LuxT controls specific quorum-sensing-regulated behaviors in Vibrionaceae spp. via repression of qrr1, encoding a small regulatory RNA.

Quorum sensing (QS) is a process of chemical communication bacteria use to transition between individual and collective behaviors. QS depends on the production, release, and synchronous response to signaling molecules called autoinducers (AIs). The marine bacterium Vibrio harveyi monitors AIs using a signal transduction pathway that relies on five small regulatory RNAs (called Qrr1-5) that post-transcriptionally control target genes. Curiously, the small RNAs largely function redundantly making it difficult to understand the necessity for five of them. Here, we identify LuxT as a transcriptional repressor of qrr1. LuxT does not regulate qrr2-5, demonstrating that qrr genes can be independently controlled to drive unique downstream QS gene expression patterns. LuxT reinforces its control over the same genes it regulates indirectly via repression of qrr1, through a second transcriptional control mechanism. Genes dually regulated by LuxT specify public goods including an aerolysin-type pore-forming toxin. Phylogenetic analyses reveal that LuxT is conserved among Vibrionaceae and sequence comparisons predict that LuxT represses qrr1 in additional species. The present findings reveal that the QS regulatory RNAs can carry out both shared and unique functions to endow bacteria with plasticity in their output behaviors.

Population genomics of Vibrionaceae isolated from an endangered oasis reveals local adaptation after an environmental perturbation.

BACKGROUND: In bacteria, pan-genomes are the result of an evolutionary "tug of war" between selection and horizontal gene transfer (HGT). High rates of HGT increase the genetic pool and the effective population size (Ne), resulting in open pan-genomes. In contrast, selective pressures can lead to local adaptation by purging the variation introduced by HGT and mutation, resulting in closed pan-genomes and clonal lineages. In this study, we explored both hypotheses, elucidating the pan-genome of Vibrionaceae isolates after a perturbation event in the endangered oasis of Cuatro Ciénegas Basin (CCB), Mexico, and looking for signals of adaptation to the environments in their genomes. RESULTS: We obtained 42 genomes of Vibrionaceae distributed in six lineages, two of them did not showed any close reference strain in databases. Five of the lineages showed closed pan-genomes and were associated to either water or sediment environment; their high Ne estimates suggest that these lineages are not from a recent origin. The only clade with an open pan-genome was found in both environments and was formed by ten genetic groups with low Ne, suggesting a recent origin. The recombination and mutation estimators (r/m) ranged from 0.005 to 2.725, which are similar to oceanic Vibrionaceae estimations. However, we identified 367 gene families with signals of positive selection, most of them found in the core genome; suggesting that despite recombination, natural selection moves the Vibrionaceae CCB lineages to local adaptation, purging the genomes and keeping closed pan-genome patterns. Moreover, we identify 598 SNPs associated with an unstructured environment; some of the genes associated with these SNPs were related to sodium transport. CONCLUSIONS: Different lines of evidence suggest that the sampled Vibrionaceae, are part of the rare biosphere usually living under famine conditions. Two of these lineages were reported for the first time. Most Vibrionaceae lineages of CCB are adapted to their micro-habitats rather than to the sampled environments. This pattern of adaptation is concordant with the association of closed pan-genomes and local adaptation.

Enterovibrio baiacu sp. nov.

We report here the novel species to encompass the isolate A649T (=CBAS 716T = CBRVS P1061T) obtained from viscera of the healthy pufferfish Sphoeroides spengleri (Family Tetraodontidae). Genomic taxonomy analysis demonstrates that the novel strain A649T had < 95% average amino acid identity/average nucleotide identity (AAI/ANI) and < 70% similarity of genome-to-genome distance (GGDH) towards its closest neighbors which places A649T into a new Enterovibrio species (Enterovibrio baiacu sp nov.). In silico phenotyping disclosed several features that may be used to differentiate related Enterovibrio species. The nearly complete genome assembly of strain A649T consisted of 5.4 Mbp and 4826 coding genes.

A structure-function analysis of interspecies antagonism by the 2-heptyl-4-alkyl-quinolone signal molecule from Pseudomonas aeruginosa.

