Integrons are genetic elements that acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism. Characterized gene cassettes consist of a target recombination sequence (attC site) usually associated with a single open reading frame coding for an antibiotic resistance determinant. The affiliation of multiresistant integrons (MRIs), which contain various combinations of antibiotic resistance gene cassettes, with transferable elements underlies the rapid evolution of multidrug resistance among diverse Gram-negative bacteria. Yet the origin of MRIs remains unknown. Recently, a chromosomal super-integron (SI) harboring hundreds of cassettes was identified in the Vibrio cholerae genome. Here, we demonstrate that the activity of its associated integrase is identical to that of the MRI integrase, IntI1. We have also identified equivalent integron superstructures in nine distinct genera throughout the gamma-proteobacterial radiation. Phylogenetic analysis revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures. The attC sites of the 63 antibiotic-resistance gene cassettes identified thus far in MRIs are highly variable. Strikingly, one-fifth of these were virtually identical to the highly related yet species-specific attC sites of the SIs described here. Furthermore, antimicrobial resistance homologues were identified among the thousands of genes entrapped by these SIs. Because the gene cassettes of SIs are substrates for MRIs, these data identify SIs as the source of contemporary MRIs and their cassettes. However, our demonstration of the metabolic functions, beyond antibiotic resistance and virulence, of three distinct SI gene cassettes indicates that integrons function as a general gene-capture system for bacterial innovation.

The virulence regulatory protein ToxR of Vibrio cholerae is unique in that it contains a cytoplasmic DNA-binding-transcriptional activation domain, a transmembrane domain, and a periplasmic domain. Although ToxR and other transmembrane transcriptional activators have been discovered in other bacteria, little is known about their mechanism of activation. Utilizing degenerate oligonucleotides and PCR, we have amplified internal toxR gene sequences from seven Vibrio and Photobacterium species and subspecies, demonstrating that toxR is an ancestral gene of the family Vibrionaceae. Sequence alignment of all available ToxR amino acid sequences revealed a region between the transcriptional activation and transmembrane domains that displays wide divergence among Vibrio species. We hypothesize that this region merely tethers the transcriptional activation domain to the cytoplasmic membrane and thus can tolerate wide divergence and multiple insertions and deletions. The divergence in the tether region at the nucleotide level may provide a useful tool for the distinction of Vibrio and Photobacterium species.

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.

Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus-account for the majority of Vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus). The assay was tested on a sample of 309 Vibrio isolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional four V. parahaemolyticus isolates among the other 119 isolates. Sequence analysis based on rpoB was used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticus sequences. The rpoB sequences for 12 of 15 previously unidentified isolates clustered with other Vibrio species in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibrio species. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibrio pathogens in clinical samples, and rpoB sequencing provides an additional identification tool for other species in the genus Vibrio.

We had previously reported that Vibrio hollisae produces aerobactin in response to iron starvation. In the present study, we identified in V. hollisae ATCC33564 the aerobactin system cluster which consists of eight genes, hatCDB, iucABCD and iutA. The hatCDB genes encode proteins homologous to components of bacterial ATP binding cassette transport systems for ferric aerobactin. The iucABCD and iutA orthologs code for aerobactin biosynthesis enzymes and the ferric aerobactin receptor, respectively. In accordance with their iron-regulated expression, putative Fur box sequences were found within the respective promoter regions of hatC, iucA and iutA. The monocistronic iutA transcript was detected by northern blotting. Moreover, phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions that were expected for the respective operons and genes on the basis of the homology search. The arrangement of the aerobactin gene clusters thus far found in Vibrio and enterobacterial species was compared and discussed from an evolutionary point of view.

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.

Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.

This study analysed the usefulness of recA gene sequences as an alternative phylogenetic and/or identification marker for vibrios. The recA sequences suggest that the genus Vibrio is polyphyletic. The high heterogeneity observed within vibrios was congruent with former polyphasic taxonomic studies on this group. Photobacterium species clustered together and apparently nested within vibrios, while Grimontia hollisae was apart from other vibrios. Within the vibrios, Vibrio cholerae and Vibrio mimicus clustered apart from the other genus members. Vibrio harveyi- and Vibrio splendidus-related species formed compact separated groups. On the other hand, species related to Vibrio tubiashii appeared scattered in the phylogenetic tree. The pairs Vibrio coralliilyticus and Vibrio neptunius, Vibrio nereis and Vibrio xuii and V. tubiashii and Vibrio brasiliensis clustered completely apart from each other. There was a correlation of 0.58 between recA and 16S rDNA pairwise similarities. Strains of the same species have at least 94 % recA sequence similarity. recA gene sequences are much more discriminatory than 16S rDNA. For 16S rDNA similarity values above 98 % there was a wide range of recA similarities, from 83 to 99 %.

