Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus

PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.     

Draft genome sequence of Vibrio fischeri SR5, a strain isolated from the light organ of the Mediterranean squid Sepiola robusta

Here, we describe the draft genome sequence of Vibrio fischeri SR5, a squid symbiotic isolate from Sepiola robusta in the Mediterranean Sea. This 4.3-Mbp genome sequence represents the first V. fischeri genome from an S. robusta symbiont and the first from outside the Pacific Ocean.     

Genome Sequence and Comparative Genomics Analysis of a Vibrio cholerae O1 Strain Isolated from a Cholera Patient in Malaysia

The genome sequence analysis of a clinical Vibrio cholerae VC35 strain from an outbreak case in Malaysia indicates multiple genes involved in host adaptation and a novel Na⁺-driven multidrug efflux pump-coding gene in the genome of Vibrio cholerae with the highest similarity to VMA_001754 of Vibrio mimicus VMA223.     

Comparison of genome structures of vibrios,bacteria possessing two chromosomes

Vibrios are gram-negative gamma-proteobacteria which are ubiquitous in marine and estuarine environments. Recently, we demonstrated that some, if not all, Vibrio species have two circular chromosomes. The whole genome sequence of Vibrio cholerae N16961 has been reported. In this study, we constructed a physical and genetic map of the genome of Kanagawa phenomenon-positive Vibrio parahaemolyticus strain KX-V237 and compared it with those of V. parahaemolyticus AQ4673 and V. cholerae N16961. The genome of KX-V237 comprised two circular chromosomes (3.3 and 1.9 Mb), similar to the structure of the AQ4673 genome. The relative positions of the genes on the genomes were well conserved in the two strains, but a large inversion on the large chromosomes, probably symmetric around the replication origin, was suggested. Although the sizes of the large chromosomes of KX-V237 and V. cholerae N16961 were similar, the sizes of the small chromosomes were very different. Unlike N16961, the superintegron of KX-V237 was located on the large chromosome. Comparison of the genetic maps of the chromosomes of KX-V237 and V. cholerae N16961 revealed that most of the open reading frames (ORFs) present on the large chromosome of the V. cholerae strain had homologues on the large chromosome of the V. parahaemolyticus strain and that most of the ORFs on the small chromosome of N16961 were present on the small chromosome of KX-V237. The difference in the orders of the ORFs on the chromosomes of N16961 and KX-V237 implies that numerous and frequent genetic exchanges have occurred intrachromosomally rather than interchromosomally.     

Genome sequence of Vibrio sp. strain EJY3, an agarolytic marine bacterium metabolizing 3,6-anhydro-L-galactose as a sole carbon source

The metabolic fate of 3,6-anhydro-L-galactose (L-AHG) is unknown in the global marine carbon cycle. Vibrio sp. strain EJY3 is an agarolytic marine bacterium that can utilize L-AHG as a sole carbon source. To elucidate the metabolic pathways of L-AHG, we have sequenced the complete genome of Vibrio sp. strain EJY3.     

Evidence that AphB, essential for the virulence of Vibrio vulnificus, is a global regulator

The Vibrio vulnificus aphB mutant was significantly less virulent than the wild type and was impaired in motility and adherence to host cells. Microarray analysis revealed that AphB of V. vulnificus (AphB(Vv)) influences the expression of over 10% of the V. vulnificus genome. The combined results indicated that AphB(Vv) is a global regulator contributing to the pathogenesis of V. vulnificus.     

Genome sequence of Vibrio splendidus: an abundant planctonic marine species with a large genotypic diversity

Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrate's mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of Gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.     