In recent years, the alkyl-quinolone molecular framework has already provided a rich source of bioactivity for the development of novel anti-infective compounds. Based on the quorum-sensing signalling molecules 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS) from the nosocomial pathogen Pseudomonas aeruginosa, modifications have been developed with markedly enhanced anti-biofilm bioactivity towards important fungal and bacterial pathogens, including Candida albicans and Aspergillus fumigatus. Here we show that antibacterial activity of HHQ against Vibrionaceae is species-specific and it requires an exquisite level of structural fidelity within the alkyl-quinolone molecular framework. Antibacterial activity was demonstrated against the serious human pathogens Vibrio vulnificus and Vibrio cholerae as well as a panel of bioluminescent squid symbiont Allivibrio fischeri isolates. In contrast, Vibrio parahaemolyticus growth and biofilm formation was unaffected in the presence of HHQ and all the structural variants tested. In general, modification to almost all of the molecule except the alkyl-chain end, led to loss of activity. This suggests that the bacteriostatic activity of HHQ requires the concerted action of the entire framework components. The only exception to this pattern was deuteration of HHQ at the C3 position. HHQ modified with a terminal alkene at the quinolone alkyl chain retained bacteriostatic activity and was also found to activate PqsR signalling comparable to the native agonist. The data from this integrated analysis provides novel insights into the structural flexibility underpinning the signalling activity of the complex alkyl-quinolone molecular communication system.

Regulatory context drives conservation of glycine riboswitch aptamers.

In comparison to protein coding sequences, the impact of mutation and natural selection on the sequence and function of non-coding (ncRNA) genes is not well understood. Many ncRNA genes are narrowly distributed to only a few organisms, and appear to be rapidly evolving. Compared to protein coding sequences, there are many challenges associated with assessment of ncRNAs that are not well addressed by conventional phylogenetic approaches, including: short sequence length, lack of primary sequence conservation, and the importance of secondary structure for biological function. Riboswitches are structured ncRNAs that directly interact with small molecules to regulate gene expression in bacteria. They typically consist of a ligand-binding domain (aptamer) whose folding changes drive changes in gene expression. The glycine riboswitch is among the most well-studied due to the widespread occurrence of a tandem aptamer arrangement (tandem), wherein two homologous aptamers interact with glycine and each other to regulate gene expression. However, a significant proportion of glycine riboswitches are comprised of single aptamers (singleton). Here we use graph clustering to circumvent the limitations of traditional phylogenetic analysis when studying the relationship between the tandem and singleton glycine aptamers. Graph clustering enables a broader range of pairwise comparison measures to be used to assess aptamer similarity. Using this approach, we show that one aptamer of the tandem glycine riboswitch pair is typically much more highly conserved, and that which aptamer is conserved depends on the regulated gene. Furthermore, our analysis also reveals that singleton aptamers are more similar to either the first or second tandem aptamer, again based on the regulated gene. Taken together, our findings suggest that tandem glycine riboswitches degrade into functional singletons, with the regulated gene(s) dictating which glycine-binding aptamer is conserved.

Characterization of a chitinase from Salinivibrio sp. BAO-1801 as an antifungal activity and a biocatalyst for producing chitobiose.

Salinivibrio genus is commonly found in salted seafood products. In this study, chitinase produced by Salinivibrio sp. BAO-1801 isolated from salted fermented shrimp was purified and subsequently characterized. The molecular weight of BAO-1801 chitinase was approximately 94.2 kDa by SDS-PAGE analysis. It was classified as a chitinase C based on homology analysis of its N-terminal amino acid residues. This strain BAO-1801 chitinase was then used for synthesis of (GlcNAc)2. Degradation of colloidal chitin and N-acetyl chitooligosaccharides by BAO-1801 chitinase was then analyzed and (GlcNAc)2 was identified as the main product by thin layer chromatography and high-performance liquid chromatography. Effects of temperature and pH on activity and stability of BAO-1801 chitinase were also investigated. Furthermore, this enzyme inhibited fungal growth in a dose-dependent manner. Taken together, these results suggest that this Salinivibrio or its chitinase can be used for the enzymatic degradation of chitin to produce chitobiose in industrial process.

