Mimicus Strains Research Articles

Sialic acid catabolism contributes to Vibrio mimicus virulence

Vibrio mimicus is an emerging pathogen primarily associated with catfish vibriosis. Infectious caused by V. mimicus in aquatic animals have been characterized by a short infection course and high mortality rate, resulting in significant economic losses. The utilization of sialic acid has been shown to be correlated with bacterial virulence, particularly in highly sialylated environments. In this study, we investigated virulence-determining phenotypes of V. mimicus strain SCCF01 (WT) and the sialic acid catabolism (SaCS) defect strain ΔSaCS, and revealed their potential molecular connections by comparative proteomics analysis. Our results showed that abrogation of sialic acid catabolism ability resulted in bacterial morphological changes, a decrease in biofilm formation, but an increase in cell adherence ability. Animal experiments confirmed that SaCS contributes to V. mimicus pathogenicity, as the LD50 of ΔSaCS strain was 27-fold lower compared to WT strain. Proteomics analysis revealed that differentially expressed proteins between the culture supernatants of WT and ΔSaCS were mainly enriched in pentose phosphate pathway, purine metabolism and bacterial chemotaxis. The ΔSaCS lacked several extracellular proteins and virulence factors, providing additional explanation for its attenuating effect.

Second extracellular protease mediating maturation of Vibrio mimicushemolysin.

Vibrio mimicus is a bacterium that causes gastroenteritis in humans. This pathogen produces an enterotoxic hemolysin called V. mimicus hemolysin (VMH), which is secreted extracellularly as an inactive 80-kDa protoxin and converted to a 66-kDa mature toxin through cleavage between Arg151 and Ser152. The 56-kDa serine protease termed V. mimicus trypsin-like protease (VmtA) is known to mediate this maturating process. However, some strains including strain ES-20 does not possess the vmtA gene. In the present study, the vmtA-negative strains were found to have a replaced gene that encodes a 43-kDa (403 aa) precursor of a serine protease designated by VmtX (V. mimicus trypsin-like protease X). To examine whether VmtX is also involved in the maturation of VMH, VmtX was isolated from the culture supernatant of V. mimicus strain NRE-20, a metalloprotease-negative mutant constructed from strain ES-20. Concretely, the culture supernatant was fractionated with 70% saturated ammonium sulfate and subjected to affinity column chromatography using a HiTrap Benzamidine FF column. The analysis of the N-terminal amino acid sequences of the proteins in the obtained VmtX preparation indicated that the 39-kDa protein was active VmtX consisting of 371 aa (Ile33-Ser403). The VmtX preparation was found to activate pro-VMH through generation of the 66-kDa protein. Additionally, treatment of the VmtX preparation with serine protease inhibitors, such as leupeptin and phenylmethylsulfonyl fluoride, significantly suppressed the activities to hydrolyze the specific peptide substrate and to synthesize the 66-kDa toxin. These findings indicate that VmtX is the second protease that mediats the maturation of VMH.

Characterization of Potential Virulence Factors of Vibrio mimicus Isolated from Fishery Products and Water.

Vibrio mimicus is a Gram-negative bacterium that is closely related to V. cholerae and causes gastroenteritis in humans due to contaminated fish consumption and seafood. This bacterium was isolated and identified from 238 analyzed samples of sea water, oysters, and fish. Twenty strains were identified as V. mimicus according to amplification of the vmhA gene, which is useful as a marker of identification of the species. The production of lipases, proteases, and nucleases was detected; 45% of the strains were able to produce thermonucleases and 40% were capable of producing hydroxamate-type siderophores, and the fragment of the iuT gene was amplified in all of the V. mimicus strains. Seventy-five percent of V. mimicus strains showed cytopathic effect on Chinese hamster ovary (CHO) cells and destruction of the monolayer, and 100% of the strains were adherent on the HEp-2 cell line with an aggregative adherence pattern. The presence of virulence factors in V. mimicus strains obtained from fishery products suggests that another member of the Vibrio genus could represent a risk to the consumer due to production of different metabolites that allows it to subsist in the host.

Core and Accessory Genome Analysis of Vibrio mimicus.

