained by digestion with restriction endonucleases were cloned into cosmid vectors and transferred into Escherichia coli. The OM2 protein was synthesized in E. coli, demonstrating that it is actually encoded by the pJM1 plasmid. Mobilization of the recombinant plasmids to V. anguillarum was accomplished by using the transfer factor pRK2013. A V. anguillarum exconjugant harboring the recombinant derivative pJHC-T7 and synthesizing the OM2 protein took up 55Fe3+ and grew under iron-limiting conditions, only in presence of the pJM1-mediated siderophore. Exconjugants harboring recombinant plasmids, such as pJHC-T2 which did not encode the OM2 protein, were transport negative. Membrane protein iodination experiments, together with protease treatment of whole cells, indicated that the OM2 protein is exposed to the outside environment of the V. anguillarum cells.