Emended description of Salinivibrio proteolyticus, including Salinivibrio costicola subsp. vallismortis and five new isolates.

We carried out a comparative taxonomic study of Salinivibrio proteolyticus and Salinivibrio costicola subsp. vallismortis, as well as of five halophilic strains (IB574, IB872, PR5, PR919 and PR932), isolated from salterns in Spain and Puerto Rico that were closely related to these bacteria. Multilocus sequence analysis of concatenated gyrB, recA, rpoA and rpoD housekeeping genes showed that they constituted a single cluster separate from the other species and subspecies of Salinivibrio. Experimental and in silico DNA-DNA hybridization studies indicated that they are members of the same species, with relatedness of 100-74 % and 97.8-70.0 %, respectively. The average nucleotide identity (ANI) determined for these strains was 99.7-95.6 % for ANIb and 99.7-95.7 % for OrthoANI. However, the ANI values for S. costicolasubsp.vallismortis DSM 8285T with respect to S. costicolasubsp.costicola DSM 11403T and S. costicolasubsp.alcaliphilus DSM 16359T were 78.7 and 78.9 % (ANIb) and 79.4 and 79.4 % (OrthoANI), respectively. The phylogenomic tree based on 1072 concatenated orthologous single-copy core genes confirmed that S. proteolyticus, S. costicolasubsp.vallismortis and the five new isolates constitute a coherent single phylogroup, separated from the other species and subspecies of Salinivibrio. All these data indicate that S. costicolasubsp.vallismortis is a heterotypic synonym of S. proteolyticus and we propose an emended description of this species.

Corallibacterium pacifica gen. nov., sp. nov., a Novel Bacterium of the Family Vibrionaceae Isolated from Hard Coral.

A gram-negative, rod-shaped, motile, oxidase- and catalase-positive, non-pigmented marine bacterium, designated strain OS-11M-2T, was isolated from a coral sample collected from the Osakura coastal area in Micronesia. Phylogenetic analysis based on 16S ribosomal RNA (rRNA) gene sequences indicated that strain OS-11M-2T is a member of the family Vibrionaceae, its closest neighbors being Photobacterium damselae subsp. piscicida NCIMB 2058T (94.9%), Photobacterium damselae subsp. damselae CIP 102761T (94.75%), Grimontia marina IMCC5001T (94.5%), Enterovibrio coralii LMG 22228T (94.5%), and Grimontia celer 96-237T (94.5%). The major cellular fatty acids were summed feature 3 (21.4%), summed feature 8 (18.5%), iso-C16:0 (13.8%), and C16:0 (11.9%). The major respiratory quinone of the bacterium was ubiquinone-8 (Q-8) and its major polar lipid phosphatidylethanolamine. Six amino lipids, two phospholipids, and one polar lipid, all unidentified, were detected. The DNA G+C content was 49.7 mol%. The 16S rRNA gene sequence of OS-11M-2T was registered in GenBank under accession number MF359550. On the basis of phenotypic, genotypic, and phylogenetic analyses, strain OS-11M-2T represents a novel genus of the family Vibrionaceae, for which we propose the name Corallibacterium pacifica gen. nov., sp. nov., with the type strain of the type species being OS-11M-2T (= KCCM 43265T). The digital protologue database (DPD) taxon number for strain OS-11M-2T is GA00041.

Distribution of siderophore gene systems on a Vibrionaceae phylogeny: Database searches, phylogenetic analyses and evolutionary perspectives.

