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1 Laboratory of Microbiology, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate 041, Japan
2 Laboratory of Microbiology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium
3 UMR 1931 CNRS/Goëmar, Place Georges Teissier B.P. 74, F29682 Roscoff cedex, France
4 UMR 6543 CNRS – Université de Nice Sophia Antipolis, Centre de Biochimie, Parc Valrose, F06108 Nice cedex 2, France
Correspondence
Tomoo Sawabe
sawabe{at}fish.hokudai.ac.jp
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ABSTRACT |
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-galactosidase test and assimilation of three carbon compounds) and from Vibrio superstes by 16 traits.
Published online ahead of print on 12 December 2003 as DOI 10.1099/ijs.0.02804-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of V. gallicus are AY257972 (CIP 107863T=LMG 21878T=strain HT2-1T), AY257971 (CIP 107864=strain HT1-3), AY257973 (CIP 107865=strain HT1-12), AY257974 (CIP 107866=strain HT2-6) and AY257975 (CIP 107867=strain HT3-3).
A table of phenotypic characteristics is available as supplementary material in IJSEM Online.
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, 2002), South Africa (Sawabe et al., 2003) and Australia (Hayashi et al., 2003). The hypothesized roles of V. halioticoli include its contribution to the digestion of alginate, which is a major polysaccharide in Japanese and South African kelps that is ingested by these animals, and its conversion into volatile short-chained fatty acids via fermentation (Sawabe et al., 2003). On the other hand, V. superstes may play a minor role in Australian abalone, due to a small population in the gut of the abalone (Hayashi et al., 2003). We have examined the presence of V. halioticoli-like bacteria in the gut of abalones; recently, we isolated a set of five strains, which were most similar phenotypically to V. halioticoli, from the gut of French abalones (Haliotis tuberculata). DNA–DNA hybridization experiments, phenotypic characterization and phylogenetic and genetic analyses demonstrated that these strains represent a so far unknown species of the genus Vibrio.
Five strains of Vibrio gallicus sp. nov. (CIP 107863T=LMG 21878T=HT2-1T, LMG 21329=CIP 107864=HT1-3, CIP 107865=HT1-12, CIP 107866=HT2-6 and CIP 107867=HT3-3) were isolated from the gut of French abalones (H. tuberculata). These were collected at the coastal area of Brest (Brittany, France) by Scuba diving in February 2001. Strains were cultured on ZoBell 2216E agar (Oppenheimer & ZoBell, 1952) and stored at –80 °C in ZoBell broth that contained 10 % glycerol.
About 1400 bp of the 16S rRNA gene sequences for strains CIP 107863T, CIP 107864, CIP 107865, CIP 107866 and CIP 107867 was determined according to Sawabe et al. (1998), by using six sequence primers (24F, 530F, 1100F, 520R, 920R and 1540R). Sequences of V. gallicus CIP 107863T, CIP 107864, CIP 107865, CIP 107866 and CIP 107867 were used for phylogenetic analyses. The 16S rRNA gene sequences of V. gallicus were used in a BLAST search of GenBank/EMBL and NRupdates (28 July 2003), in order to retrieve the 50 most closely related sequences. New sequences were included and aligned in a local database of 85 000 already aligned bacterial 16S rRNA gene sequences. Selection of sequences for the final analysis was according to previous phylogenetic analyses of the entire database and the results of the BLAST query. Thirty-one sequences were retained, excluding unidentified species and non-type species. Phylogenetic trees were constructed by using three different methods [BIONJ (neighbour-joining), maximum-likelihood (ML) and maximum-parsimony (MP)]. For neighbour-joining (NJ) analysis, distance matrices were calculated by using the Kimura two-parameter correction. BIONJ was according to Gascuel (1997) and ML and MP were from PHYLIP (Phylogeny Inference Package, version 3.573c; distributed by J. Felsenstein, Department of Genetics, UW, Seattle, WA, USA). For the final tree, almost-entire sequences (corresponding to positions 67–1401 of the type strain of V. gallicus) were used (parts of sequences that were available for all strains and showed no obvious homoplasy). Phylogenetic trees were drawn by using NJPLOT (Perrière & Gouy, 1996). When several sequences were available for a type species, all sequences were included in a preliminary analysis (they often differed by a few nucleotides) and a single one was chosen for the tree that is presented. The distance bar in Fig. 1 corresponds to distances that were corrected as indicated above and has no meaning in terms of sequence similarities.
