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1 Coastal Marine Laboratory/Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR
2 Department of Biology, Chinese University of Hong Kong, Shatin, N. T., Hong Kong SAR
Correspondence
Pei-Yuan Qian
boqianpy{at}ust.hk
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ABSTRACT |
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7c and 18 : 19c, altogether representing 82.9 % of the total. Phylogenetic analysis based on the 16S rRNA gene sequence placed UST050418-052T in a distinct lineage within the Roseobacter clade in the family Rhodobacteraceae, with 95.0–95.8 % sequence similarity to members of the nearest genus Thalassobius. The DNA–DNA relatedness between UST050418-052T and Thalassobius gelatinovorus IAM 12617T was 9 %. Strain UST050418-052T could be differentiated from closely related members of the Roseobacter clade by a number of chemotaxonomic and phenotypic characteristics such as its distinct fatty acid profile, ability to reduce nitrate to nitrite and inability to utilize citrate, succinate, L-arginine and pyruvate. Based on the phylogenetic, chemotaxonomic and phenotypic evidence presented in this study, we suggest that strain UST050418-052T represents a novel genus in the family Rhodobacteraceae. The name Thalassococcus halodurans gen. nov., sp. nov., is thus proposed. The type strain of Thalassococcus halodurans is UST050418-052T (=JCM 13833T =NRRL B-41465T).
Scanning electron micrographs of cells of strain UST050418-052T are available as supplementary material with the online version of this paper.
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-3 subgroup of the Proteobacteria and is one of the nine dominant marine clades that comprise marine microbial communities (Giovannoni & Rappé, 2000
). Members of this clade represent one of the most readily cultivated groups of marine lineages and are isolated from diverse marine environments ranging from coastal to open waters, sea floor to sea ice, in the form of free-living or particle-attached cells or in association with marine phytoplankton, invertebrates or vertebrates (see review by Buchan et al., 2005 and references therein). The first genus within this clade, Roseobacter, was established in 1991, in which two species, Roseobacter litoralis and Roseobacter denitrificans, were described (Shiba, 1991). Since then, the number of species and genera described has been increasing steadily. By 2004, this clade contained 35 species with validly published names from 16 genera: Antarctobacter, Jannaschia, Ketogulonicigenium, Leisingera, Loktanella, Oceanibulbus, Oceanicola, Octadecabacter, Roseobacter, Roseovarius, Ruegeria, Sagittula, Salipiger, Silicibacter, Staleya and Sulfitobacter (Schaefer et al., 2002; Allgaier et al., 2003; Cho & Giovannoni, 2004; Martínez-Cánovas et al., 2004; Wagner-Döbler et al., 2004; Van Trappen et al., 2004). At the time of writing, about 45 species have been described in 24 genera (Yi & Chun, 2006). This paper deals with the description of another novel bacterial strain in the Roseobacter clade, strain UST050418-052T.
Strain UST050418-052T was isolated from the surface of a colony of the marine sponge Halichondria panicea collected from Friday Harbor, San Juan Island, WA, USA, in April 2005. A freshly collected sponge colony was rinsed with autoclaved 0.22 µm-filtered seawater (AFSW) to remove loosely attached bacteria and its surface was then swabbed with a sterile cotton bud, which was then placed in a culture tube containing 1 ml AFSW for subsequent bacterial isolation and purification procedures. By using the standard dilution plating technique on a marine agar medium containing 3 g yeast extract (Oxoid), 5 g peptone (Oxoid) and 12 g bacteriological agar (Oxoid) in 1 l AFSW at 32 
salinity and repeated purification and incubation at 28 °C for 48 h, pure colonies of the strain were obtained. Unless otherwise specified, all characteristics described hereafter were based on cultures grown on marine agar under the same conditions. Pure colonies were observed under a light microscope (Leica MZ6; x40 magnification) as milky, raised and circular (1.1–1.4 mm in diameter) with an entire edge and a smooth surface. Gram staining was determined using light microscopy according to Smibert & Krieg (1994) and cell morphology was examined using scanning electron microscopy (6700F; JEOL) according to Neu et al. (2001). Swimming and gliding motilities were observed under a phase-contrast light microscope (Olympus BX51; x100 magnification) after growing the strain on one-quarter-strength marine broth 2216 (Oxoid) solidified with 0.4 or 1.2 % agar, respectively (Bowman, 2000). Strain UST050418-052T appeared as Gram-negative, non-motile and ovoid-shaped cells (see Supplementary Fig. S1 available in IJSEM Online).