Siderophores are small molecules synthesized and secreted by bacteria and fungi to scavenge iron. Extracellular ferri-siderohores are recognized by cognate receptors on the cell surface for transport over membranes. Several siderophore systems from Vibrionaceae representatives are known and well understood, e.g., the molecular structure of the siderophore, the biosynthesis gene cluster and pathway, and the gene expression pattern. Less is known about how these systems are distributed among the ~140 Vibrionaceae species, and which evolutionary processes contributed to the present-day distribution. In this work, we compiled existing knowledge on siderophore biosynthesis systems and siderophore receptors from Vibrionaceae and used phylogenetic analyses to investigate their organization, distribution, origin and evolution. Through literature searches, we identified nine different siderophore biosynthesis systems and thirteen siderophore receptors in Vibrionaceae. Homologs were identified by BLAST searches, and the results were mapped onto a Vibrionaceae phylogeny. We identified 81 biosynthetic systems distributed in 45 Vibrionaceae species and 16 unclassified Vibrionaceae strains, and 409 receptors in 89 Vibrionaceae species and 49 unclassified Vibrionaceae strains. The majority of taxa are associated with at least one type of siderophore biosynthesis system, some (e.g., aerobactin and vibrioferrin) of which are widely distributed in the family, whereas others (i.e., bisucaberin and vibriobactin) are found in one lineage. Cognate receptors are found more widespread. Phylogenetic analysis of three siderophore systems (piscibactin, vibrioferrin and aerobactin) show that their present-day distribution can be explained by an old insertion into Vibrionaceae, followed mainly by stable vertical evolution and extensive loss, and some cases of horizontal gene transfers. The present work provides an up to date overview of the distribution of siderophore-based iron acquisition systems in Vibrionaceae, and presents phylogenetic analysis of these systems. Our results suggest that the present-day distribution is a result of several evolutionary processes, such as old and new gene acquisitions, gene loss, and both vertical and horizontal gene transfers.

Salinivibrio kushneri sp. nov., a moderately halophilic bacterium isolated from salterns.

Ten Gram-strain-negative, facultatively anaerobic, moderately halophilic bacterial strains, designated AL184T, IB560, IB563, IC202, IC317, MA421, ML277, ML318, ML328A and ML331, were isolated from water ponds of five salterns located in Spain. The cells were motile, curved rods and oxidase and catalase positive. All of them grew optimally at 37°C, at pH 7.2-7.4 and in the presence of 7.5% (w/v) NaCl. Based on phylogenetic analyses of the 16S rRNA, the isolates were most closely related to Salinivibrio sharmensis BAGT (99.6-98.2% 16S rRNA gene sequence similarity) and Salinivibrio costicola subsp. costicola ATCC 35508T (99.0-98.1%). According to the MLSA analyses based on four (gyrB, recA, rpoA and rpoD) and eight (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA and topA) concatenated gene sequences, the most closely relatives were S. siamensis JCM 14472T (96.8-95.4% and 94.9-94.7%, respectively) and S. sharmensis DSM 18182T (94.0-92.6% and 92.9-92.7%, respectively). In silico DNA-DNA hybridization (GGDC) and average nucleotide identity (ANI) showed values of 23.3-44.8% and 80.2-91.8%, respectively with the related species demonstrating that the ten isolates constituted a single novel species of the genus Salinivibrio. Its pangenome and core genome consist of 6041 and 1230 genes, respectively. The phylogeny based on the concatenated orthologous core genes revealed that the ten strains form a coherent phylogroup well separated from the rest of the species of the genus Salinivibrio. The major cellular fatty acids of strain AL184T were C16:0 and C18:1. The DNA G+C content range was 51.9-52.5mol% (Tm) and 50.2-50.9mol% (genome). Based on the phylogenetic-phylogenomic, phenotypic and chemotaxonomic data, the ten isolates represent a novel species of the genus Salinivibrio, for which the name Salinivibrio kushneri sp. nov. is proposed. The type strain is AL184T (=CECT 9177T=LMG 29817T).

Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, Japan.

Two phylogenetically distinct Vibrionaceae strains C4II189T and C4V358T isolated from reef seawater off Ishigaki Island, Japan, in 2014 were studied with advanced genome-based taxonomy approaches. All aspects of phylogenetic (16S rRNA phylogeny, MLSA), phenotypic and genetic (ANI, DDH, AAI, and the number of core genes) cohesions between the two identified species were high enough to propose them as members of a new genus within the family Vibrionaceae. Consequently, an eighth genus Thaumasiovibrio gen. nov. is proposed that contains two new species Thaumasiovibrio occultus sp. nov. strain C4II189T (=DSM 101554T=JCM 31629T) (type species) and Thaumasiovibrio subtropicus sp. nov. strain C4V358T (=DSM 101555T=JCM 31630T). Thaumasiovibrio species were phylogenetically distinct from the other Vibrionaceae species based on pyrH gene sequences. The combination of catalase negative, sensitivity to vibriostatic agent O/129, and green colony formation on TCBS for the phylogenetically affiliated strains was the diagnostic features for the current tentative identification of this genus.