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3 subgroup of the -Proteobacteria (Garrity & Holt, 2000). The closest phylogenetic neighbours of the five French abalone strains were species such as V. halioticoli, V. superstes, Vibrio fischeri and a number of other species, as shown in Fig. 1
, but no single known species could be grouped significantly with V. gallicus in a robust clade. The five strains of V. gallicus had 98·0 % or less 16S rRNA gene sequence similarity with V. superstes LMG 21323T, 97·8 % with V. halioticoli IAM 14596T, 97·2 % with Vibrio agarivorans and 97·1 % or less with Vibrio penaeicida, but none of these species was a sister species to V. gallicus. All other Vibrio species, including V. fischeri, Vibrio salmonicida, Vibrio logei and Vibrio wodanis (sister species), showed similarity levels of <97 %. Phylogenetic analysis of 16S rRNA gene sequences demonstrates clearly that the five new strains belong to the genus Vibrio and suggests that they should be assigned to a novel species, as they do not form a robust clade with any single recognized Vibrio species.
In total, 78 phenotypic characteristics of V. gallicus, V. halioticoli, V. superstes and V. fischeri strains were determined as described previously (Leifson, 1963; Hidaka & Sakai, 1968; West et al., 1977; Ostle & Holt, 1982; Baumann & Schubert, 1984; Holt et al., 1994). Phenotypic characterization was done under the same conditions at 20 °C. All available phenotypic traits of sister species (V. salmonicida, V. logei and V. wodanis) and V. agarivorans (Baumann & Schubert, 1984; Egidius et al., 1986; Lunder et al., 2000; Macián et al., 2001) were compared to those of V. gallicus.
The five French abalone strains have the main phenotypic features of the genus Vibrio (except for the absence of flagella). The strains are non-motile, Gram-negative and fermentative (Sawabe et al., 1998). No flagellated cells were observed by transmission electron microscopic observations. These strains require salt for their growth, do not accumulate poly--hydroxybutyrate and are oxidase-positive (see Supplementary Table, available in IJSEM Online). No peritrichous cells were observed when the strains were cultivated on solid media. Other phenotypic features of V. gallicus are shown in the Supplementary Table (available in IJSEM Online). The mesophilic growth temperature of V. gallicus is a key phenotypic trait that differentiates it from its psychrophilic sister species V. salmonicida, V. logei and V. wodanis. The five abalone isolates were most similar phenotypically to V. halioticoli IAM 14596T, although the strains differed by four traits out of 78 tested (see Supplementary Table, available in IJSEM Online).
DNA of bacterial strains was prepared by using a Promega Wizard DNA extraction kit for fluorescence amplified fragment length polymorphism (FAFLP) analysis, and by the procedures of Marmur (1961) for measurement of G+C content and DNA hybridization experiments. FAFLP analysis was performed as described previously (Thompson et al., 2001; Sawabe et al., 2002). Clustering of the patterns was done by using the Dice coefficient (SD) and the Ward algorithm (Sneath & Sokal, 1973). DNA G+C contents were determined by HPLC (Tamaoka & Komagata, 1984). DNA–DNA hybridization experiments were performed in microdilution wells by using a fluorometric direct binding method, as described by Ezaki et al. (1988, 1989). DNA homology values are indicated as means of triplicate measurements.
