Initiator protein dimerization plays a key role in replication control of Vibrio cholerae chromosome 2

RctB, the initiator of replication of Vibrio cholerae chromosome 2 (chr2), binds to the origin of replication to specific 12-mer sites both as a monomer and a dimer. Binding to 12-mers is essential for initiation. The monomers also bind to a second kind of site, 39-mers, which inhibits initiation. Mutations in rctB that reduce dimer binding increase monomer binding to 12-mers but decrease monomer binding to 39-mers. The mechanism of this paradoxical binding behavior has been unclear. Using deletion and alanine substitution mutants of RctB, we have now localized to a 71 amino acid region residues important for binding to the two kinds of DNA sites and for RctB dimerization. We find that the dimerization domain overlaps with both the DNA binding domains, explaining how changes in the dimerization domain can alter both kinds of DNA binding. Moreover, dimerization-defective mutants could be initiation-defective without apparent DNA binding defect. These results suggest that dimerization might be important for initiation beyond its role in controlling DNA binding. The finding that determinants of crucial initiator functions reside in a small region makes the region an attractive target for anti-V. cholerae drugs.     

RpoS Plays a Central Role in the SOS Induction by Sub-Lethal Aminoglycoside Concentrations in Vibrio cholerae

Bacteria encounter sub-inhibitory concentrations of antibiotics in various niches, where these low doses play a key role for antibiotic resistance selection. However, the physiological effects of these sub-lethal concentrations and their observed connection to the cellular mechanisms generating genetic diversification are still poorly understood. It is known that, unlike for the model bacterium Escherichia coli, sub-minimal inhibitory concentrations (sub-MIC) of aminoglycosides (AGs) induce the SOS response in Vibrio cholerae. SOS is induced upon DNA damage, and since AGs do not directly target DNA, we addressed two issues in this study: how sub-MIC AGs induce SOS in V. cholerae and why they do not do so in E. coli. We found that when bacteria are grown with tobramycin at a concentration 100-fold below the MIC, intracellular reactive oxygen species strongly increase in V. cholerae but not in E. coli. Using flow cytometry and gfp fusions with the SOS regulated promoter of intIA, we followed AG-dependent SOS induction. Testing the different mutation repair pathways, we found that over-expression of the base excision repair (BER) pathway protein MutY relieved this SOS induction in V. cholerae, suggesting a role for oxidized guanine in AG-mediated indirect DNA damage. As a corollary, we established that a BER pathway deficient E. coli strain induces SOS in response to sub-MIC AGs. We finally demonstrate that the RpoS general stress regulator prevents oxidative stress-mediated DNA damage formation in E. coli. We further show that AG-mediated SOS induction is conserved among the distantly related Gram negative pathogens Klebsiella pneumoniae and Photorhabdus luminescens, suggesting that E. coli is more of an exception than a paradigm for the physiological response to antibiotics sub-MIC.     

Ecotin and LamB in Escherichia coli influence the susceptibility to Type VI secretion-mediated interbacterial competition and killing by Vibrio cholerae

BackgroundA prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet.MethodsEscherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses.ResultsThe T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition.ConclusionsOur study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae.General significanceWe envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.     

Inhibition of in vitro growth of enteropathogens by new Lactobacillus isolates of human intestinal origin

Three human Lactobacillus strains, coded B21060, B21070 and B21190, have recently been isolated. The strains show a series of features (acid and bile resistance, adhesion to various types of mucosal cell) which make them particularly promising for the preparation of probiotic products. In the present study, the ability of the strains to inhibit the growth of pathogens in coculture was investigated. Lactobacilli were incubated simultaneously or after one overnight growth with enterotoxigenic Escherichia coli, Salmonella enteritidis or Vibrio cholerae. After 24 and 48 h, bacterial counts of the pathogens and of the lactobacilli were performed. The results showed that these Lactobacillus strains inhibited the in vitro growth of E. coli and S. enteritidis under both conditions. Moreover, a cumulative effect was observed for mixtures of lactobacilli. In contrast, no significant inhibition of Vibrio cholerae growth was observed, provided that the pH of the medium was kept constant. The presence of the pathogens did not affect the growth of the Lactobacillus strains. Moreover, each of the Lactobacillus strains showed coaggregation ability with two pathogenic E. coli strains, namely ATCC 25922 and ATCC 35401.     