Specific DNA probes for the identification of the fish pathogen,Renibacterium salmoninarum

To obtain specific DNA probes for the identification of the fish pathogen, Renibacterium salmoninarum, a discriminatory recombinant DNA library was constructed using selective fragments of the bacterial genome. Three renibacterial clones, pMAM29, pMAM46 and pMAM77, containing 149, 73, and 154 bp respectively, were isolated and characterized. The specificity of the probes was confirmed by dot-blot and Southern hybridization analyses. Bacterial hybridization experiments revealed that pMAM29 discriminates the R. salmoninarum genome from that of other fish pathogens such as Aeromonas salmonicida, Yersinia ruckeri, Flexibacter columnaris, Lactobacillus piscicola, Vibrio ordalii, Vibrio anguillarum and Aeromonas hydrophila. Thus, this probe may provide a new means to diagnose bacterial kidney disease in asymptomatic fish and ova.The authors are with the instituto de Bioquímica, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile     

Draft genome sequence of the shrimp pathogen Vibrio harveyi CAIM 1792

Vibrio harveyi is a Gram-negative bacterium found in tropical and temperate marine environments as a free-living organism or in association with aquatic animals. We report the first sequenced genome of a Vibrio harveyi strain, CAIM 1792, the etiologic agent of the "bright red" syndrome of the Pacific white shrimp Litopenaeus vannamei.     

Genome Sequence of the Human-Pathogenic Bacterium Vibrio vulnificus Type Strain ATCC 27562

Vibrio vulnificus, which is the causative agent of cholera, is a Gram-negative, curved, motile, and rod-shaped bacterium. Here, we present the draft genome sequence of the type strain, ATCC 27562, which was the first isolated Vibrio vulnificus strain.     

Cloning,functional expression in Escherichia coli and primary characterization of a new NA+/H+ antiporter,NhaD, of Vibrio cholerae

Vibrio cholerae is the infectious agent of the deadly diarrheal disease, cholera. Na+ ion homeostasis is believed to play a key role in both physiology and pathogenicity of this bacterium. However, molecular mechanisms of sodium exchange in V. cholerae are still poorly understood. In the present work a gene encoding an unusual Na+/H+ antiporter, nhaD, was identified in the V. cholerae genome. nhaD was cloned from Vibrio cholerae and expressed in Escherichia coli. The antiporter functioned in an E. coli nhaAnhaB mutant strain to confer resistance to LiCl and NaCl. When assayed in inside-out subbacterial vesicles, V. cholerae NhaD demonstrated high affinity for Na+ ions (1.1 mM Na+ was required for the half-maximal response at the pH-optimum). The most striking feature of Vc-NhaD is a unique pH-profile of its activity with a sharp maximum at pH 8.0, different from that of any bacterial sodium-proton antiporter described so far. The difference is rationalized as being the result of a His to Arg substitution in a putative pH sensing residue.     

The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force

Vibrio cholerae is a highly motile bacterium which possesses a single polar flagellum as a locomotion organelle. Motility is thought to be an important factor for the virulence of V. cholerae. The genome sequencing project of this organism is in progress, and the genes that are highly homologous to the essential genes of the Na+-driven polar flagellar motor of Vibrio alginolyticus were found in the genome database of V. cholerae. The energy source of its flagellar motor was investigated. We examined the Na+ dependence and the sensitivity to the Na+ motor-specific inhibitor of the motility of the V. cholerae strains and present the evidence that the polar flagellar motor of V. cholerae is driven by an Na+ motive force.     

Genome size reduction through multiple events of gene disintegration in Buchnera APS.

The evolution of the endosymbiont Buchnera during its adaptation to intracellular life involved a massive reduction in its genome. By comparing the orthologous genes of Buchnera, Escherichia coli and Vibrio cholerae, we show that the minimal genome size of Buchnera arose from multiple events of gene disintegration dispersed over the whole genome. The elimination of the genes was a continuous process that began with gene inactivation and progressed until the DNA corresponding to the pseudogenes were completely deleted.     

Evolution of alkaline phosphatase in marine species of Vibrio.

