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.

The family Vibrionaceae is considered to be one of the most diverse and well-studied groups of bacteria. Here, evolution is assessed within the Vibrionaceae to determine whether multiple origins of eukaryotic associations have occurred within this diverse group of bacteria. Analyses were based on a large molecular dataset, along with a matrix that consisted of 100 biochemical and restriction digest characters. By using direct optimization methods to analyse both datasets individually and in combination, a total-evidence cladogram has been produced, which supports the hypothesis that several important symbionts (both mutualistic and pathogenic) within the Vibrionaceae are not monophyletic. This leads us to consider that symbiosis (and subsequently, associations with Eukarya) has evolved multiple times within the Vibrionaceae lineage.

We synthesized population structure data from three studies that assessed the fine-scale distribution of Vibrionaceae among temporally and spatially distinct environmental categories in coastal seawater and animals. All studies used a dynamic model (AdaptML) to identify phylogenetically cohesive and ecologically distinct bacterial populations and their predicted habitats without relying on a predefined genetic cutoff or relationships to previously named species. Across the three studies, populations were highly overlapping, displaying similar phylogenetic characteristics (identity and diversity), and were predominantly congruent with taxonomic Vibrio species previously characterized as genotypic clusters by multilocus sequence analysis (MLSA). The environmental fidelity of these populations appears high, with 9 out of 12 reproducibly associating with the same predicted (micro)habitats when similar environmental categories were sampled. Overall, this meta-analysis provides information on the habitat predictability and structure of previously described species, demonstrating that MLSA-based taxonomy can, at least in some cases, serve to approximate ecologically cohesive populations.

The evolutionary relationship among Vibrio fischeri isolates obtained from the light organs of Euprymna scolopes collected around Oahu, Hawaii, were examined in this study. Phylogenetic reconstructions based on a concatenation of fragments of four housekeeping loci (recA, mdh, katA, pyrC) identified one monophyletic group ('Group-A') of V. fischeri from Oahu. Group-A V. fischeri strains could also be identified by a single DNA fingerprint type. V. fischeri strains with this fingerprint type had been observed to be at a significantly higher abundance than other strains in the light organs of adult squid collected from Maunalua Bay, Oahu, in 2005. We hypothesized that these previous observations might be related to a growth/survival advantage of the Group-A strains in the Maunalua Bay environments. Competition experiments between Group-A strains and non-Group-A strains demonstrated an advantage of the former in colonizing juvenile Maunalua Bay hosts. Growth and survival assays in Maunalua Bay seawater microcosms revealed a reduced fitness of Group-A strains relative to non-Group-A strains. From these results, we hypothesize that there may exist trade-offs between growth in the light organ and in seawater environments for local V. fischeri strains from Oahu. Alternatively, Group-A V. fischeri may represent an example of rapid, evolutionarily significant, specialization of a horizontally transmitted symbiont to a local host population.

The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N-acyl-homoserine lactone (AHL), the autoinducer-2 (AI-2) and the cholerae autoinducer-1-like (CAI-1-like) molecules in 25 Vibrionaceae strains. The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty-one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin-layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI-2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI-1-like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. The results indicated that the QS signal molecules, especially AHL and AI-2 molecules, were widespread in the family Vibrionaceae. In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.

Although animal-associated microbial communities (microbiomes) are increasingly recognized to influence health, the extent to which animals represent highly selective habitats for microbes leading to predominance of high host specificity remains poorly understood. Here, we show that vibrios, which are well-known commensals and opportunistic pathogens of marine animals, overall display little host preference, likely because of efficient dispersal-colonization dynamics mediated by food items. We isolated 1753 strains from water and animal samples, which are linked in a food chain and display different degrees of similarity (respiratory and digestive tract of mussels and crabs, live and dead zooplankton, and whole water samples). Multilocus sequence data served as input for modelling and statistical analysis of spatiotemporal population structure. These data showed that the majority of populations occurred broadly within and among hosts, with the dominant population being a near perfect generalist with regard to seasons, host taxa and body regions. Zooplankton harboured the fewest and most specific populations, while crabs and mussels contained the highest diversity with little evidence for host preferences. Most mussel- and crab-associated populations were detected in water samples at similar frequencies, particularly in filter-feeding mussels where populations were also evenly distributed across host individuals. The higher variation among individuals observed in crabs and zooplankton is consistent with stochastic clonal expansions. These patterns suggest that evolution of a high degree of host specificity is surprisingly rare even though these animals represent long-lived habitats, and vibrios are consistent members of their microbiome. Instead, many of the populations show stronger association with planktonic (micro)habitats while the microbiome may be a fairly open system for vibrios in which high rates of immigration can outpace selection for specialization.

The "Vibrio fischeri species group" recently was reclassified as a new genus, Aliivibrio, comprising four species, Aliivibrio fischeri, Aliivibrio logei, Aliivibrio salmonicida, and Aliivibrio wodanis. Only limited phylogenetic analysis of strains within Aliivibrio has been carried out, however, and taxonomic ambiguity is evident within this group, especially for phenotypically unusual strains and certain strains isolated from bioluminescent symbioses. Therefore, to examine in depth the evolutionary relationships within Aliivibrio and redefine the host affiliations of symbiotic species, we examined several previously identified and newly isolated strains using phylogenetic analysis based on multiple independent loci, gapA, gyrB, pyrH, recA, rpoA, the luxABE region, and the 16S rRNA gene. The analysis resolved Aliivibrio as distinct from Vibrio, Photobacterium, and other genera of Vibrionaceae, and resolved A. fischeri, A. salmonicida, A. logei, and A. wodanis as distinct, well-supported clades. However, it also revealed that several previously reported strains are incorrectly identified and that substantial unrecognized diversity exists in this genus. Specifically, strain ATCC 33715 (Y-1) and several other strains having a yellow-shifted luminescence were not members of A. fischeri. Furthermore, no strain previously identified as A. logei grouped with the type strain (ATCC 29985(T)), and no bona-fide strain of A. logei was identified as a bioluminescent symbiont. Several additional strains identified previously as A. logei group instead with the type strain of A. wodanis (ATCC BAA-104(T)), or are members of a new clade. Two strongly supported clades were evident within A. fischeri, a phylogenetic structure that might reflect differences in the host species or differences in the ecological incidence of strains. The results of this study highlight the importance of basing taxonomic conclusions on examination of type strains.