Salinivibrio costicola GL6, a Novel Isolated Strain for Biotransformation of Caffeine to Theobromine Under Hypersaline Conditions.

The present study has been conducted towards isolation of moderately halophilic bacteria capable of transforming caffeine into theobromine. A total of 45 caffeine-degrading moderate halophiles were enriched from hypersaline lakes and examined for the biotransformation of caffeine to theobromine by thin-layer chromatography (TLC) and high-performance liquid chromatography analyses. Strain GL6, giving the highest yield of theobromine, was isolated from the Hoz Soltan Lake, 20 % w/v salinity, central Iran, and identified as Salinivibrio costicola based on morphological and biochemical features as well as its 16S rRNA gene sequence analysis (GeneBank Accession No. KT378066) and DNA-DNA relatedness. The biotransformation of caffeine with strain GL6 leads to the formation of two metabolites, identified as theobromine and paraxanthine, but the yield of paraxanthine was much lower. Further study on the production of theobromine from caffeine under resting cell experiment was carried out subsequently. The optimal yield of theobromine (56 %) was obtained after a 32-h incubation using 5 mM of caffeine and 15 g l-1 (wet weight) of biomass in 0.1 M saline phosphate buffer (pH 7.0 and 10 % w/v NaCl) under agitation 180 rpm at 30 °C. The biotransformed theobromine was purified by preparative TLC and subjected to FTIR and mass spectroscopy for chemical identification. This is the first evidence for biotransformation of caffeine into theobromine by strains of the genus Salinivibrio.

Genome Evolution in the Obligate but Environmentally Active Luminous Symbionts of Flashlight Fish.

The luminous bacterial symbionts of anomalopid flashlight fish are thought to be obligately dependent on their hosts for growth and share several aspects of genome evolution with unrelated obligate symbionts, including genome reduction. However, in contrast to most obligate bacteria, anomalopid symbionts have an active environmental phase that may be important for symbiont transmission. Here we investigated patterns of evolution between anomalopid symbionts compared with patterns in free-living relatives and unrelated obligate symbionts to determine if trends common to obligate symbionts are also found in anomalopid symbionts. Two symbionts, "Candidatus Photodesmus katoptron" and "Candidatus Photodesmus blepharus," have genomes that are highly similar in gene content and order, suggesting genome stasis similar to ancient obligate symbionts present in insect lineages. This genome stasis exists in spite of the symbiont's inferred ability to recombine, which is frequently lacking in obligate symbionts with stable genomes. Additionally, we used genome comparisons and tests of selection to infer which genes may be particularly important for the symbiont's ecology compared with relatives. In keeping with obligate dependence, substitution patterns suggest that most symbiont genes are experiencing relaxed purifying selection compared with relatives. However, genes involved in motility and carbon storage, which are likely to be used outside the host, appear to be under increased purifying selection. Two chemoreceptor chemotaxis genes are retained by both species and show high conservation with amino acid sensing genes, suggesting that the bacteria may actively seek out hosts using chemotaxis toward amino acids, which the symbionts are not able to synthesize.

Paraphotobacterium marinum gen. nov., sp. nov., a member of the family Vibrionaceae, isolated from surface seawater.