Vibrio mimicus is an emerging pathogen, mainly associated with contaminated seafood consumption. However, little is known about its evolution, biodiversity, and pathogenic potential. This study analyzes the pan-, core, and accessory genomes of nine V. mimicus strains. The core genome yielded 2424 genes in chromosome I (ChI) and 822 genes in chromosome II (ChII), with an accessory genome comprising an average of 10.9% of the whole genome for ChI and 29% for ChII. Core genome phylogenetic trees were obtained, and V. mimicus ATCC-33654 strain was the closest to the outgroup in both chromosomes. Additionally, a phylogenetic study of eight conserved genes (ftsZ, gapA, gyrB, topA, rpoA, recA, mreB, and pyrH), including Vibrio cholerae, Vibrio parilis, Vibrio metoecus, and Vibrio caribbenthicus, clearly showed clade differentiation. The main virulence genes found in ChI corresponded with type I secretion proteins, extracellular components, flagellar proteins, and potential regulators, while, in ChII, the main categories were type-I secretion proteins, chemotaxis proteins, and antibiotic resistance proteins. The accessory genome was characterized by the presence of mobile elements and toxin encoding genes in both chromosomes. Based on the genome atlas, it was possible to characterize differential regions between strains. The pan-genome of V. mimicus encompassed 3539 genes for ChI and 2355 genes for ChII. These results give us an insight into the virulence and gene content of V. mimicus, as well as constitute the first approach to its diversity.

Complete genome analysis of Vibrio mimicus strain SCCF01, a highly virulent isolate from the freshwater catfish

ABSTRACT Vibrio mimicus is a foodborne pathogen, which is widely distributed in the aquatic environment. Moreover, it is often involved in aquatic animal diseases. In recent years, V. mimicus is an emerging pathogen in some species of Siluriformes. The strain SCCF01 was isolated from yellow catfish (Pelteobagrus fulvidraco). In this study, we aimed to perform genomic analysis of V. mimicus strain SCCF01 to identify genetic features and evolutionary relationships. Information on gene function and classification was obtained by functional annotation, and circular graph of strain SCCF01 genome, which was created by Circos v0.64. Information on virulence genes (adhesion, flagellum system, exotoxin, and secretory system, etc.) was obtained by virulence genes annotation. Genome element prediction showed that most of the mobile elements were distributed in chromosome I. Therefore, chromosome I of SCCF01 genome has more plasticity than chromosome II and might be larger in size. Genomic linear relationship between the strain of V. mimicus and strain SCCF01 was analyzed by linear pairwise comparison but was unable to determine the relationship. Gene family analysis predicted that the evolutionary direction of strain SCCF01 was: clinical strain → environmental strain → SCCF01 strain. Phylogenetic analysis showed that the strain SCCF01 was more closely related to environmental strains. According to gene family analysis and phylogenetic analysis, we speculated that strain SCCF01 has probably diverged from environmental strains.

Long-distance transmission of pathogenic Vibrio species by migratory waterbirds: a potential threat to the public health

A potential mechanism for the global distribution of waterborne pathogens is through carriage by the migratory waterbirds. However, this mode of transmission has yet been confirmed epidemiologically. Here, we conducted whole genome sequencing of Vibrio spp. collected from waterbirds, sediments, and mollusks in the estuary of the Liaohe River in China to investigate this transmission mode. We found that a V. parahaemolyticus strain isolated from a waterbird was clonally related to the other V. parahaemolyticus strains obtained from the sediments and mollusks, and three V. mimicus strains isolated from bird feces were genomically related to those found in the mollusks and upstream groundwater, suggesting that the bird-carried Vibrio strains were acquired through the direct predation of the local mollusks. Surprisingly, two bird-carried V. parahaemolyticus strains belonging to the same clone were identified in Panjin and Shanghai, which are over 1,150 km apart, and another two were found at two locations 50 km apart, further supporting that waterbirds are capable of carrying and disseminating these pathogens over long distances. Our results provide the first evidence of direct transmission from mollusks to waterbirds and confirm that waterbirds act as disseminating vehicles of waterborne pathogens. Effective surveillance of migratory waterbirds along their routes will be valuable for predicting future epidemics of infectious diseases.

