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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
Correspondence
Pei-Yuan Qian
boqianpy{at}ust.hk
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ABSTRACT |
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9c, i17 : 0 3-OH and summed feature 3, comprising i15 : 0 2-OH and/or 16 : 17c (altogether representing 76·2 % of the total). MK-6 was the only respiratory quinone. Flexirubin-type pigments were not produced. Phylogenetic analysis based on 16S rRNA gene sequences indicated that Gramella echinicola KMM 6050T (the only species in the genus) was the closest relative of UST040801-001T, sharing 98·0 % sequence similarity. The DNA–DNA relatedness between UST040801-001T and Gramella echinicola KMM 6050T was 13 %. Strain UST040801-001T can be distinguished from G. echinicola by means of 11 phenotypic traits. The results of molecular and phenotypic analyses suggested that UST040801-001T represents a novel species of Gramella. The name Gramella portivictoriae sp. nov. is proposed for this bacterium, with UST040801-001T (=NRRL 41137T=JCM 13192T) as the type strain.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain UST040801-001T is DQ002871.
An electron micrograph of cells of strain UST040801-001T, the results of API 20E, API 20NE and API ZYM tests and details of the carbon sources tested are available as supplementary material in IJSEM Online.
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), was created recently by Nedashkovskaya et al. (2005) and currently consists of a single species, Gramella echinicola, originating from the sea urchin Strongylocentrotus intermedius in the Sea of Japan. In this study, we propose a novel species of Gramella originating from marine sediment. During the characterization of a microbial community in marine sediment collected from Victoria Harbour, Hong Kong, the bacterial strain UST040801-001T was isolated on an agar medium composed of 5 g peptone l–1, 3 g yeast extract l–1 (both Oxoid) and 0·22-µm-filtered sea water (referred to as marine agar hereafter). After 48 h incubation at 30 °C on marine agar, UST040801-001T appeared as yellow, convex, circular colonies (2–4 mm in diameter) with smooth surfaces and entire translucent margins. No diffusible pigment was observed. Unless otherwise specified, all characteristics described hereafter were based on cultures grown on marine agar for 48 h at 30 °C.
The nearly-complete 16S rRNA gene sequence of UST040801-001T (1468 bp) was obtained using a dye terminator method, as described elsewhere (Lau et al., 2004). Fragments of DNA sequence obtained from individual primers with at least six replicates were each assembled using the SEQUENCHER software package (Gene Codes). Comparison of the nearly-complete 16S rRNA gene sequence of UST040801-001T with those available from GenBank revealed that UST040801-001T was a member of the family Flavobacteriaceae and was closely related to G. echinicola KMM 6050T (98·0 % sequence similarity) and to the uncharacterized marine bacteria KT0803 and KMM 6048 (97·8 and 98·4 % sequence similarity, respectively). A neighbour-joining phylogenetic tree constructed using the ARB software package (Ludwig et al., 2004) showed that the four bacteria formed a distinctive clade (Fig. 1). Within this clade, UST040801-001T formed a branch with the marine bacterium KMM 6048 without significant bootstrap support. Trees based on maximum-parsimony and maximum-likelihood methods in the ARB software package showed identical topology (Fig. 1). The level of DNA–DNA relatedness between UST040801-001T and G. echinicola KMM 6050T was 13 %, which clearly indicated that UST040801-001T should belong to a novel species of the genus Gramella. DNA–DNA hybridization experiments were performed by the BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Universiteit Gent (Gent, Belgium) using photobiotin-labelled probes as described by Ezaki et al. (1989).