Vibrio cholerae VciB promotes iron uptake via ferrous iron transporters

Vibrio cholerae uses a variety of strategies for obtaining iron in its diverse environments. In this study we report the identification of a novel iron utilization protein in V. cholerae, VciB. The vciB gene and its linked gene, vciA, were isolated in a screen for V. cholerae genes that permitted growth of an Escherichia coli siderophore mutant in low-iron medium. The vciAB operon encodes a predicted TonB-dependent outer membrane receptor, VciA, and a putative inner membrane protein, VciB. VciB, but not VciA, was required for growth stimulation of E. coli and Shigella flexneri strains in low-iron medium. Consistent with these findings, TonB was not needed for VciB-mediated growth. No growth enhancement was seen when vciB was expressed in an E. coli or S. flexneri strain defective for the ferrous iron transporter Feo. Supplying the E. coli feo mutant with a plasmid encoding either E. coli or V. cholerae Feo, or the S. flexneri ferrous iron transport system Sit, restored VciB-mediated growth; however, no stimulation was seen when either of the ferric uptake systems V. cholerae Fbp and Haemophilus influenzae Hit was expressed. These data indicate that VciB functions by promoting iron uptake via a ferrous, but not ferric, iron transport system. VciB-dependent iron accumulation via Feo was demonstrated directly in iron transport assays using radiolabeled iron. A V. cholerae vciB mutant did not exhibit any growth defects in either in vitro or in vivo assays, possibly due to the presence of other systems with overlapping functions in this pathogen.     

A Functional Homolog of Escherichia coli NhaR in Vibrio cholerae

Escherichia coli NhaR controls expression of a sodium/proton (Na⁺/H⁺) antiporter, NhaA. The Vibrio cholerae NhaR protein shows over 60% identity to those of Escherichia coli and Salmonella enteritidis. V. cholerae NhaR complements an E. coli nhaR mutant for growth in 100 mM LiCl–33 mM NaCl, pH 7.6, and enhances the Na⁺-dependent induction of an E. coli chromosomal nhaA::lacZ fusion. These findings indicate functional homology to E. coli NhaR. Two V. cholerae nhaR mutants were constructed by using kanamycin resistance cartridge insertion at different sites to disrupt the gene. Both mutants showed sensitivity to growth in 120 mM LiCl, pH 9.2, compared with the wild-type strain and could be complemented by the introduction of V. cholerae nhaR on a low-copy-number plasmid. An nhaR mutation had no detectable effect on the virulence of the V. cholerae strain in the infant mouse model, suggesting that the antiporter system involved is not required in vivo, at least in this animal model.     

Characterization of the small untranslated RNA RyhB and its regulon in Vibrio cholerae

Numerous small untranslated RNAs (sRNAs) have been identified in Escherichia coli in recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved in Vibrio cholerae, and both identification and characterization of sRNAs in V. cholerae remain at a preliminary stage. We have characterized one of the few sRNAs conserved between E. coli and V. cholerae: RyhB. Sequence conservation is limited to the central region of the gene, and RyhB in V. cholerae is significantly larger than in E. coli. As in E. coli, V. cholerae RyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB in V. cholerae and E. coli appear to differ, although some overlap is evident. Analysis of gene expression in V. cholerae in the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host by V. cholerae.     

