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Salegentibacter flavus sp. nov.

¹ Swinburne University of Technology, PO Box 218, Hawthorn, Victoria 3122, Australia
² Pacific Institute of Bioorganic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, Pr. 100 Let Vladivostoku 159, Russia
³ Department of Agricultural Science, University of Tasmania, Hobart, Tasmania, Australia
⁴ UMR6543 CNRS – Université de Nice Sophia Antipolis, Centre de Biochimie, Parc Valrose 06108 Nice cedex 2, France
⁵ Institute of Marine Biology of the Far-Eastern Branch of the Russian Academy of Sciences, 690041 Vladivostok, Russia
⁶ Institute of Microbiology of the Russian Academy of Sciences, 117811 Moscow, Russia
⁷ Pacific Oceanological Institute of the Far-Eastern Branch of the Russian Academy of Sciences, Baltiiskaya Str. 43, 690017 Vladivostok, Russia

Correspondence
Elena P. Ivanova
eivanova{at}swin.edu.au

	ABSTRACT

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A yellow-pigmented, non-motile, Gram-negative bacterium, designated Fg 69^T, was isolated from a sediment sample collected in Chazhma Bay (Sea of Japan). The novel organism grew at 10–35 °C, was neutrophilic and required 3–10 % NaCl for optimal growth. Strain Fg 69^T was able to degrade starch and to hydrolyse gelatin and Tween 80 weakly but not casein or agar. Predominant cellular fatty acids comprised n-C₁₅ and n-C₁₆ branched-chain and straight-chain saturated and unsaturated fatty acids, including iso-C_{15 : 0} (5 %), anteiso-C_{15 : 0} (11 %), C_{15 : 0} (9 %), iso-C_{15 : 1} (5 %), iso-C_{16 : 0} (8 %), C_{16 : 0} (5 %) and C_{16 : 1}7 (5 %) and iso- and anteiso-branched 2-OH and 3-OH C_{15 : 0} to C_{17 : 0} fatty acids (26 % in total). The G+C content of the DNA was 40·4 mol%. 16S rRNA gene sequence data indicated that strain Fg 69^T belonged to the genus Salegentibacter but was distinct from recognized Salegentibacter species (94–95 % sequence similarity). Based on these results, a novel species, Salegentibacter flavus sp. nov., is proposed. The type strain is Fg 69^T (=KMM 6000^T=CIP 107843^T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain Fg 69^T is AY682200.

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The genus Salegentibacter, belonging to the family Flavobacteriaceae, was created to accommodate moderately halophilic, yellow-pigmented, non-gliding bacteria that were isolated from a hypersaline meromictic lake in Antarctica (McCammon & Bowman, 2000). At the time of writing, the genus comprises three recognized species, Salegentibacter salegens (Dobson et al., 1993; McCammon & Bowman, 2000), Salegentibacter holothuriorum (Nedashkovskaya et al., 2004) and Salegentibacter mishustinae (Nedashkovskaya et al., 2005). We report here the characterization and description of a novel Flavobacterium-like bacterium, strain Fg 69^T, isolated from a sediment sample collected in Chazhma Bay (Sea of Japan). On the basis of the results obtained, we propose that this organism be classified as a novel species of the genus Salegentibacter.

This work formed part of a taxonomic survey of free-living microbial populations of a coastal area of the north-west Pacific Ocean, where sediments have become contaminated by radionuclides as a result of an accident on a nuclear submarine. Sediment samples were collected from a sub-seafloor depth of 0·5 m in October–November 2000 from Chazhma Bay, Gulf of Peter the Great, Sea of Japan (Ivanova et al., 2005). Samples were kept at 4 °C and processed within 4–8 h. Sample handling and isolation procedures were as described elsewhere (Ivanova et al., 1996, 2004, 2005). Inoculated plates were incubated aerobically at room temperature (about 22–25 °C) for 5–10 days. Strains were stored at –80 °C in marine broth 2216 (Difco) supplemented with 20 % (v/v) glycerol.