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), was 39·9±0·2 mol% (three replicates). This value was similar to that for G. echinicola KMM 6050T (39·6 mol%). The dominant cellular fatty acids of UST040801-001T were a15 : 0, i15 : 0, i15 : 0 3-OH, i17 : 19c, i17 : 0 3-OH 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 (MIDI) according to the manufacturer's protocol (Table 1). MK-6 was the only respiratory quinone, as determined using an HPLC method according to Collins (1994). Menaquinones extracted from Cellulophaga lytica (Johansen et al., 1999) and Pedobacter heparinus (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 (7600F; JEOL) according to Neu et al. (2001) (see Supplementary Fig. S1 available in IJSEM Online). The reaction to Gram stain was determined using light microscopy according to Smibert & Krieg (1994). Gliding motility was determined using phase-contrast light microscopy (Olympia) after growth on quarter-strength marine 2216 medium (Oxoid) solidified with 1 % agar according to Bowman (2000). Susceptibility to antibiotics was tested as described by Acar (1980). Flexirubin-type pigment production and carboxymethylcellulose hydrolysis were determined as described by Bernardet et al. (2002). Casein hydrolysis was determined as described by Norris et al. (1985); Tween 20, 40 and 80 hydrolysis and chitin were tested as described by Baumann & Baumann (1988). Spore formation, oxidase and catalase activities and the hydrolysis of agar, DNA and starch were tested as described by Smibert & Krieg (1994). Other enzymic activities, the substrate-utilization profile, nitrate reduction and the production of H2S, indole and acetoin were tested using the commercial systems API 20E, API 20NE, API 50 CH and API ZYM (all from 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). Growth on D-arabinose, D-galactose, D-glucose, glycerol, D-mannitol, D-melibiose, D-sorbitol, starch and sucrose as sole carbon sources was additionally tested using a medium that contained 0·2 g NaNO3 l–1, 0·2 g NH4Cl l–1, 0·05 g yeast extract l–1 and 4 % (w/v) carbon source in a solution of sea-water mix at 35 salinity (Nedashkovskaya et al., 2003). The phenotypic characteristics of UST040801-001T are given in the species description (and in Supplementary Table S1, available in IJSEM Online).
Strain UST040801-001T can be distinguished from G. echinicola by (i) the absence of 
-galactosidase activity; (ii) the ability to hydrolyse Tween 20, to produce acetoin and to utilize D-mannitol and D-sorbitol; and (iii) the inability to grow in 15 % NaCl and to hydrolyse casein and DNA (Table 2). The results of molecular analysis, together with the phenotypic characteristics, suggest that strain UST040801-001T represents a novel species of the genus Gramella.
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Cells are Gram-negative, rod-shaped (0·6–3·6 µm), glide slowly and do not form spores. Colonies on marine agar are yellow in colour without diffusible pigment, circular, 2·0–4·0 mm in diameter and convex with smooth surfaces and entire translucent margins after 48 h at 30·0 °C. Growth is strictly aerobic and occurs between 4·0 and 36·0 °C (28·0–30·0 °C optimum), at pH 6·0–10·0 (pH 7·0–8·0 optimum) and at 1·0–6·0 % NaCl. The DNA G+C content is 39·9 mol%. The predominant cellular fatty acids are a15 : 0, i15 : 0, i15 : 0 3-OH, i17 : 19c, i17 : 0 3-OH and summed feature 3, comprising i15 : 0 2-OH and/or 16 : 17c (altogether representing 76·2 % of the total). The major quinone is MK-6. Flexirubin-type pigments are not produced. Susceptible to ampicillin, chloramphenicol, penicillin and tetracycline, but not to kanamycin or streptomycin. Acetoin is produced, but indole and H2S are not. Citrate is not utilized. Nitrate is not reduced. Aesculin ferric citrate, gelatin, starch and Tweens 20, 40 and 80 are hydrolysed, but agar, casein, carboxymethylcellulose, chitin and DNA are not hydrolysed. Positive for acid phosphatase, alkaline phosphatase, 
-chymotrypsin, catalase, cystine arylamidase, leucine arylamidase, valine arylamidase, esterase (C4), esterase lipase (C8), lipase (C14), oxidase, -galactosidase, -glucosidase, -glucosidase, trypsin and naphthol-AS-BI-phosphohydrolase activities. Negative for N-acetyl--glucosaminidase, arginine dihydrolase, -fucosidase, -galactosidase, -glucuronidase, -mannosidase, lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease activities. Utilization of D-arabinose, D-galactose, D-glucose, glycerol, D-mannitol, D-melibiose, D-sorbitol, starch and sucrose as sole carbon sources is observed on agar medium supplemented with 4 % (w/v) carbon source. Utilization of 
-hydroxybutyric acid is observed with the MicroLog 3 system. However, no growth or acid production is observed from the carbon sources in the API 50 CH, API 20E and API 20NE test systems (lists of carbon sources included in the API and MicroLog 3 test systems are available in Supplementary Tables S2 and S3, respectively, in IJSEM Online.)
The type strain is UST040801-001T (=NRRL 41137T=JCM 13192T), isolated from sediment in Victoria Harbour, Hong Kong.
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ACKNOWLEDGEMENTS |
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