Quantitative Microbial Risk Assessment of Pathogenic Vibrios in Marine Recreational Waters of Southern California

This study investigated the occurrence of three types of vibrios in Southern California recreational beach waters during the peak marine bathing season in 2007. Over 160 water samples were concentrated and enriched for the detection of vibrios. Four sets of PCR primers, specific for Vibrio cholerae, V. parahaemolyticus, and V. vulnificus species and the V. parahaemolyticus toxin gene, respectively, were used for the amplification of bacterial genomic DNA. Of 66 samples from Doheny State Beach, CA, 40.1% were positive for V. cholerae and 27.3% were positive for V. parahaemolyticus, and 1 sample (1.5%) was positive for the V. parahaemolyticus toxin gene. Of the 96 samples from Avalon Harbor, CA, 18.7% were positive for V. cholerae, 69.8% were positive for V. parahaemolyticus, and 5.2% were positive for the V. parahaemolyticus toxin gene. The detection of the V. cholerae genetic marker was significantly more frequent at Doheny State Beach, while the detection of the V. parahaemolyticus genetic marker was significantly more frequent at Avalon Harbor. A probability-of-illness model for V. parahaemolyticus was applied to the data. The risk for bathers exposed to recreational waters at two beaches was evaluated through Monte Carlo simulation techniques. The results suggest that the microbial risk from vibrios during beach recreation was below the illness benchmark set by the U.S. EPA. However, the risk varied with location and the type of water recreation activities. Surfers and children were exposed to a higher risk of vibrio diseases. Microbial risk assessment can serve as a useful tool for the management of risk related to opportunistic marine pathogens.     

Pathogenomics of the Virulence Plasmids of Escherichia coli

Summary: Bacterial plasmids are self-replicating, extrachromosomal elements that are key agents of change in microbial populations. They promote the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to antimicrobial agents, and metabolism of rare substances. Escherichia coli, perhaps the most studied of microorganisms, has been found to possess a variety of plasmid types. Included among these are plasmids associated with virulence. Several types of E. coli virulence plasmids exist, including those essential for the virulence of enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli, and extraintestinal pathogenic E. coli. Despite their diversity, these plasmids belong to a few plasmid backbones that present themselves in a conserved and syntenic manner. Thanks to some recent research, including sequence analysis of several representative plasmid genomes and molecular pathogenesis studies, the evolution of these virulence plasmids and the implications of their acquisition by E. coli are now better understood and appreciated. Here, work involving each of the E. coli virulence plasmid types is summarized, with the available plasmid genomic sequences for several E. coli pathotypes being compared in an effort to understand the evolution of these plasmid types and define their core and accessory components.     

The coordinated replication of Vibrio cholerae’s two chromosomes required the acquisition of a unique domain by the RctB initiator

Vibrio cholerae, the pathogenic bacterium that causes cholera, has two chromosomes (Chr1, Chr2) that replicate in a well-orchestrated sequence. Chr2 initiation is triggered only after the replication of the crtS site on Chr1. The initiator of Chr2 replication, RctB, displays activities corresponding with its different binding sites: initiator at the iteron sites, repressor at the 39m sites, and trigger at the crtS site. The mechanism by which RctB relays the signal to initiate Chr2 replication from crtS is not well-understood. In this study, we provide new insights into how Chr2 replication initiation is regulated by crtS via RctB. We show that crtS (on Chr1) acts as an anti-inhibitory site by preventing 39m sites (on Chr2) from repressing initiation. The competition between these two sites for RctB binding is explained by the fact that RctB interacts with crtS and 39m via the same DNA-binding surface. We further show that the extreme C-terminal tail of RctB, essential for RctB self-interaction, is crucial for the control exerted by crtS. This subregion of RctB is conserved in all Vibrio, but absent in other Rep-like initiators. Hence, the coordinated replication of both chromosomes likely results from the acquisition of this unique domain by RctB.     