The nearly complete 16S rRNA gene sequence of strain UST050418-052T (1383 bp) was obtained bidirectionally with three replicates as described by Lau et al. (2004). Comparative analysis of the 16S rRNA gene sequence with sequences deposited in GenBank using BLAST indicated that the strain fell within the Roseobacter clade in the -subgroup of the Proteobacteria and shared the highest sequence similarities, 99.6–99.8 %, with five uncharacterized Roseobacter strains (strains JL-129, JL-131, JL-132, JL-135 and JL-137). The most closely related characterized relatives of UST050418-052T are members of the genera Thalassobius (Arahal et al., 2005; Yi & Chun, 2006), Phaeobacter (Martens et al., 2006), Marinovum (Martens et al., 2006) and Leisingera (Schaefer et al., 2002), with 95.0–95.8 % sequence similarity. The 16S rRNA gene sequence was aligned automatically and then manually with a database of >30 000 already-aligned 16S rRNA gene sequences using the ARB software package (Ludwig et al., 2004). Phylogenetic trees showing the relatedness of strain UST050418-052T and other close members in the Roseobacter clade were then constructed using three tree-drawing methods: neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Fitch, 1971). The neighbour-joining tree showed that strain UST050418-052T formed a distinct cluster with the five uncharacterized Roseobacter strains, and this cluster was distantly related to another cluster comprising the members of Thalassobius (Fig. 1). This close phylogenetic relationship was also revealed in the maximum-parsimony and maximum-likelihood trees (data not shown). Strain UST050418-052T also shared the same lineage with the members of Phaeobacter, Marinovum and Leisingera. DNA–DNA hybridizations were performed by the BCCM/LMG Bacteria Collection (Laboratorium voor Microbiologie, University Gent, Gent, Belgium) as described by Ezaki et al. (1989) to determine the relatedness of UST050418-052T with its close relatives. The strain showed DNA relatedness with Thalassobius gelatinovorus IAM 12617T and Phaeobacter inhibens T5T of 9 and 7 %, respectively, which clearly indicated that UST050418-052T does not belong to the genera Thalassobius or Phaeobacter. These results support the conclusion that UST050418-052T represents a novel genus within the Roseobacter clade.
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as 57.8±0.1 mol% (n=3). This value is within the range of the G+C contents (57–61 mol%) observed among the members of closely related genera (Table 2). Extraction of respiratory quinones followed Minnikin et al. (1984) and the presence of ubiquinone Q-10, the primary isoprenoid quinone found in Thalassobius, Phaeobacter, Marinovum and Leisingera, was verified using an HPLC method according to Collins (1994). Ubiquinones extracted from Agrobacterium tumefaciens ATCC 23308T as described above served as a reference for Q-10. The cellular fatty acid profile of the strain was determined using the Sherlock Microbial Identification System (MIDI) according to the manufacturer's protocol. Strain UST050418-052T had a relatively simple cellular fatty acid profile which was dominated by the monounsaturated fatty acids 18 : 17c (37.1 %) and 18 : 19c (21.0 %), followed by the saturated fatty acids 16 : 0 (13.9 %) and 18 : 0 (10.9 %), altogether representing 82.9 % of the total fatty acids (Table 1). The most dominant fatty acid in UST050418-052T, 18 : 17c, was also found in members of Thalassobius, Phaeobacter and Leisingera, but the relative proportions were drastically different; the other dominant fatty acids, 18 : 19c, 16 : 0 and 18 : 0, were found in very small proportions or even not detected in its close relatives (Table 1). On the other hand, the polyunsaturated fatty acid 20 : 26,9c was found in large proportions in Phaeobacter, Leisingera and Marinovum (70.3–86.7 %) but not in UST050418-052T. Results from fatty acid analysis therefore differentiated UST050418-052T from closely related genera.
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. Hydrolysis of casein and cellulose was tested according to Norris et al. (1985) and Bowman (2000), respectively. Hydrolysis of Tweens 20, 40 and 80 and chitin was tested as described by Baumann & Baumann (1981). Hydrolysis of agar, DNA and starch and production of oxidase and catalase were determined according to Smibert & Krieg (1994). Other enzyme activities and the ability to reduce nitrate, produce H2S, indole and acetoin and utilize different carbon sources were assessed using commercial API 20E, API 20NE, API 50CH and API ZYM systems (bioMérieux) and MicroLog 3 (Biolog) according to the manufacturers' manuals. Cells for inoculating into the API 20E system were suspended in sterile seawater at 22 salinity before inoculation (MacDonell et al., 1982). Growth on glycerol, D-glucose, sucrose, D-mannitol, D-galactose, starch, D-sorbitol, D-arabinose and D-melibiose as sole carbon sources was also examined on a 1.2 % agar medium containing 0.2 g NaNO3, 0.2 g NH4Cl, 0.05 g yeast extract and 4 % (w/v) carbon source per litre seawater at 35 salinity (Nedashkovskaya et al., 2003). Detailed physiological and biochemical characteristics of UST050418-052T are given in the species description.
