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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, New Territories, Hong Kong SAR
3 Center for Marine Science, University of North Carolina at Wilmington, Wilmington, NC, USA
Correspondence
Pei-Yuan Qian
boqianpy{at}ust.hk
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ABSTRACT |
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Tables detailing the results of API 20E, 20NE and 50CH tests and MicroLog 3 tests for strains UST030701-097T and UST030701-084T and scanning electron micrographs of cells of the two strains are available as supplementary material in IJSEM Online.
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TOPABSTRACTMAIN TEXTREFERENCES |
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). These bacteria can be maternal in origin or captured from sea water as the sponges filter feed (Imhoff & Stohr, 2003). The bacteria associated with sponges are believed to play essential roles in their survival and fitness. For example, the bioactive metabolites of bacteria may defend sponges against epibiosis (Chelossi et al., 2004) and the extracellular enzymes of the bacteria may mobilize food resources that are otherwise indigestible by sponges (Wilkinson et al., 1999). During the course of studying bacterial communities associated with the marine sponge Tedania ignis in the Bahamas, bacterial strains UST030701-097T and UST030701-084T were isolated. The strains appeared as pink-pigmented, circular, convex colonies (2–4 mm in diameter) with a smooth surface and an entire margin after 48 h of cultivation at 30 °C on an agar medium composed of 5 g peptone l–1, 3 g yeast extract l–1 (both obtained from Oxoid) and 0.22 µm-filtered sea water. This agar medium is hereafter referred to as marine agar. Unless otherwise specified, all the characteristics described hereafter are based on cultures grown on marine agar at 30 °C for 48 h. Based on the polyphasic taxonomic data obtained in the present study, we propose that strain UST030701-097T represents a novel genus and that strain UST030701-084T represents a novel species within the family ‘Flexibacteraceae’ of the phylum Bacteroidetes.
The nearly complete 16S rRNA gene sequences of strains UST030701-097T (1412 bp) and UST030701-084T (1387 bp) were obtained bidirectionally with replications (n=3) as described elsewhere (Lau et al., 2004). Phylogenetic analysis based on nearly complete 16S rRNA gene sequences indicated that the two strains shared 95.0 % sequence similarity and were members of the family ‘Flexibacteraceae’ in the phylum Bacteroidetes. Strain UST030701-084T was most closely related to members of the genera Marinicola (Yoon et al., 2005) and Roseivirga (Nedashkovskaya et al., 2005a, b), with 95.8–96.0 % sequence similarity. Strain UST030701-097T was most closely related to two uncharacterized bacteria (strains PM13 and DG1129), with 94.8–96.5 % sequence similarity, and to the members of the genera Marinicola and Roseivirga, with 93.1–93.3 % sequence similarity. A neighbour-joining phylogenetic tree constructed using the ARB software package (Ludwig et al., 2004) indicated that strains UST030701-097T and UST030701-084T, the uncharacterized strains PM13 and DG1129 and members of the genera Marinicola and Roseivirga belonged to the same lineage. Within this lineage, strain UST030701-097T and the two uncharacterized bacteria constituted one branch, whereas strain UST030701-084T and the members of the genera Marinicola and Roseivirga constituted another (Fig. 1). This tree topology is supported by high bootstrap values within the lineage (>84 %, 500 replicates) and by its recurrence in maximum-parsimony and maximum-likelihood trees as determined using the ARB software package (Fig. 1).
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proposed that the genus Marinicola is a later heterotypic synonym of the genus Roseivirga due to many common genomic, chemotaxonomic and phenotypic features seen between members of the two genera. However, a formal proposal to reclassify [Marinicola] seohaensis, the only member of the genus Marinicola, has not yet been made. The results of the phylogenetic analysis in the study support the reclassification of [Marinicola] seohaensis to the genus Roseivirga. We thus propose that [Marinicola] seohaensis be renamed as Roseivirga seohaensis comb. nov.