The mechanoelectrical response of the cytoplasmic membrane of Vibrio cholerae

Persistence of Vibrio cholerae in waters of fluctuating salinity relies on the capacity of this facultative enteric pathogen to adapt to varying osmotic conditions. In an event of osmotic downshift, osmolytes accumulated inside the bacterium can be quickly released through tension-activated channels. With the newly established procedure of giant spheroplast preparation from V. cholerae, we performed the first patch-clamp characterization of its cytoplasmic membrane and compared tension-activated currents with those in Esherichia coli. Saturating pressure ramps revealed two waves of activation belonging to the ∼1-nS mechanosensitive channel of small conductance (MscS)-like channels and ∼3-nS mechanosensitive channel of large conductance (MscL)-like channels, with a pressure midpoint ratio p_0.5MscS/p_0.5MscL of 0.48. We found that MscL-like channels in V. cholerae present at a density three times higher than in E. coli, and yet, these vibrios were less tolerant to large osmotic downshocks. The Vibrio MscS-like channels exhibit characteristic inward rectification and subconductive states at depolarizing voltages; they also adapt and inactivate at subsaturating tensions and recover within 2 s upon tension release, just like E. coli MscS. Trehalose, a compatible internal osmolyte accumulated under hypertonic conditions, significantly shifts activation curves of both MscL- and MscS-like channels toward higher tensions, yet does not freely partition into the channel pore. Direct electrophysiology of V. cholerae offers new avenues for the in situ analysis of membrane components critical for osmotic survival and electrogenic transport in this pathogen.     

On the Origins of a Vibrio Species

Thirty-two genome sequences of various Vibrionaceae members are compared, with emphasis on what makes V. cholerae unique. As few as 1,000 gene families are conserved across all the Vibrionaceae genomes analysed; this fraction roughly doubles for gene families conserved within the species V. cholerae. Of these, approximately 200 gene families that cluster on various locations of the genome are not found in other sequenced Vibrionaceae; these are possibly unique to the V. cholerae species. By comparing gene family content of the analysed genomes, the relatedness to a particular species is identified for two unspeciated genomes. Conversely, two genomes presumably belonging to the same species have suspiciously dissimilar gene family content. We are able to identify a number of genes that are conserved in, and unique to, V. cholerae. Some of these genes may be crucial to the niche adaptation of this species.     

Biotinylation Facilitates the Uptake of Large Peptides by Escherichia coli and Other Gram-Negative Bacteria

Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.     

Cell Separation in Vibrio cholerae Is Mediated by a Single Amidase Whose Action Is Modulated by Two Nonredundant Activators

Synthesis and hydrolysis of septal peptidoglycan (PG) are critical processes at the conclusion of cell division that enable separation of daughter cells. Cleavage of septal PG is mediated by PG amidases, hydrolytic enzymes that release peptide side chains from the glycan strand. Most gammaproteobacteria, including Escherichia coli, encode several functionally redundant periplasmic amidases. However, members of the Vibrio genus, including the enteric pathogen Vibrio cholerae, encode only a single PG amidase, AmiB. Here, we show that V. cholerae AmiB is crucial for cell division and growth. Genetic and biochemical analyses indicated that AmiB is regulated by two activators, EnvC and NlpD, at least one of which is required for AmiB''s localization to the cell division site. Localization of the activators (and thus of AmiB) is dependent upon the cell division protein FtsN. These factors mediate septal PG cleavage in E. coli as well; however, their precise roles vary between the two organisms in a number of ways. Notably, even though V. cholerae EnvC and NlpD appear to be functionally redundant under most growth conditions tested, NlpD is specifically required for intestinal colonization in the infant mouse model of cholera and for V. cholerae resistance against bile salts, perhaps due to environmental regulation of AmiB or its activators. Collectively, our findings reveal that although the cellular components that enable cleavage of septal PG appear to be generally conserved between E. coli and V. cholerae, they can be combined into diverse functional regulatory networks.     

A Family of Stability Determinants in Pathogenic Bacteria

A novel segregational stability system was identified on plasmid R485, which originates from Morganella morganii. The system is composed of two overlapping genes, stbD and stbE, which potentially encode proteins of 83 and 93 amino acids, respectively. Homologs of the stbDE genes were identified on the enterotoxigenic plasmid P307 from Escherichia coli and on the chromosomes of Vibrio cholerae and Haemophilus influenzae biogroup aegyptius. The former two homologs also promote plasmid stability in E. coli. Furthermore, the stbDE genes share homology with components of the relBEF operon and with the dnaT gene of E. coli. The organization of the stbDE cassette is reminiscent of toxin-antitoxin stability cassettes.     

