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1 Coastal Marine Laboratory/Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR
2 Department of Biology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR
3 Center for Marine Science, University of North Carolina at Wilmington, USA
Correspondence
Pei-Yuan Qian
boqianpy{at}ust.hk
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ABSTRACT |
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9c and summed feature 3, comprising i15 : 0 2-OH and/or 16 : 17c. MK-6 was the only respiratory quinone. Flexirubin-type pigments were not produced. Phylogenetic analysis based on 16S rRNA gene sequences placed UST030701-156T within a distinct lineage in the family Flavobacteriaceae, with 93·3 % sequence similarity to the nearest neighbour, Nonlabens tegetincola. The DNA G+C content of UST030701-156T was 41·0 mol% and was much higher than that of N. tegetincola (33·6 mol%). Strain UST030701-156T can be distinguished from other members of the Flavobacteriaceae by means of a number of chemotaxonomic and phenotypic characteristics. It is proposed, therefore, that UST030701-156T represents a novel taxon designated Stenothermobacter spongiae gen. nov., sp. nov. The type strain is UST030701-156T (=NRRL B-41138T=JCM 13191T). Carbon-source utilization by N. tegetincola was re-examined and an emended description is therefore included.
A neighbour-joining dendrogram for strain UST030701-156T and related species and the results of API 20E, API 20NE, API ZYM, API 50 CH and MicroLog 3 tests are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). For example, Salegentibacter holothuriorum is from a sea cucumber (Nedashkovskaya et al., 2004), Mesonia algae and Formosa algae are from macroalgae (Nedashkovskaya et al., 2003; Ivanova et al., 2004), current members of the genus Winogradskyella are from macroalgae and sponges (Nedashkovskaya et al., 2005a; Lau et al., 2005a) and Gramella echinicola, Leeuwenhoekiella aequorea KMM 6066 and Salegentibacter mishustinae are from the sea urchin Strongylocentrotus intermedius (Nedashkovskaya et al., 2005b, c, d). On the basis of the polyphasic taxonomic data from the present study, we propose that the bacterial strain UST030701-156T, originating from a marine sponge, represents a novel genus of the Flavobacteriaceae. The bacterial strain UST030701-156T was isolated from tissue of the marine sponge Lissodendoryx isodictyalis in the Bahamas after 48 h cultivation at 30 °C on an agar medium composed of 5 g peptone l–1, 3 g yeast extract l–1 and 0·22-µm-filtered sea water (hereafter referred to as marine agar). Colonies of UST030701-156T were circular, 2–4 mm in diameter and convex with smooth surfaces, entire margins and a non-diffusible orange pigment. Unless otherwise specified, all characteristics described hereafter are based on cultures grown on marine agar for 48 h at 30 °C. The optimum temperature for the growth of UST030701-156T was 28–30 °C.
The nearly complete 16S rRNA gene sequence of UST030701-156T (1399 bp) was obtained bidirectionally with replications (n=3) as described elsewhere (Lau et al., 2004). Comparison of the nearly complete 16S rRNA gene sequence of UST030701-156T with those available from GenBank revealed that UST030701-156T is a member of the family Flavobacteriaceae. UST030701-156T shared 93·3 and 93·6 % sequence similarity with the two most closely related species, Nonlabens tegetincola (Lau et al., 2005b) and Donghaena dokdonensis (Yoon et al., 2006), respectively. The sequence similarity to other species was 
90·9 %. A neighbour-joining phylogenetic tree constructed using the ARB software package (Ludwig et al., 2004) showed that UST030701-156T belonged to a distinct branch, which clustered robustly (93 %, 500 replications) with N. tegetincola and D. dokdonensis (Fig. 1). Trees based on maximum-parsimony and maximum-likelihood methods showed essentially the same topology (Fig. 1). The results of phylogenetic analysis suggest that UST030701-156T represents a novel genus within the family Flavobacteriaceae. The distinction of UST030701-156T from the closest relative, N. tegetincola, is further supported by the substantial difference in their DNA G+C contents: the value for UST030701-156T is 41·0±0·2 mol% (three replicates) while that for N. tegetincola is 33·6±0·2 mol% (Lau et al., 2005b). The DNA G+C content was determined by using an HPLC method according to Mesbah et al. (1989).