The DNA G+C contents of strains UST030701-097T and UST030701-084T were 42.5±0.3 mol% (three replicates) and 43.7±0.6 mol% (three replicates), respectively, as determined by an HPLC method according to Mesbah et al. (1989). MK-7 was the major respiratory quinone in both strains as determined using an HPLC method described by Collins (1994). Menaquinones extracted from Cellulophaga lytica (Johansen et al., 1999) and Pedobacter heparinus (Steyn et al., 1998) were used as references for MK-6 and MK-7, respectively. Fatty acid contents of strains UST030701-097T and UST030701-084T were determined using the Sherlock Microbial Identification System (MIDI) according to the manufacturer's protocol and are given in Table 1. The fatty acid profile of the two strains differed mainly by the presence/absence of i14 : 0, i14 : 0 3-OH, 15 : 0 3-OH, 16 : 0 3-OH, i16 : 1 and i17 : 1
9c and by the quantity of i15 : 0 3-OH, a15 : 0, i16 : 0 3-OH, 17 : 0 2-OH, i17 : 0 3-OH and summed feature 3 (SF3; comprising i15 : 0 2-OH and/or 16 : 17c) (Table 1). The fatty acid profile of strain UST030701-097T differed from those described for the members of the Marinicola and Roseivirga mainly by having larger quantities of i15 : 0 3-OH, i16 : 0 3-OH and SF3 and by the additional presence of i14 : 0 3-OH, 15 : 0 2-OH and 15 : 0 3-OH (Table 1). The fatty acid profile of strain UST030701-084T could be distinguished from those of species of the genera Marinicola and Roseivirga mainly by having different quantities of i13 : 0, i15 : 1, i16 : 0 3-OH, 17 : 0 2-OH, i17 : 0 3-OH and i17 : 19c and by the additional presence of 15 : 0 2-OH (Table 1).
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). Cell morphology was examined using scanning electron microscopy (7600F; JEOL) according to the procedures described in Neu et al. (2001) (see Supplementary Fig. S1 in IJSEM Online). Reaction to Gram-stain was determined using light microscopy according to Smibert & Krieg (1994). Gliding motility was determined using phase-contrast light microscopy (Olympus) after growth on quarter-strength marine 2216 medium solidified with 1 % agar according to Bowman (2000). Susceptibility to antibiotics was tested by the routine disc-diffusion plate method according to Acar (1980). Flexirubin-type pigment production and carboxymethylcellulose 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 performed by using the method of Baumann & Baumann (1988). Oxidase and catalase activities and the hydrolysis of agar, DNA and starch were tested according to Smibert & Krieg (1994). Other enzyme activities, growth on carbon sources, acid production from carbon sources, nitrate reduction and the production of H2S, indole and acetoin were tested using the API 20E, API 20NE, API 50CH, API ZYM (bioMérieux) and MicroLog 3 (Biolog) commercial systems. Cells for inoculation into the API systems were suspended in a sterile solution of a seawater mixture at 22 
salinity (MacDonell et al., 1982). The phenotypic characteristics of strains UST030701-097T and UST030701-084T are given in the genus/species descriptions. Results obtained from the API and MicroLog 3 systems are detailed in Supplementary Tables S1–S3 in IJSEM Online.