A novel Gram-stain-negative bacterium, named strain NSCS20N07DT, was isolated from surface seawater of the South China Sea. Cells of this strain contained poly-ß-hydroxybutyrate granules. Growth was observed at 15-35 °C with optimum of 30 °C, at a salinity range of 1-6 % (w/v) NaCl with optimum of 3 % and at pH 5-8 with optimum of pH 5. The full-length 16S rRNA gene sequence of strain NSCS20N07DT showed highest similarity to Photobacterium iliopiscarium ATCC 51760T of 96.0 %, followed by Photobacterium kishitanii pjapo.1.1T, Photobacterium phosphoreum ATCC 11040T and Photobacterium aquimaris LC2-065T of 96.0, 95.8 and 95.7 %, respectively. Phylogenetic analysis showed that strain NSCS20N07DT formed a separate clade distinct from species of the genus Photobacterium and other genera within the family Vibrionaceae, indicating that strain NSCS20N07DT represented a novel genus affiliated with this family. The genome size of strain NSCS20N07DT was 2.5 Mb, which was much smaller than those of related species in the family Vibrionaceae. The predominant fatty acids were C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c, C18 : 0 and summed feature 2 (C14 : 0 3-OH/iso-C16 : 1 I). The respiratory quinone was Q-8. The polar lipids were identified as phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and four unidentified lipids. The DNA G+C content was 30.7 mol%. Combined, these results suggest that strain NSCS20N07DT represents a novel species of a new genus, for which the name Paraphotobacterium marinum gen. nov., sp. nov. is proposed. The type strain of Paraphotobacterium marinum is NSCS20N07DT (=KCTC 52126T=MCCC 1A01886T=CIP 111031T).

Grimontia celer sp. nov., from sea water.

Strain 96-237T, a Gram-reaction-negative, curved- to spiral-shaped motile bacterium, isolated from coastal marine water, was found to be related to species of the genus Grimontia by 16S rRNA gene sequence comparison, sharing 98.3 % similarity to Grimontia marina CECT 8713Tand 98.8 % to 'Grimontiaindica' AK16. Phenotypic analysis revealed that strain 96-237T is slightly halophilic, mesophilic and facultatively anaerobic, fermenting d-glucose, d-ribose, d-mannose, d-mannitol, maltose and sucrose. It was positive for oxidase and indole production and negative for arginine dihydrolase and lysine and ornithine decarboxylases. Its major fatty acids were C16 : 1ω7c/C16 : 1ω6c (SF3), C18 : 1ω7c and C16 : 0. Its DNA G+C content was 48.4 mol%. The strain was different at the species level from all other species of the genusGrimontia, with average nucleotide identity indices of 79.6 % to Grimontia. hollisae CECT 8713T, 87.8 % to G. marina CECT 5069T and 89.1 % to 'G. indica' AK16 genomes. Thus, the strain represents a novel species for which we propose the name Grimontia celer and 96-237T (=CECT 9029T =KCTC 42960T =LMG 29238T) as the type strain.

Enterovibrio pacificus sp. nov., isolated from seawater, and emended descriptions of Enterovibrio coralii and the genus Enterovibrio.

A Gram-stain-negative, elliptical and facultatively anaerobic strain, designated SW014T, motile by means of a single polar flagellum and positive for poly-ß-hydroxybutyrate accumulation, was isolated from surface seawater of the South Pacific Gyre, during the Integrated Ocean Drilling Program Expedition 329. The strain was able to grow at 10-37 °C (optimum 28 °C). Growth was observed at NaCl concentrations (w/v) of 1-7 % (optimum 3-4 %). The pH range for growth was 7.0-9.0 (optimum pH 8.0). Phylogenetic analysis based on 16S rRNA gene sequences and multilocus sequence analysis indicated that strain SW014T belongs to the genus Enterovibrio within the family Vibrionaceae and is related most closely to Enterovibrio coralii LMG 22228T with 96.3, 83.7, 95.0, 77.1, 84.1 and 85.8 % sequence similarity based on 16S rRNA, recA, rpoA, rpoD, pyrH and ftsZ genes, respectively. The predominant cellular fatty acids were C16 : 1ω7c and/or C16 : 1ω6c, C16 : 0, and C18 : 1ω7c and/or C18 : 1ω6c. The respiratory quinone was ubiquinone-8 (Q-8). The polar lipids of strain SW014T comprised phosphatidylethanolamine, glycolipid, two unidentified aminolipids, two unidentified phospholipids and two unidentified polar lipids. The DNA G+C content was 44.8 mol%. Combining phylogenetic analysis, phenotypic characteristics and chemotaxonomic studies, strain SW014T represents a novel species of the genus Enterovibrio, for which the name Enterovibrio pacificus sp. nov. is proposed. The type strain is SW014T ( = KCTC 42425T = MCCC 1K00500T). Emended descriptions of Enterovibrio coralii and of the genus Enterovibrio are also provided.