Unless otherwise indicated, the phenotypic properties used for characterization of Flavobacterium-related species were tested following established procedures (McMeekin et al., 1971; Smibert & Krieg, 1994; Ivanova et al., 1996; Bernardet et al., 2002). To test for spreading growth and gliding motility, strains were grown on medium B (Ivanova et al., 1996) with reduced peptone content (0·2 g l^–1). Gliding motility was verified by using phase-contrast microscopy (Nikon) of hanging drop preparations. In order to detect flexirubin-type pigments, the bathochromic shift test utilizing 20 % (w/v) KOH was used (Fautz & Reichenbach, 1980). Cellulose hydrolysis was tested both by using cellulose overlay plates (1 % CM-cellulose) and by examining strips of filter paper in liquid bacterial cultures for dissolution (Smibert & Krieg, 1994). The following physiological and biochemical properties were examined using the methods described by Smibert & Krieg (1994): oxidation/fermentation of glucose; oxidase and catalase activity; gelatin liquefaction; sodium chloride requirement [0, 0·5, 1, 3, 6, 8, 10 and 12 % (w/v) NaCl]; acetoin, urea, indole and H₂S production; and the ability to hydrolyse starch, Tween 80 and casein. Denitrification was assessed using the procedure of Azegami et al. (1987). Growth at different temperatures was tested in marine broth 2216 (Difco) at 2, 4, 6, 9, 29, 30, 35 and 37 °C. Carbon source utilization was tested on medium containing 0·2 g NaNO₃, 0·2 g NH₄Cl, 0·05 g yeast extract (Difco) and 0·4 % (w/v) carbon source in 1 litre artificial sea water as described by Nedashkovskaya et al. (2003). The following carbon sources were examined: arabinose, glucose, lactose, raffinose, sucrose, inositol, sorbitol, mannitol, rhamnose, salicin, xylose and adonitol.

Phenotypic analysis showed that the organism had characteristics reported for genera of the family Flavobacteriaceae: it was Gram-negative, strictly aerobic, oxidase- and catalase-positive, did not produce H₂S or indole and was weakly positive for nitrate reduction. Detailed morphological and physiological properties are shown in Table 1 and are given in the species description.

For analysis of fatty acids, the strain was grown at 28 °C on marine agar 2216. Cells were harvested after 48 h. Bacterial biomass was treated with 5 % HCl in methanol at 80 °C for 180 min to produce fatty acid methyl esters (FAMEs) (Christie, 1982). FAMEs were analysed by flame-ionization detector (FID)-GC (Shimadzu GC-17) with a fused silica capillary column (30 mx0·25 mm), coated with Supelcowax 10, at 210 °C. Helium was used as a carrier gas. FAMEs were identified by comparing the retention times with those of known standards and using equivalent chain-length measurements. To ensure correct identification, FAMEs were further analysed by GC-MS using a model GCMS-QP5050A (Shimadzu) fitted with an MDN-5S capillary column (30 mx0·25 mm). The column temperature was programmed as follows: 1 min hold at 170 °C, followed by an increase to 240 °C at 2 °C min^–1 and a hold at 240 °C for 20 min. The temperature of the injector and detector was 250 °C. The FAMEs formed by the novel organism were (as percentages of whole-cell fatty acids): C_{12 : 0} (1·2 %), iso-C_{14 : 0} (1·5 %), C_{14 : 0} (2·7 %), iso-C_{15 : 0} (5·4 %), anteiso-C_{15 : 0} (11·1 %), iso-C_{15 : 1} (5·4 %), C_{15 : 0} (8·6 %), C_{15 : 1}6 (2·9 %), iso-C_{16 : 0} (7·7 %), anteiso-C_{16 : 1} (3·5 %), C_{16 : 0} (5·0 %), C_{16 : 1}7 (5·1 %), C_{16 : 1}9 (2·4 %), iso-C_{17 : 1} (1·8 %), anteiso-C_{17 : 1} (1·2 %), C_{17 : 0} (0·6 %), C_{17 : 1}6 (2·8 %), C_{17 : 1}8 (1·5 %), C_{18 : 0} (1·8 %), C_{18 : 1}9 (1·7 %), C_{14 : 0} 3-OH (0·6 %), iso-C_{15 : 0} 2-OH (3·6 %), anteiso-C_{15 : 0} 2-OH (3·2 %), iso-C_{15 : 0} 3-OH (1·0 %), iso-C_{16 : 0} 2-OH (4·3 %), iso-C_{16 : 0} 3-OH (6·0 %), iso-C_{17 : 0} 3-OH (3·2 %) and anteiso-C_{17 : 0} 3-OH (4·4 %). The fatty acid profile of the new isolate exhibited features characteristic of the family Flavobacteriaceae and of the genus Salegentibacter, e.g. the combination of n-C₁₅ and n-C₁₆ branched-chain saturated and unsaturated cellular fatty acids accounted for 50–70 % of the total. A significantly higher proportion of iso- and anteiso-branched 2-OH and 3-OH n-C_{14 : 0} to C_{17 : 0} fatty acids (26 % in total) was found for strain Fg 69^T than for S. salegens, S. holothuriorum or S. mishustinae (Nedashkovskaya et al., 2004, 2005).