FAFLP patterns of V. gallicus CIP 107863T and CIP 107864 consisted respectively of 87 and 103 bands. The similarity between these two band patterns was 79 % (Fig. 2). The FAFLP pattern similarity of V. gallicus to V. halioticoli, Vibrio rumoiensis and Vibrio harveyi was 48, 47 and 44 %, respectively. FAFLP pattern similarity of CIP 107863T and CIP 107864 to other Vibrio species, including the sister species of V. gallicus (V. wodanis, V. logei and V. salmonicida), was <44 %, indicating clearly that this novel species is different from other vibrios (Thompson et al., 2001). The FAFLP results are supported by our DNA–DNA hybridization experiments, which showed that the five strains of V. gallicus, CIP 107863T, CIP 107864, CIP 107865, CIP 107866 and CIP 107867, were conspecific strains that were clearly separate from V. halioticoli, V. superstes, V. fischeri, V. agarivorans and V. logei (See Supplementary Table in IJSEM Online).
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We have found three species of vibrios, V. halioticoli, V. superstes and V. gallicus, that are associated with abalone. These species are related phylogenetically (Fig. 1), but are different phenotypically (see Supplementary Table, available in IJSEM Online). The ecological niches of these species are similar (Sawabe et al., 1998, 2003; Hayashi et al., 2003). A recent phylogenetic study that used internal transcribed spacer region sequences of 19 species of the genus Haliotis (Coleman & Vacquier, 2002) revealed three subclades in the genus, each encompassing low variation. North Pacific species group together, whereas Australian and European species are different subclades. A single species, Haliotis iris from New Zealand, is quite distant from the remaining species of Haliotis. Haliotis midae and Haliotis diversicolor supertexta (Taiwan) both diverged basal to European and Australian species. Assuming co-evolution of the symbiotic relationships between vibrios and Haliotis species, the taxonomic status of V. gallicus and V. superstes as close relatives of V. halioticoli is in accordance with the phylogeny of Haliotis species. Comparative whole-genome analyses among these three species are needed to clarify the evolutionary history. Ecological studies of V. gallicus are also needed, in order to better understand its interactions in the gut of marine herbivores, particularly abalones.
Description of Vibrio gallicus sp. nov.
Vibrio gallicus (gal'li.cus. L. masc. adj. gallicus from France).
Gram-negative, facultatively anaerobic, non-motile and non-flagellated. Cells in ZoBell 2216E broth are rod-shaped with rounded ends (0·5–0·6x1·2–3·0 µm). No endospores or capsules are formed. Flagellation is not observed when the organism is cultivated on solidified media and/or in liquid media. Colonies on ZoBell 2216E agar are beige, circular, smooth and convex with an entire edge. Sodium ions are essential for growth. Mesophilic and neutrophilic chemo-organotroph that grows at temperatures between 15 and 30 °C. No growth occurs at 37 °C. Positive for acid production from glucose, nitrate reduction, hydrolysis of alginate, oxidase and catalase and assimilation of D-fructose, D-glucose, maltose, D-mannitol and alginate. The following tests are negative: gas production from glucose, acetoin production, lysine decarboxylase, ornithine decarboxylase, arginine dehydrolase, -galactosidase test, luminescence, pigmentation, requirement for organic growth factors, hydrolysis of starch, gelatin, chitin, Tween 80 and agar, accumulation of poly--hydroxybutyrate, assimilation of D-mannose, sucrose, D-gluconate, D-sorbitol, 2-oxoglutarate, D-galactose, cellobiose, melibiose, lactose, D-glucuronate, trehalose, putrescine, -aminobutyrate, acetate, pyruvate, L-tyrosine, propionate, D-glucosamine, fumarate, succinate, meso-erythritol, D-xylose, L-arabinose, citrate, DL-malate, 
-aminovarate and aconitate. DNA G+C content is 43·6–44·3 mol%.
The type strain is CIP 107863T=LMG 21878T. Isolated from the gut of the French abalone Haliotis tuberculata.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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