The collagenase gene was cloned from Grimontia (Vibrio) hollisae 1706B, and its complete nucleotide sequence was determined. Nucleotide sequencing showed that the open reading frame was 2,301 bp in length and encoded an 84-kDa protein of 767 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH motif. G. hollisae collagenase showed 60 and 59% amino acid sequence identities to Vibrio parahaemolyticus and Vibrio alginolyticus collagenase, respectively. In contrast, this enzyme showed < 20% sequence identity with Clostridium histolyticum collagenase. When the recombinant mature collagenase, which consisted of 680 amino acids with a calculated molecular mass of 74 kDa, was produced by the Brevibacillus expression system, a major gelatinolytic protein band of ~ 60 kDa was determined by zymographic analysis. This result suggested that cloned collagenase might undergo processing after secretion. Moreover, the purified recombinant enzyme was shown to possess a specific activity of 5,314 U/mg, an ~ 4-fold greater activity than that of C. histolyticum collagenase.

The gene (designated as Vh-tdh) of Vibrio hollisae 9041 encoding a hemolysin similar to the thermostable direct hemolysin (TDH) of V. parahaemolyticus contained a 567-base-pair open reading frame (ORF), which was 93.3-93.5% homologous to those of the tdh genes of V. parahaemolyticus, V. cholerae non-01, and V. mimicus encoding TDH or similar hemolysins. Comparative analysis of the nucleotide sequence containing the Vh-tdh ORF with published nucleotide and amino acid sequences suggested that the Vh-tdh gene and other tdh genes diverged from a common ancestral gene, that the divergence was closely associated with the evolutionary divergence of V. hollisae from other species of genus Vibrio, and that strain-to-strain variation of the Vh-tdh gene exists in V. hollisae.

We investigated the use of atpA gene sequences as alternative phylogenetic and identification markers for vibrios. A fragment of 1322 bp (corresponding to approximately 88% of the coding region) was analysed in 151 strains of vibrios. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. For instance, the Vibrio cholerae, Vibrio halioticoli, Vibrio harveyi and Vibrio splendidus species groups appeared in the atpA gene phylogenetic analyses, suggesting that these groups may be considered as separate genera within the current Vibrio genus. Overall, atpA gene sequences appeared to be more discriminatory for species differentiation than 16S rRNA gene sequences. 16S rRNA gene sequence similarities above 97% corresponded to atpA gene sequences similarities above 80%. The intraspecies variation in the atpA gene sequence was about 99% sequence similarity. The results showed clearly that atpA gene sequences are a suitable alternative for the identification and phylogenetic study of vibrios.

Microbial taxonomy is essential in all areas of microbial science. The 16S rRNA gene sequence is one of the main phylogenetic species markers; however, it does not provide discrimination in the family Vibrionaceae, where other molecular techniques allow better interspecies resolution. Although multilocus sequence analysis (MLSA) has been used successfully in the identification of Vibrio species, the technique has several limitations. They include the fact that several locus amplifications and sequencing have to be performed, which still sometimes lead to doubtful identifications. Using an in silico approach based on genomes from 103 Vibrionaceae strains, we demonstrate here the high resolution of the fur gene in the identification of Vibrionaceae species and its usefulness as a phylogenetic marker. The fur gene showed within-species similarity higher than 95%, and the relationships inferred from its use were in agreement with those observed for 16S rRNA analysis and MLSA. Furthermore, we developed a fur PCR sequencing-based method that allowed identification of Vibrio species. The discovery of the phylogenetic power of the fur gene and the development of a PCR method that can be used in amplification and sequencing of the gene are of general interest whether for use alone or together with the previously suggested loci in an MLSA.

Vibrio hollisae produces a hemolysin (Vh-rTDH) that is related to the thermostable direct hemolysin of Vibrio parahaemolyticus (Vp-TDH). Although both hemolysins are essentially similar biologically and immunologically, they differ markedly in heat stability; Vp-TDH is heat stable, whereas Vh-rTDH is heat labile. To elucidate the relationships between their characteristics and molecular structures, we analyzed the amino acid sequence of Vh-rTDH and compared it with that of Vp-TDH. Vh-rTDH consisted of 165 residues, of which 23 residues, spread over the peptide chain, differed from those of Vp-TDH.