Phenotypic characterization of pore mutants of the Vibrio cholerae porin OmpU

General-diffusion porins form large β-barrel channels that control the permeability of the outer membrane of gram-negative bacteria to nutrients, some antibiotics, and external signals. Here, we have analyzed the effects of mutations in the OmpU porin of Vibrio cholerae at conserved residues that are known to affect pore properties in the Escherichia coli porins OmpF and OmpC. Various phenotypes were investigated, including sensitivity to β-lactam antibiotics, growth on large sugars, and sensitivity to and biofilm induction by sodium deoxycholate, a major bile component that acts as an external signal for multiple cellular responses of this intestinal pathogen. Overall, our results indicate that specific residues play different roles in controlling the passage of various compounds. Mutations of barrel wall arginine residues that protrude in the pore affect pore size and growth in the presence of large sugars or sodium deoxycholate. Sensitivity to large cephalosporins is mostly affected by D116, located on the L3 loop, whose homolog in E. coli, OmpF, is a known binding determinant for these drugs. L3 loop residues also affect biofilm induction. The results are interpreted in terms of a homology model based on the structures of E. coli porins.     

In Vivo Transmission of an IncA/C Plasmid in Escherichia coli Depends on Tetracycline Concentration,and Acquisition of the Plasmid Results in a Variable Cost of Fitness

IncA/C plasmids are broad-host-range plasmids enabling multidrug resistance that have emerged worldwide among bacterial pathogens of humans and animals. Although antibiotic usage is suspected to be a driving force in the emergence of such strains, few studies have examined the impact of different types of antibiotic administration on the selection of plasmid-containing multidrug resistant isolates. In this study, chlortetracycline treatment at different concentrations in pig feed was examined for its impact on selection and dissemination of an IncA/C plasmid introduced orally via a commensal Escherichia coli host. Continuous low-dose administration of chlortetracycline at 50 g per ton had no observable impact on the proportions of IncA/C plasmid-containing E. coli from pig feces over the course of 35 days. In contrast, high-dose administration of chlortetracycline at 350 g per ton significantly increased IncA/C plasmid-containing E. coli in pig feces (P < 0.001) and increased movement of the IncA/C plasmid to other indigenous E. coli hosts. There was no evidence of conjugal transfer of the IncA/C plasmid to bacterial species other than E. coli. In vitro competition assays demonstrated that bacterial host background substantially impacted the cost of IncA/C plasmid carriage in E. coli and Salmonella. In vitro transfer and selection experiments demonstrated that tetracycline at 32 μg/ml was necessary to enhance IncA/C plasmid conjugative transfer, while subinhibitory concentrations of tetracycline in vitro strongly selected for IncA/C plasmid-containing E. coli. Together, these experiments improve our knowledge on the impact of differing concentrations of tetracycline on the selection of IncA/C-type plasmids.     

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps.     

Wide-Host-Range Plasmids Function in the Genus Thiobacillus

Plasmids S-a, RP4, R388, and several RP4 derivatives (pMD101, pDT387, and pDT566) were transmissible by conjugation to Thiobacillus novellus from Escherichia coli. Genetic markers were expressed in T. novellus, with the exception of chloramphenicol resistance and ampicillin resistance. Plasmids were not transmissible by conjugation from E. coli donors to Thiobacillus intermedius, T. perometabolis, T. neapolitanus, or T. acidophilus recipients, although they could be mated into these strains from T. novellus. All Thiobacillus species tested could transfer plasmids back to E. coli, with the exception of T. acidophilus. The donor-specific bacteriophages PRR1 and PRD1 were incapable of initiating the lytic cycle in RP4-bearing strains of T. novellus. The cosmid cloning vehicle pVK100 could be mobilized from E. coli to T. novellus with the aid of the “helper” plasmid pRK2013. pVK100 is stable in T. novellus, but pRK2013 is not maintained in this species. pRK2013 was also used to mobilize another cloning vector, R300B, to T. novellus. A previously unreported cryptic plasmid of approximately 24 megadaltons was observed in T. intermedius. No native plasmids were demonstrated in the other Thiobacillus species except in T. acidophilus, which contained cryptic plasmids ranging in size from 7.6 to 56 megadaltons (molecular mass).     

Phylogeny and Molecular Identification of Vibrios on the Basis of Multilocus Sequence Analysis

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.