Chemotaxonomic and phenotypic characteristics that distinguish strain UST030701-084T from other members of the genera Marinicola and Roseivirga are given in Tables 1 and 2
. Characteristics that distinguish strain UST030701-097T from other genera in the phylum Bacteroidetes are detailed in Table 3. Most notably, strains UST030701-097T and UST030701-084T differ from their close relatives by being more halotolerant, but not requiring NaCl for growth. Moreover, strain UST030701-097T has a distinctive curved cell shape and strain UST030701-084T has a range of hydrolytic and enzyme activities not found in other members of the genera Marinicola and Roseivirga. Strains UST030701-097T and UST030701-084T differ from each other by: (i) cell shape, (ii) halo- and thermotolerance levels, (iii) gelatin hydrolysis and arginine dihydrolase, 
-galactosidase, 
-galactosidase and -mannosidase activities and (iv) susceptibility to ampicillin, penicillin, streptomycin and tetracycline. Strain UST030701-097T is able to utilize a variety of sole carbon sources in the API and MicroLog 3 systems, while strain UST030701-084T can only utilize aesculin ferric citrate in the API 50CH system and -ketovaleric acid in the MicroLog 3 system (see Supplementary Tables S2 and S3 in IJSEM Online). The small number of carbon sources utilized by strain UST030701-084T is a feature also found in the members of the genus Roseivirga. Molecular evidence, together with chemotaxonomic and phenotypic characteristics, suggest that strain UST030701-097T represents a novel genus and that strain UST030701-084T represents a novel species within the phylum Bacteroidetes. The name Fabibacter halotolerans gen. nov., sp. nov., is proposed for strain UST030701-097T. Strain UST030701-084T is proposed as Roseivirga spongicola sp. nov.
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Cells are Gram-negative, curved rods (1.5 µm long x0.5 µm wide). Strictly aerobic and chemo-organotrophic. The major respiratory quinone is MK-7. Flexirubin-type pigments are not produced. Oxidase- and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequence indicates that the genus is a member of the family ‘Flexibacteraceae’ in the phylum Bacteroidetes.
Currently, the genus contains one species, the type species Fabibacter halotolerans.
Description of Fabibacter halotolerans sp. nov.
Fabibacter halotolerans (ha.lo.to'le.rans. Gr. masc. n. hals salt; L. part. adj. tolerans tolerating; N.L. part. adj. halotolerans salt-tolerating).
Displays the following properties in addition to those given in the genus description. Colonies on marine agar are pink, circular, 2.0–4.0 mm in diameter, convex with a smooth surface and an entire margin. No diffusible pigment. Has fast-gliding motility. Growth occurs between 12 and 36 °C (optimum of 28–30 °C) and between pH 5.0 and 10.0. Does not require sodium for growth, but can tolerate up to 12 % NaCl. In disc-diffusion tests, susceptible to ampicillin (1 µg), chloramphenicol (1 µg), penicillin (1 µg), streptomycin (0.1 µg) and tetracycline (5 µg), but not to kanamycin (tested up to 100 µg). DNA G+C content is 42.5±0.3 mol%. Predominant fatty acids (>5 %) are i15 : 0, i15 : 1, i15 : 0 3-OH, i16 : 0 3-OH, i17 : 0 3-OH and SF 3(comprising i15 : 0 2-OH and/or 16 : 17c). These fatty acids represent 80.7 % of the total. Produces acetoin, but not indole or H2S. Nitrate is not reduced. DNA and Tweens 20, 40 and 80 are hydrolysed, but not agar, casein, carboxymethylcellulose, chitin or gelatin. Starch is weakly hydrolysed. N-Acetyl--glucosaminidase, acid phosphatase, alkaline phosphatase, arginine dihydrolase, -galactosidase, -galactosidase, -glucosidase, -glucosidase, -chymotrypsin, cystine arylamidase, leucine arylamidase, valine arylamidase, esterase (C4), esterase lipase (C8), lipase (C14), -mannosidase, trypsin and naphthol-AS-BI-phosphohydrolase activities are positive. No activities of -fucosidase, -glucuronidase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase or urease. Growth occurs on the following sole carbon sources in the API 20E, 20NE and 50CH systems: D-cellobiose, D-lactose, D-maltose and starch. Acid is produced from the following sole carbon sources in the API 20E and 50CH systems: amygdalin, arbutin, D-cellobiose, aesculin ferric citrate, D-galactose, D-glucose, gentiobiose, maltose, methyl -D-glucopyranoside, D-raffinose, salicin, sucrose, starch and D-trehalose. Utilizes the following carbon sources in the MicroLog 3 system: L-alaninamide, L-alanine, L-alanyl glycine, L-aspartic acid, D-cellobiose, dextrin, D-galacturonic acid, gentiobiose, -D-glucose, D-glucose 6-phosphate, L-glutamic acid, glycogen, glycyl L-aspartic acid, glycyl L-glutamic acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, DL-lactic acid, -D-lactose, lactulose, maltose, D-melibiose, methyl -D-glucoside, L-ornithine, L-proline, L-pyroglutamic acid, D-raffinose, succinamic acid, sucrose, D-trehalose, turanose and L-threonine. A full list of carbon sources included in the API and MicroLog 3 systems is given in Supplementary Tables S2 and S3 in IJSEM Online.