The fur gene as a new phylogenetic marker for Vibrionaceae species identification.

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.

Phylogenetic divergence between the obligate luminous symbionts of flashlight fishes demonstrates specificity of bacteria to host genera.

The luminous bacterial symbionts of anomalopid flashlight fishes, which appear to be obligately dependent on their hosts for growth, share several evolutionary patterns with unrelated obligate bacteria. However, only one flashlight fish symbiont species has been characterized in detail, and it is therefore not known if the bacteria from other anomalopid species are highly divergent (a pattern common to obligate symbionts). Unlike most obligate symbionts, the bacteria symbiotic with anomalopids are extracellular and spend time outside their hosts in the environment, from which they are thought to colonize new host generations. Environmental acquisition might decrease the likelihood of bacterial divergence between host species. We used phylogenetic analysis to determine the relatedness of symbionts from different anomalopid host species. The symbionts of hosts in the genus Photoblepharon were resolved as a new species, for which we propose the name 'Candidatus Photodesmus blepharus'. Furthermore, different genera of anomalopids were found to harbour different species of bacteria, even when the hosts overlapped in geographic range. This finding suggests that the divergence between bacterial species is not the result of geographic isolation. The specificity of symbionts to host genera is consistent with obligate dependence on the host and has implications for symbiont transmission.

Pyrosequencing-based characterization of gastrointestinal bacteria of Atlantic salmon (Salmo salar L.) within a commercial mariculture system.

AIMS: The relationship of Atlantic salmon gastrointestinal (GI) tract bacteria to environmental factors, in particular water temperature within a commercial mariculture system, was investigated. METHODS AND RESULTS: Salmon GI tract bacterial communities commercially farmed in south-eastern Tasmania were analysed, over a 13-month period across a standard commercial production farm cycle, using 454 16S rRNA-based pyrosequencing. Faecal bacterial communities were highly dynamic but largely similar between randomly selected fish. In postsmolt, the faecal bacteria population was dominated by Gram-positive fermentative bacteria; however, by midsummer, members of the family Vibrionaceae predominated. As fish progressed towards harvest, a range of different bacterial genera became more prominent corresponding to a decline in Vibrionaceae. The sampled fish were fed two different commercial diet series with slightly different protein, lipid and digestible energy level; however, the effect of these differences was minimal. CONCLUSIONS: The overall data demonstrated dynamic hind gut communities in salmon that were related to season and fish growth phases but were less influenced by differences in commercial diets used routinely within the farm system studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides understanding of farmed salmon GI bacterial communities and describes the relative impact of diet, environmental and farm factors.

Vibrio trends in the ecology of the Venice lagoon.

Vibrio is a very diverse genus that is responsible for different human and animal diseases. The accurate identification of Vibrio at the species level is important to assess the risks related to public health and diseases caused by aquatic organisms. The ecology of Vibrio spp., together with their genetic background, represents an important key for species discrimination and evolution. Thus, analyses of population structure and ecology association are necessary for reliable characterization of bacteria and to investigate whether bacterial species are going through adaptation processes. In this study, a population of Vibrionaceae was isolated from shellfish of the Venice lagoon and analyzed in depth to study its structure and distribution in the environment. A multilocus sequence analysis (MLSA) was developed on the basis of four housekeeping genes. Both molecular and biochemical approaches were used for species characterization, and the results were compared to assess the consistency of the two methods. In addition, strain ecology and the association between genetic information and environment were investigated through statistical models. The phylogenetic and population analyses achieved good species clustering, while biochemical identification was demonstrated to be imprecise. In addition, this study provided a fine-scale overview of the distribution of Vibrio spp. in the Venice lagoon, and the results highlighted a preferential association of the species toward specific ecological variables. These findings support the use of MLSA for taxonomic studies and demonstrate the need to consider environmental information to obtain broader and more accurate bacterial characterization.