Multiplex genome editing by natural transformation in Vibrio mimicus with potential application in attenuated vaccine development

Vibrio mimicus (V. mimicus) is a significant pathogen in freshwater catfish, though knowledge of virulence determinants and effective vaccine is lacking. Multiplex genome editing by natural transformation (MuGENT) is an easy knockout method, which has successfully used in various bacteria except for V. mimicus. Here, we found V. mimicus strain SCCF01 can uptake exogenous DNA and insert it into genome by natural transformation assay. Subsequently, we exploited this property to make five mutants (△Hem, △TS1, △TS2, △TS1△TS2, and △II), and removed the antibiotic resistance marker by Flp-recombination. Finally, all of the mutants were identified by PCR and RT-PCR. The results showed that combination of natural transformation and FLP-recombination can be applied successfully to generate targeted gene disruptions without the antibiotic resistance marker in V. mimicus. In addition, the five mutants showed mutant could be inherited after several subcultures and a 668-fold decrease in the virulence to yellow catfish (Pelteobagrus fulvidraco). This study provides a convenient method for the genetic manipulation of V. mimicus. It will facilitate the identification and characterization of V. mimicus virulence factors and eventually contribute to a better understanding of V. mimicus pathogenicity and development of attenuated vaccine.

Isolation and identification ofVibriospecies in the Rio Bravo/Grande and water bodies from Reynosa, Tamaulipas

Water quality is associated with public health, as it plays an important role in the transmission and epidemiology of pathogens such as Vibrio, since this species have been responsible for human diseases around the world. This study demonstrated the presence of toxigenic Vibrio species in water bodies in Reynosa surroundings, indicating that water bodies may be a source of public health risk.

Construction of Vibrio mimicus ghosts as a novel inactivated vaccine candidate and its protective efficacy against ascites disease in grass carps (Ctenopharyngodon idella)

Vibrio mimicus (V. mimicus) is an important bacterial pathogen causing high mortality rates for fish and shellfish species. Prevention of vibriosis still remains a challenge, and an efficacious and safe vaccine that confers protection against V. mimicus needs to be developed. Here, with the purpose of generating V. mimicus bacterial ghosts (VmBGs), the bacteriophage PhiX174 lysis gene E was cloned into the shuttle plasmid pBV220 for construction of the recombinant temperature-sensitive lysis plasmid pBV220-lysisE. The pBV220-lysisE was then electroporated into V. mimicus strain 04–14, and the resultant transformants were used for production of VmBGs by inducing lysis gene E expression. The VmBGs were characterized by observing their morphology and structure on electron microscopy, and then the safety and vaccine potential of VmBGs were further evaluated in grass carps (Ctenopharyngodon idella), The results showed that the efficiency of VmBGs induction was about 99.91%, and no living bacteria survived after lyophilization. The obvious transmembrane lysis pores with loss of cytoplasmic materials were observed in VmBGs by electron microscopy. Fish vaccinated with VmBGs via intraperitoneal route showed significantly up-regulated expression of several immune-related genes (G-type lysozyme, IL-1β, MHC-I and MHC-IIβ) in the spleens, and higher serum agglutinating antibody level, serum bactericidal activity as well as peripheral blood lymphocytes proliferation responses compared to formalin-killed cells of V. mimicus (FKC) vaccinated fish and PBS control fish. More importantly, strong protection with relative survival rate of 76.67% was recorded in the VmBGs-vaccinated fish after challenge with V. mimicus strain 04–14, whereas the FKC vaccine and PBS control showed 40% and 0% survival rate, respectively. Overall, these findings suggest that the newly constructed VmBGs may be used as a promising vaccine candidate for the control of V. mimicus infection in fish.

Virulence-associated factors in Vibrio cholerae non-O1/non-O139 and V.mimicus strains isolated in ornamental fish species.

During recent decades, ornamental fish have proven to be one of the fastest growing categories of pets in Europe. In this framework, we evaluated both the potential pathogenic and zoonotic risks caused by 53 Vibrio cholerae non-O1/non-O139 and a Vibrio mimicus strain isolated from ornamental fish species mostly originating from South-East Asia countries between 2000 and 2015 in Italy. All the strains were firstly identified at species level by biochemical, phylogenetic and mass spectrometry (matrix-assisted laser desorption ionization time of flight) methods, and then studied to reveal the presence of the main virulence and colonization-associated factors, as ctxA, ace, zot, stn/sto, toxR, rtxA, hlyA and tcpA by multiplex and single endpoint PCR assays. Findings showed that 21 of 54 strains harboured at least one virulence factor with a predominance for the toxR+ , rtxA+ and hlyAET+ genotype. Interestingly, the V.mimicus strain harboured the colonization factor and the CTX prophage receptor, tcpA, indicating the ability to capture and integrate it in its genome increasing its pathogenicity. Although these enterotoxins can sporadically cause gastroenteritis, the results highlight their probable involvement in causing severe implications for public health, suggesting the need for an European microbiological monitoring.