The type strain, UST030701-097T (=NRRL B-41220T=JCM 13334T), was isolated from the marine sponge Tedania ignis in the Bahamas.
Description of Roseivirga spongicola sp. nov.
Roseivirga spongicola [spon.gi'co.la. Late L. n. spongos -i sponge; L. masc./fem. suffix n. -cola (from incola) inhabitant; N.L. nom. n. (in apposition) spongicola inhabitant of sponges].
Cells are Gram-negative rods, 2.0 µm long x0.5 µm wide, with gliding motility. Colonies on marine agar are pink, circular, 2.0–4.0 mm in diameter, convex with a smooth surface and an entire margin. No diffusible pigment. Strictly aerobic and chemo-organotrophic. Growth occurs between 12 and 44 °C (optimum is 20–30 °C) and between pH 5.0 and 10.0. Does not require sodium for growth, but can tolerate up to 16 % NaCl. The major respiratory quinone is MK-7. In disc diffusion tests, susceptible to chloramphenicol (100 µg), but not to ampicillin, kanamycin, penicillin, streptomycin or tetracycline (each tested up to 100 µg). Flexirubin-type pigments are not produced. DNA G+C content is 43.7±0.6 mol%. Predominant fatty acids (>5 %) are a15 : 0, i15 : 0, i15 : 1, 17 : 0 2-OH, i17 : 0 3-OH, i17 : 19c and SF 3 (comprising i15 : 0 2-OH and/or 16 : 17c). These fatty acids represent 88.3 % of the total. Produces acetoin, but not indole or H2S. Nitrate is not reduced. DNA, gelatin and Tweens 20, 40 and 80 are hydrolysed, but not agar, casein, carboxymethylcellulose, chitin or starch. N-Acetyl--glucosaminidase, catalase, cystine arylamidase, leucine arylamidase, valine arylamidase, -chymotrypsin, oxidase, -glucosidase, -glucosidase, esterase (C4), esterase lipase (C8), acid phosphatase, alkaline phosphatase, lipase (C14), naphthol-AS-BI-phosphohydrolase and trypsin activities are positive. No activities of arginine dihydrolase, -fucosidase, -galactosidase, -galactosidase, -glucuronidase, lysine decarboxylase, -mannosidase, ornithine decarboxylase, tryptophan deaminase or urease. Utilizes only aesculin ferric citrate in the API 50CH system and -ketovaleric acid in the MicroLog 3 system as sole carbon sources. No acid production from the sole carbon sources in the API 20E and 50CH systems. A full list of carbon sources included in the API and MicroLog 3 systems is provided in Supplementary Tables S2 and S3 in IJSEM Online.
The type strain, UST030701-084T (=NRRL B-41219T=JCM 13337T), was isolated from the marine sponge Tedania ignis in the Bahamas.
Description of Roseivirga seohaensis comb. nov.
Roseivirga seohaensis (seo.ha.en'sis. N.L. fem. adj. seohaensis of Seohae, the Korean name for the Yellow Sea in Korea, from where the type strain was isolated).
Basonym: Marinicola seohaensis Yoon et al. 2005
The description is identical to that given for Marinicola seohaensis by Yoon et al. (2005). The type strain is SW-152T (=KCTC 12312T=JCM 12600T).
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ACKNOWLEDGEMENTS |
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50 % (500 replicates) are indicated at the nodes. GenBank accession numbers are shown in parentheses. Bar, 1 nucleotide substitution per 100 nucleotides.