UST050418-052T differs from most of its close relatives by its ability to grow at salt concentrations higher than 9 % and at 40 °C and to reduce nitrate to nitrite and its inability to utilize citrate, succinate, L-arginine and pyruvate (Table 2
). Apart from these characteristics, the strain can be further differentiated from its closest relative Thalassobius aestuarii JC2049T by its ability to grow at 8 % NaCl, produce valine arylamidase, - and 
-galactosidases and to utilize D-fructose, D-mannitol, D-sorbitol, D-cellobiose, sucrose and trehalose as sole carbon sources and its inability to grow at 4 °C, to reduce nitrite to nitrogen, to hydrolyse gelatin, to produce lysine decarboxylase and ornithine decarboxylase and to utilize D-ribose. It also differs from other close relatives by a number of phenotypic characteristics as shown in Table 2. Based on the phylogenetic evidence and chemotaxonomic and phenotypic characteristics presented in this study, we suggest that strain UST050418-052T represents a novel member in the Roseobacter clade and propose the name Thalassococcus halodurans gen. nov., sp. nov.
Description of Thalassococcus gen. nov.
Thalassococcus (Tha.las.so.coc'cus. Gr. n. thalassa the ocean; L. n. coccus from Gr. n. kokkos berry, coccus; N.L. masc. n. Thalassococcus coccus from the ocean).
Cells are Gram-negative, ovoid-shaped, non-pigmented, devoid of swimming and gliding motility, strictly aerobic and halophilic. Oxidase- and catalase-positive. The major respiratory quinone is ubiquinone Q-10. The major fatty acids are 16 : 0, 18 : 0, 18 : 19c, 18 : 17c (altogether representing 82.9 % of the total in the type strain of the type species) and summed feature 5 (18 : 26,9c and/or anteiso-18 : 0). Phylogenetic analysis based on 16S rRNA gene sequences suggests that Thalassococcus is a member of the Roseobacter clade in the family Rhodobacteraceae. The genus currently contains one species, Thalassococcus halodurans, the type species.
Description of Thalassococcus halodurans sp. nov.
Thalassococcus halodurans (ha.lo.du'rans. Gr. n. hals salt, L. part. pres. durans withstanding, N.L. part. adj. halodurans withstanding salt).
Exhibits the following properties in addition to those given in the genus description. Cells are 0.7–1.0 µm in diameter and colonies are milky, 1.1–1.4 mm in diameter, raised and circular with a smooth surface and an entire edge when cultivated on marine agar at 28 °C for 48 h. Cells do not produce flexirubin-type or diffusible pigments. Growth occurs at pH 6–10 and 12–44 °C, with optimal growth at 28–36 °C; no growth below pH 5 or at 4 or 52 °C. Cells require a minimum of 2.0 % NaCl for growth and tolerate up to 18.0 % NaCl (optimum growth at 2.0–6.0 %). The DNA G+C content is 58.8 mol%. Cells are susceptible to 1.0 µg benzylpenicillin, chloramphenicol and ampicillin, 5.0 µg kanamycin and tetracycline and 10.0 µg streptomycin. Does not hydrolyse starch, casein, chitin, cellulose, agar, gelatin or Tween 20, 40 or 80. Acetoin is produced but not indole or H2S. Nitrate is reduced to nitrite but not to nitrogen gas. Citrate is not utilized. Positive for activities of DNase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, - and -galactosidases, - and -glucosidases and arginine dihydrolase. Negative for activities of urease, lipase (C14), cystine arylamidase, trypsin, -chymotrypsin, -glucuronidase, N-acetyl--glucosaminidase, -mannosidase, -fucosidase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase. Utilizes glycerol, D-glucose, sucrose, D-mannitol, D-galactose, starch, D-sorbitol, D-arabinose and D-melibiose as sole carbon sources on agar medium supplemented with 4 % (w/v) carbon source, glycerol, L-arabinose, D-xylose, D-galactose, D-glucose, D-fructose, inositol, D-mannitol, D-cellobiose, maltose, D-melibiose, sucrose, trehalose and potassium 2-ketogluconate in the API 50 CH system and D-sorbitol in the MicroLog 3 system. Utilization of other carbon sources included in the MicroLog 3, API 20 NE and 50 CH systems is not observed. Acid is produced from glycerol, L-arabinose, D-xylose, D-galactose, D-glucose, D-fructose, inositol, D-mannitol, D-sorbitol, D-melibiose, sucrose, D-fucose and potassium 2-ketogluconate in API 50 CH, but no acid production is observed in the API 20E system.
The type strain is UST050418-052T (=JCM 13833T =NRRL B-41465T), isolated from the surface of the marine sponge Halichondria panicea collected from Friday Harbor, San Juan Island, WA, USA.
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ACKNOWLEDGEMENTS |
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