Efficiency of Indigenous <i>Pseudomonas aeruginosa</i> as Biocontrol Agent against <i>Vibrio</i> Infection in Shrimp (<i>Penaeus monodon</i>) Culture

The efficacy of Pseudomonas aeruginosa as a biocontrol agent against Vibrio mimicus infection in shrimp ( Penaeus monodon ) culture was evaluated. The antagonistic Pseudomonas sp. Ps2 and Vibrio sp. V1 were previously isolated from healthy and moribund shrimp intestines respectively. The two strains were identified using molecular method. Four-day acute pathogenicity tests were carried out on shrimp ( Penaeus monodon ) postlarvae using different concentrations of the strains. Histopathological examination of the hepatopancreas of the postlarvae was carried out after each pathogenicity test. Probiotic trial of the antagonistic Pseudomonas aeruginosa on culture of shrimp ( Penaeus monodon ) challenged with pathogenic Vibrio mimicus by immersion was carried out for 12 days. Water quality parameters (pH, temperature, ammonia, nitrite, nitrate, salinity and dissolved oxygen) were determined during shrimp culture. Molecular analysis conducted on Pseudomonas sp. Ps2 and Vibrio sp. V1 revealed that they are closely related to Pseudomonas aeruginosa strain XyC5 and Vibrio mimicus strain XQ respectively. The use of Pseudomonas aeruginosa had no pathogenicity effect on shrimp since no shrimp mortality was recorded. The LC 50 of Vibrio mimicus was calculated to be 3.2 x 10 4 cfu/ml. Vibrio mimicus addition resulted in 54% mortality and unhealthy hepatopancreas. No addition of bacteria resulted in 22% mortality and healthy hepatopancreas while Pseudomonas aeruginosa plus Vibrio mimicus addition resulted in zero mortality and healthy hepatopancreas. The water quality parameters were within the optimum ranges throughout the culture period. The results suggest that the indigenous antagonistic Pseudomonas aeruginosa strain XyC5 could be beneficial as biological control agent for management of vibriosis in shrimp aquaculture.

Regulation of Vibrio mimicus metalloprotease (VMP) production by the quorum-sensing master regulatory protein, LuxR.

Vibrio mimicus is an estuarine bacterium, while it can cause severe diarrhea, wound infection, and otitis media in humans. This pathogen secretes a relatively important toxin named V. mimicus metalloprotease (VMP). In this study, we clarified regulation of the VMP production according to the quorum-sensing master regulatory protein named LuxR. First, the full length of luxR gene, encoding LuxR, was detected in V. mimicus strain E-37, an environmental isolate. Next, the putative consensus binding sequence of LuxR protein could be detected in the upstream (promoter) region of VMP encoding gene, vmp. Finally, the effect of disruption of luxR gene on the expression of vmp and production of VMP was evaluated. Namely, the expression of vmp was significantly diminished by luxR disruption and the production of VMP was severely altered. Taken together, here we report that VMP production is under the positive regulation of the quorum-sensing master regulatory protein, LuxR.

Identification of capsule, biofilm, lateral flagellum, and type IV pili in Vibrio mimicus strains

Vibrio mimicus is a bacterium that causes gastroenteritis; it is closely related to Vibrio cholerae, and can cause acute diarrhea like cholera- or dysentery-type diarrhea. It is distributed worldwide. Factors associated with virulence (such as hemolysins, enterotoxins, proteases, phospholipases, aerobactin, and hemagglutinin) have been identified; however, its pathogenicity mechanism is still unknown. In pathogenic Vibrio species such as V. cholerae, Vibrio. parahaemolyticus and Vibrio vulnificus, capsule, biofilms, lateral flagellum, and type IV pili are structures described as essential for pathogenicity. These structures had not been described in V. mimicus until this work. We used 20 V. mimicus strains isolated from water (6), oyster (9), and fish (5) samples and we were able to identify the capsule, biofilm, lateral flagellum, and type IV pili through phenotypic tests, electron microscopy, PCR, and sequencing. In all tested strains, we observed and identified the presence of capsular exopolysaccharide, biofilm formation in an in vitro model, as well as swarming, multiple flagellation, and pili. In addition, we identified homologous genes to those described in other bacteria of the genus in which these structures have been found. Identification of these structures in V. mimicus is a contribution to the biology of this organism and can help to reveal its pathogenic behavior.