The evolution of alkaline phosphatase was studied in marine species of Vibrio. Two antisera prepared against purified alkaline phosphatases from Vibrio splendidus and Vibrio harveyi were used to estimate the amino acid sequence divergence of this enzyme in 51 strains belonging to nine species. The methods used were the quantitative microcomplement fixation technique and the Ouchterlony double-diffusion procedure. There was a high degree of congruence between the measurement of the amino acid sequence divergence of alkaline phosphatase and the percentage of deoxyribonucleic acid homology of the different organisms relative to both reference strains (correlation coefficient of -0.89) as well as between the amino acid sequence divergence of alkaline phosphatase and superoxide dismutase (correlation coefficient of 0.92) relative to V. splendidus. These findings supported the view that the evolution of marine species of Vibrio is primarily vertical and that horizontal evolution (involving genetic exchange between species), if significant, is restricted to a minor fraction of the bacterial genome.     

Genomic and functional analysis of Vibrio phage SIO-2 reveals novel insights into ecology and evolution of marine siphoviruses

We report on a genomic and functional analysis of a novel marine siphovirus, the Vibrio phage SIO-2. This phage is lytic for related Vibrio species of great ecological interest including the broadly antagonistic bacterium Vibrio sp. SWAT3 as well as notable members of the Harveyi clade (V.harveyi ATTC BAA-1116 and V.campbellii ATCC 25920). Vibrio phage SIO-2 has a circularly permuted genome of 80598 bp, which displays unusual features. This genome is larger than that of most known siphoviruses and only 38 of the 116 predicted proteins had homologues in databases. Another divergence is manifest by the origin of core genes, most of which share robust similarities with unrelated viruses and bacteria spanning a wide range of phyla. These core genes are arranged in the same order as in most bacteriophages but they are unusually interspaced at two places with insertions of DNA comprising a high density of uncharacterized genes. The acquisition of these DNA inserts is associated with morphological variation of SIO-2 capsid, which assembles as a large (80 nm) shell with a novel T=12 symmetry. These atypical structural features confer on SIO-2 a remarkable stability to a variety of physical, chemical and environmental factors. Given this high level of functional and genomic novelty, SIO-2 emerges as a model of considerable interest in ecological and evolutionary studies.     

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

霍乱弧菌分型噬菌体VP3蛋白的双向电泳分析

目的:噬菌体-生物分型方案具有区分霍乱弧菌潜在致病力的作用,VP3是5个霍乱弧菌分型噬菌体之一。对VP3成熟颗粒的蛋白组成进行测定和分析,以补充基因组注释信息。方法:在全基因组测序及生物信息学分析的基础上,利用双向电泳技术及质谱鉴定,对纯化的成熟VP3噬菌体的结构蛋白进行分离及鉴定。结果:双向电泳分离得到近20个蛋白点,质谱鉴定出了其中的10个,对应于4个VP3蛋白和4个霍乱弧菌蛋白。结论:VP3结构蛋白的组成和T7具有很高的相似性。与噬菌体颗粒一起被纯化分离的宿主蛋白可能在VP3的转染过程中起作用。     

Genome Sequence of the Human-Pathogenetic Bacterium Vibrio vulnificus B2

Vibrio vulnificus, which can lead to rapidly expanding cellulitis or septicemia, is present in the marine environment. Here, we present the draft genome sequence of strain B2, which was isolated from a septicemia patient in 2010.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

Genome sequence of the bacterioplanktonic, mixotrophic Vibrio campbellii strain PEL22A, isolated in the Abrolhos Bank

Vibrio campbellii PEL22A was isolated from open ocean water in the Abrolhos Bank. The genome of PEL22A consists of 6,788,038 bp (the GC content is 45%). The number of coding sequences (CDS) is 6,359, as determined according to the Rapid Annotation using Subsystem Technology (RAST) server. The number of ribosomal genes is 80, of which 68 are tRNAs and 12 are rRNAs. V. campbellii PEL22A contains genes related to virulence and fitness, including a complete proteorhodopsin cluster, complete type II and III secretion systems, incomplete type I, IV, and VI secretion systems, a hemolysin, and CTXΦ.