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9c and summed feature 3 (comprising i15 : 0 2-OH and/or 16 : 17c) (altogether representing 76·2 % of the total), as determined using the Sherlock Microbial Identification System according to the manufacturer's protocol (Table 1). This fatty acid profile differed from that described for N. tegetincola by the absence of 18 : 0 and by having a smaller amount of i16 : 0 (Table 1). MK-6 was the only respiratory quinone in UST030701-156T, as determined using an HPLC method according to Collins (1994). Menaquinones extracted from Cellulophaga lytica ATCC 23178T (Johansen et al., 1999) and Pedobacter heparinus ATCC 13125T (Steyn et al., 1998) served as references for MK-6 and MK-7, respectively.
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). Cell morphology was examined using scanning electron microscopy (JEOL 7600F) according to the procedures described by Neu et al. (2001) (see Supplementary Fig. S1, available in IJSEM Online, for a scanning electron micrograph). The Gram-stain reaction was determined using light microscopy according to Smibert & Krieg (1994). Gliding motility was determined using phase-contrast light microscopy after growth on quarter-strength marine 2216 medium solidified with 1 % agar according to Bowman (2000). Susceptibility to antibiotics was tested according to Acar (1980). Flexirubin-type pigment production and carboxylmethylcellulose hydrolysis were determined according to Bernardet et al. (2002). Casein hydrolysis was determined according to Norris et al. (1985); hydrolysis of chitin and Tweens 20, 40 and 80 was determined according to Baumann & Baumann (1988). Oxidase and catalase activities and the hydrolysis of agar, DNA and starch were tested according to Smibert & Krieg (1994). Other enzymic activities, the substrate-utilization pattern, nitrate reduction and the production of H2S, indole and acetoin were determined by using the commercial systems API 20E, API 20NE, API 50 CH, API ZYM (bioMérieux) and MicroLog 3 (Biolog). Cells for inoculation to the API systems were suspended in a sterile solution of sea-water mix at 22 
salinity (MacDonell et al., 1982). The phenotypic characteristics of UST030701-156T are given in the species description. Detailed results obtained from the API and MicroLog 3 systems are provided in Supplementary Tables S1–S3. The MicroLog 3 system was also used to test for the utilization of carbon sources by N. tegetincola UST030701-324T. Results are given in the emended description of N. tegetincola and Supplementary Table S3.
Chemotaxonomic and phenotypic characteristics that distinguish UST030701-156T from other genera of the Flavobacteriaceae are given in Tables 1 and 2
. Strain UST030701-156T differs from N. tegetincola by (i) having a higher DNA G+C content, (ii) not being able to hydrolyse DNA, (iii) having gliding motility and a different cell morphology, (iv) growing in a narrower temperature range, (v) having 
-glucosidase and 
-galactosidase activities and (vi) having a different carbon-source utilization pattern (refer to Supplementary Tables S2 and S3 for a detailed comparison). Strain UST030701-156T differs from the members of Psychroflexus by (i) having different cell morphology and a different fatty acid profile, (ii) having a higher DNA G+C content, (iii) being less halotolerant and psychrotolerant and (iv) having -galactosidase activity but not -glucosidase activity. Additionally, UST030701-156T can be distinguished from the members of Mesonia, Gramella and Salegentibacter by means of the traits detailed in Table 2. Molecular evidence, together with the chemotaxonomic and phenotypic characteristics, suggests that strain UST030701-156T constitutes a novel genus within the family Flavobacteriaceae.
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Cells are Gram-negative rods (>2·5 µm in length) with tapered ends forming chains of up to four cells. Strictly aerobic. Chemo-organotrophic. MK-6 is the only respiratory quinone. Oxidase-positive. Catalase activity is very weak. Phylogenetic analysis based on the 16S rRNA gene sequence indicates that Stenothermobacter is a member of the family Flavobacteriaceae. The genus contains one species, Stenothermobacter spongiae, which is the type species.