A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin.

Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N-terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin-like substrate specificity was purified from the bacterial culture supernatant. The N-terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg(151)-Ser(152) bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.

Genome Sequencing Reveals Unique Mutations in Characteristic Metabolic Pathways and the Transfer of Virulence Genes between V. mimicus and V. cholerae

Vibrio mimicus, the species most similar to V. cholerae, is a microbe present in the natural environmental and sometimes causes diarrhea and internal infections in humans. It shows similar phenotypes to V. cholerae but differs in some biochemical characteristics. The molecular mechanisms underlying the differences in biochemical metabolism between V. mimicus and V. cholerae are currently unclear. Several V. mimicus isolates have been found that carry cholera toxin genes (ctxAB) and cause cholera-like diarrhea in humans. Here, the genome of the V. mimicus isolate SX-4, which carries an intact CTX element, was sequenced and annotated. Analysis of its genome, together with those of other Vibrio species, revealed extensive differences within the Vibrionaceae. Common mutations in gene clusters involved in three biochemical metabolism pathways that are used for discrimination between V. mimicus and V. cholerae were found in V. mimicus strains. We also constructed detailed genomic structures and evolution maps for the general types of genomic drift associated with pathogenic characters in polysaccharides, CTX elements and toxin co-regulated pilus (TCP) gene clusters. Overall, the whole-genome sequencing of the V. mimicus strain carrying the cholera toxin gene provides detailed information for understanding genomic differences among Vibrio spp. V. mimicus has a large number of diverse gene and nucleotide differences from its nearest neighbor, V. cholerae. The observed mutations in the characteristic metabolism pathways may indicate different adaptations to different niches for these species and may be caused by ancient events in evolution before the divergence of V. cholerae and V. mimicus. Horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae, rather than the seventh pandemic strains, have generated the pathogenic V. mimicus strain carrying cholera toxin genes.

Presence of genes for type III secretion system 2 in Vibrio mimicus strains

BackgroundVibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2β, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear.ResultsWe therefore examined the distribution of the genes for T3SS2 in vibrios other than V. parahaemolyticus by using a PCR assay targeting both T3SS2α and T3SS2β genes. Among the 32 Vibrio species tested in our study, several T3SS2-related genes were detected in three species, V. cholerae, V. mimicus and V. hollisae, and most of the essential genes for type III secretion were present in T3SS2-positive V. cholerae and V. mimicus strains. Moreover, both V. mimicus strains possessing T3SS2α and T3SS2β were identified. The gene organization of the T3SS2 gene clusters in V. mimicus strains was fundamentally similar to that of V. parahaemolyticus and V. cholerae in both T3SS2α- and T3SS2β-possessing strains.ConclusionsThis study is the first reported evidence of the presence of T3SS2 gene clusters in V. mimicus strains. This finding thus provides a new insight into the pathogenicity of the V. mimicus species.

Interaction between Vibrio mimicus and Acanthamoeba castellanii

Vibrio mimicus is a Gram-negative bacterium, which causes gastroenteritis and is closely related to Vibrio cholerae. The environmental reservoir of this bacterium is far from defined. Acanthamoeba as well as Vibrio species are found in diverse aquatic environments. The present study was aimed to investigate the ability of A. castellanii to host V. mimicus, the role of bacterial protease on interaction with A. castellanii and to disclose the ability of cysts to protect intracellular V. mimicus. Co-cultivation, viable counts, gentamicin assay, electron microscopy and statistical analysis showed that co-cultivation of wild type and luxO mutant of V. mimicus strains with A. castellanii did not inhibit growth of the amoeba. On the other hand co-cultivation enhanced growth and survival of V. mimicus strains. Vibrio mimicus showed intracellular behaviour because bacteria were found to be localized in the cytoplasm of amoeba trophozoites and remain viable for 14 days. The cysts protected intracellular V. mimicus from high level of gentamicin. The intracellular growth of V. mimicus in A. castellanii suggests a role of A. castellanii as a host for V. mimicus.

Genomic taxonomy of Vibrios.