The small-subunit rRNA gene of Fg 69^T was sequenced (1471 bp) as described elsewhere (Ivanova et al., 2004). The most similar 16S rRNA gene sequences were retrieved using n-BLAST searches (www.ncbi.nlm.nih.gov) and aligned for phylogenetic analysis. Analysis was based on three different approaches: neighbour-joining (Gascuel, 1997), parsimony and maximum-likelihood with global search (from PHYLIP version 3.57c; Felsenstein, 1993). The program DNADIST was used to determine sequence similarities using the Kimura two-parameter correction. Finally, a bootstrap analysis (1000 replications) was performed using neighbour-joining (Felsenstein, 1985). 16S rRNA gene sequence analysis revealed that the new isolate clearly belonged to the genus Salegentibacter, as these sequences formed a robust clade (all three methods, bootstrap value of 99·8 %; Fig. 1). The 16S rRNA gene sequence of strain Fg 69^T showed <95 % similarity with those of the three recognized Salegentibacter species. The phylogenetic analysis thus supports the inclusion of Fg 69^T within the genus Salegentibacter, while the low level of 16S rRNA gene sequence similarity indicates that it represents a novel species.

View larger version (36K): [in this window] [in a new window]   Fig. 1. Phylogenetic position of Salegentibacter flavus sp. nov. according to 16S rRNA gene sequence analysis. Unrooted tree resulting from a neighbour-joining analysis as described in the text. Percentages of 1000 bootstrap replications are indicated for branches that were also supported by parsimony and maximum-likelihood (P<0·01) analyses. Bar, 0·01 nucleotide substitutions per position.

Description of Salegentibacter flavus sp. nov.
Salegentibacter flavus (fla'vus. L. masc. adj. flavus golden yellow).

Cells are Gram-negative, microaerophilic, chemo-organotrophic, non-motile, asporogenic rods, 0·5–0·7 mm wide and 2·5–4·0 mm long. Oxidase- and catalase-positive. Colonies are circular, 1–3 mm in diameter and of low convexity when grown on marine agar 2216. Produces non-diffusible yellow pigment. Flexirubin-type pigments are absent. No growth is detected at 8 or 37 °C. Optimal temperature for growth is 22–25 °C. The pH range for growth is 5·0–10·0, with optimal growth between pH 8·0 and 8·5. Growth occurs between 3 and 10 % NaCl, with optimal growth at 3 % NaCl. No growth is observed without Na⁺. Starch, gelatin and urea are hydrolysed and Tween 80 is weakly hydrolysed. Does not decompose cellulose (CM-cellulose or filter paper), agar or casein. H₂S, indole and acetoin (Voges–Proskauer reaction) are not produced. Nitrate reduction is negative. Does not utilize citrate, arabinose, glucose, lactose, raffinose, sucrose, inositol, sorbitol, mannitol, rhamnose, salicin, xylose or adonitol. Major cellular fatty acids are iso-C_{15 : 0}, anteiso-C_{15 : 0}, C_{15 : 0}, iso-C_{16 : 0}, C_{16 : 0} and a range of C_{14 : 0} to C_{17 : 0} hydroxy fatty acids (about 64 %). The DNA G+C content is 40·4±0·5 mol%.

The type strain, strain Fg 69^T (=KMM 6000^T=CIP 107843^T), was isolated from sediments from Chazhma Bay, Sea of Japan.

	ACKNOWLEDGEMENTS

 
This study was partially supported by funds from the Australian Research Council (ARC), grants (State contracts) from the Federal Agency for Science and Innovations of the Ministry for Education and Science of the Russian Federation ‘KMM’ and ‘Scientific Schools’, grant 05-04-48211 from the Russian Foundation for Basic Research and grants from the Presidium of the Russian Academy of Sciences ‘Molecular and Cell Biology’.

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