Vibrionaceae are regarded as important marine chitin degraders, and attachment to chitin regulates important biological functions; yet, the degree of chitin pathway conservation in Vibrionaceae is unknown. Here, a core chitin degradation pathway is proposed based on comparison of 19 Vibrio and Photobacterium genomes with a detailed metabolic map assembled for V. cholerae from published biochemical, genomic, and transcriptomic results. Further, to assess whether chitin degradation is a conserved property of Vibrionaceae, a set of 54 strains from 32 taxa were tested for the ability to grow on various forms of chitin. All strains grew on N-acetylglucosamine (GlcNAc), the monomer of chitin. The majority of isolates grew on alpha (crab shell) and beta (squid pen) chitin and contained chitinase A (chiA) genes. chiA sequencing and phylogenetic analysis suggest that this gene is a good indicator of chitin metabolism but appears subject to horizontal gene transfer and duplication. Overall, chitin metabolism appears to be a core function of Vibrionaceae, but individual pathway components exhibit dynamic evolutionary histories.

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.

Sequence analysis of the bacterial luminescence (lux) genes has proven effective in helping resolve evolutionary relationships among luminous bacteria. Phylogenetic analysis using lux genes, however, is based on the assumptions that the lux genes are present as single copies on the bacterial chromosome and are vertically inherited. We report here that certain strains of Photobacterium leiognathi carry multiple phylogenetically distinct copies of the entire operon that codes for luminescence and riboflavin synthesis genes, luxCDABEG-ribEBHA. Merodiploid lux-rib strains of P. leiognathi were detected during sequence analysis of luxA. To define the gene content, organization, and sequence of each lux-rib operon, we constructed a fosmid library of genomic DNA from a representative merodiploid strain, lnuch.13.1. Sequence analysis of fosmid clones and genomic analysis of lnuch.13.1 defined two complete, physically separate, and apparently functional operons, designated lux-rib1 and lux-rib2. P. leiognathi strains lelon.2.1 and lnuch.21.1 were also found to carry lux-rib1 and lux-rib2, whereas ATCC 25521T apparently carries only lux-rib1. In lnuch.13.1, lelon.2.1, lnuch.21.1, and ATCC 25521T, lux-rib1 is flanked upstream by lumQ and putA and downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flank lux-rib2 of lnuch.13.1, and the chromosomal location of lux-rib2 apparently differs in lnuch.13.1, lelon.2.1, and lnuch.21.1. Phylogenetic analysis demonstrated that lux-rib1 and lux-rib2 are more closely related to each other than either one is to the lux and rib genes of other bacterial species, which rules out interspecies lateral gene transfer as the origin of lux-rib2 in P. leiognathi; lux-rib2 apparently arose within a previously unsampled or extinct P. leiognathi lineage. Analysis of 170 additional strains of P. leiognathi, for a total of 174 strains examined from coastal waters of Japan, Taiwan, the Philippine Islands, and Thailand, identified 106 strains that carry only a single lux-rib operon and 68 that carry multiple lux-rib operons. Strains bearing a single lux-rib operon were obtained throughout the geographic sampling range, whereas lux-rib merodiploid strains were found only in coastal waters of central Honshu. This is the first report of merodiploidy of lux or rib genes in a luminous bacterium and the first indication that a natural merodiploid state in bacteria can correlate with geography.

The lux-operon ?f the psychrophilic bioluminescent bacterium A. logei is regulated by Quorum Sensing (QS). The key components of this system are LuxI that catalyzes the synthesis of autoinducer (AI) and LuxR that activates the transcription of entire lux-operon. The lux-operon of A. logei contains two copies of luxR gene: luxR1 and luxR2. In the present study lux-operon sequence analysis from 16 strains of A. logei, isolated from cold habitats of the White, Baltic, Okhotsk and Bering Seas was carried out. The phylogenetic analysis showed that all isolated strains of A. logei have both copies of luxR genes which are homologous to luxR genes of relative A. salmonicida. LuxR1 and LuxR2 activity evaluation showed that LuxR2 remain active at significantly lower concentration of AI (10-9M) compared to LuxR1 which is activate only at high AI concentration (10-6?). As QS response is already prominent at AI concentrations as low as 10-8 - 10-9M, we conclude that LuxR2 is a main activator of the lux-operon of A. logei. The thermolabilities of LuxR1 and LuxR2 are similar and exceed that of LuxR of mesophilic bacterium A. fischeri. As oppose to LuxR2, LuxR1 is not a substrate of Lon protease and does not require the chaperonin GroEL/ES for its folding.

Here we provide a molecular description of a new psychrophilic strain, KCh11, of marine luminescent bacteria classified as Aliivibrio logei. We sequenced the entire lux operon of A. logei KCh1 and showed that it is substantially similar to the lux operon of Aliivibrio salmonicida. It was demonstrated that the reduced production of bioluminescence in A. salmonicida is most likely defined by a specific defect in its luxD gene.