BackgroundVibrio taxonomy has been based on a polyphasic approach. In this study, we retrieve useful taxonomic information (i.e. data that can be used to distinguish different taxonomic levels, such as species and genera) from 32 genome sequences of different vibrio species. We use a variety of tools to explore the taxonomic relationship between the sequenced genomes, including Multilocus Sequence Analysis (MLSA), supertrees, Average Amino Acid Identity (AAI), genomic signatures, and Genome BLAST atlases. Our aim is to analyse the usefulness of these tools for species identification in vibrios.ResultsWe have generated four new genome sequences of three Vibrio species, i.e., V. alginolyticus 40B, V. harveyi-like 1DA3, and V. mimicus strains VM573 and VM603, and present a broad analyses of these genomes along with other sequenced Vibrio species. The genome atlas and pangenome plots provide a tantalizing image of the genomic differences that occur between closely related sister species, e.g. V. cholerae and V. mimicus. The vibrio pangenome contains around 26504 genes. The V. cholerae core genome and pangenome consist of 1520 and 6923 genes, respectively. Pangenomes might allow different strains of V. cholerae to occupy different niches. MLSA and supertree analyses resulted in a similar phylogenetic picture, with a clear distinction of four groups (Vibrio core group, V. cholerae-V. mimicus, Aliivibrio spp., and Photobacterium spp.). A Vibrio species is defined as a group of strains that share > 95% DNA identity in MLSA and supertree analysis, > 96% AAI, ≤ 10 genome signature dissimilarity, and > 61% proteome identity. Strains of the same species and species of the same genus will form monophyletic groups on the basis of MLSA and supertree.ConclusionThe combination of different analytical and bioinformatics tools will enable the most accurate species identification through genomic computational analysis. This endeavour will culminate in the birth of the online genomic taxonomy whereby researchers and end-users of taxonomy will be able to identify their isolates through a web-based server. This novel approach to microbial systematics will result in a tremendous advance concerning biodiversity discovery, description, and understanding.

Identification of Vibrio cholerae and Vibrio mimicus by multilocus sequence analysis (MLSA)

Vibrio cholerae and Vibrio mimicus have similar phenotypes and genomes making rapid differentiation of these two species difficult. The first standard multilocus sequence analysis (MLSA) scheme for the identification of these species is described. A collection of 45 representative isolates from different geographical regions and hosts was examined using segments of the housekeeping genes pyrH, recA and rpoA. Overall, the closest phylogenetic neighbours of these species were Vibrio furnissii and Vibrio fluvialis. V. cholerae and V. mimicus formed separate species clusters on the basis of each gene, suggesting that these genes are useful as identification markers. These species clusters arose by the accumulation of point mutations. The pyrH gene showed the highest resolution for differentiating V. cholerae and V. mimicus. The maximum interspecies pyrH gene sequence similarity was 91 %. Clearly, V. mimicus strains were more heterogeneous than V. cholerae strains at the three loci. It is suggested that vibrio species may be defined on the basis of MLSA data. A vibrio species was defined as a group of strains forming a monophyletic group on the basis of these loci and with an intraspecific sequence similarity of at least 95 %. V. cholerae and V. mimicus isolates can be readily identified through the open database resource 'The Taxonomy of Vibrios' (http://www.taxvibrio.lncc.br/).

Proteolytic Activation of Vibrio mimicus (Vm) Major Outer Membrane Protein Haemagglutinin (HA) with Vm‐HA/Protease: Implication for Understanding Bacterial Adherence

Vibrio mimicus (Vm) haemagglutinins (HAs), such as an extracellular HA/protease (Vm-HA/protease) and a major outer membrane protein-HA (Vm-OMPHA), have been recognized as the putative adherence factors for the bacterium. However, the mechanism by which HAs coordinate the adherence function of the bacterium remains as yet unknown. We report herein the positive interaction between Vm-HA/protease and Vm-OMPHA resulting in significant enhancement of the haemagglutinating ability. In this interaction, no cleaved polypeptide was detected; however, limited proteolysis of Vm-OMPHA was confirmed by SDS-PAGE. The proteolytic activation of the native cell-associated Vm-OMPHA by limited proteolysis was also demonstrated in several V. mimicus strains. Proteolytic activation of OMPHA was also achieved with various proteases from bacterial and eukaryotic sources. These findings may indicate a novel coordination of V. mimicus HAs in the adherence of the bacterium.