Description of Stenothermobacter spongiae sp. nov.
Stenothermobacter spongiae (spon'gi.ae. L. gen. n. spongiae of a sponge, pertaining to the isolation source of the type strain).
The description is as for the genus, with the following additions. Cells are slowly motile by gliding. Colonies on marine agar are orange in colour, circular, 2·0–4·0 mm in diameter and convex with smooth surfaces and entire margins. No diffusible pigment. Flexirubin-type pigments are not produced. Growth occurs at 20·0–36·0 °C (28·0–30·0 °C optimum), at pH 6·0–10·0 and at 2·0–6·0 % NaCl. Susceptible to ampicillin, chloramphenicol, penicillin, streptomycin and tetracycline, but not to kanamycin. The DNA G+C content is 41·0 mol%. The predominant fatty acids (>5 %) are a15 : 0, i15 : 0, i15 : 0 3-OH, i17 : 0 3-OH, i17 : 19c and summed feature 3 (comprising i15 : 0 2-OH and/or 16 : 17c) (altogether representing 76·2 % of the total). Produces acetoin, but not indole or H2S. Nitrate is not reduced. Hydrolyses gelatin, starch and Tweens 20, 40 and 80, but not agar, casein, carboxylmethylcellulose, chitin or DNA. Positive for acid phosphatase, alkaline phosphatase, -galactosidase, -glucosidase, -chymotrypsin, cystine arylamidase, leucine arylamidase, valine arylamidase, esterase (C4), esterase lipase (C8), lipase (C14), trypsin and naphthol-AS-BI-phosphohydrolase activities. Negative for N-acetyl--glucosaminidase, arginine dihydrolase, -fucosidase, -galactosidase, -glucosidase, -glucuronidase, -mannosidase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease activities. Growth occurs on the following sole carbon sources in the API 20E, API 20NE and API 50 CH systems: D-arabinose, D-galactose, D-glucose, glycerol, D-mannitol, D-melibiose, D-sorbitol, starch and D-sucrose. No acid is produced from these carbon sources. The following carbon sources in the MicroLog 3 system are utilized: D-maltose, D-melibiose, D-raffinose, sucrose, monomethyl succinate, acetic acid, -ketoglutaric acid, -ketovaleric acid, propionic acid, L-alaninamide, L-alanine, L-alanyl glycine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-ornithine and L-proline. Refer to Supplementary Tables S2 and S3 for full lists of carbon sources included in the API and MicroLog 3 systems.
The type strain is UST030701-156T (=NRRL B-41138T=JCM 13191T), isolated from tissue of the marine sponge Lissodendoryx isodictyalis in the Bahamas.
Emended description of Nonlabens tegetincola
The description remains as given by Lau et al. (2005b), but with the following modifications: able to utilize 57 carbon sources in the MicroLog 3 system, including -cyclodextrin, dextrin, glycogen, adonitol, D-arabitol, L-erythritol, D-fructose, L-fucose, D-galactose, -D-glucose, myo-inositol, -D-lactose, lactulose, maltose, D-mannitol, D-mannose, D-melibiose, methyl -D-glucoside, D-psicose, D-raffinose, L-rhamnose, D-sorbitol, sucrose, D-trehalose, turanose, methylpyruvate, monomethyl succinate, acetic acid, citric acid, D-galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucosaminic acid, D-glucuronic acid, -hydroxybutyric acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, succinamic acid, glucuronamide, L-alaninamide, L-alanine, L-alanyl glycine, L-aspartic acid, L-glutamic acid, glycyl L-aspartic acid, glycyl L-glutamic acid, L-ornithine, L-proline, L-serine, L-threonine and uridine. A full list of carbon sources included in the MicroLog 3 test system is available in Supplementary Table S3.
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ACKNOWLEDGEMENTS |
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