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1 School of Biological Sciences and Institute of Microbiology, Seoul National University, Kwanak-gu, Seoul 151-742, Republic of Korea
2 Department of Biology, College of Natural Sciences, Sunchon National University, Sunchon 540-742, Republic of Korea
3 Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, Republic of Korea
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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7c fatty acids. The DNA G+C content was 39 mol%. On the basis of polyphasic evidence, it is concluded that the isolate represents a novel species within the genus Pseudoalteromonas, for which the name Pseudoalteromonas byunsanensis sp. nov. is proposed. The type strain is FR1199T (=JCM 12483T=KCTC 12274T).
Published online ahead of print on 16 September 2005 as DOI 10.1099/ijs.0.63750-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Pseudoalteromonas byunsanensis sp. nov. FR1199T is DQ011289.
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for marine aerobic, Gram-negative, non-fermentative, polarly flagellated bacteria, was later divided into two genera, Alteromonas and Pseudoalteromonas, on the basis of phylogenetic analysis (Gauthier et al., 1995). The genus Pseudoalteromonas currently comprises 33 species (Gauthier, 1976, 1977, 1982; Gauthier & Breittmayer, 1979; Novick & Tyler, 1985; Romanenko et al., 1995, 2003a; Ivanova et al., 1996, 1998, 2000, 2001, 2002a, b, c, d, 2004; Bozal et al., 1997; Bowman, 1998; Holmström et al., 1998; Sawabe et al., 1998, 2000; Venkateswaran & Dohmoto, 2000; Egan et al., 2001; Isnansetyo & Kamei, 2003; Kobayashi et al., 2003). In the course of our study on marine microbial diversity, a pseudoalteromonad, designated strain FR1199T, was isolated from a tidal flat sediment sample and was the subject of a polyphasic taxonomic investigation. On the basis of our evidence, strain FR1199T represents a novel species in the genus Pseudoalteromonas.
A marine sediment sample was collected from the Korean tidal flat of Byunsan in South Korea (35° 34' 52·3'' N 126° 30' 51·3'' E) in August 2002. The sample was diluted with sterilized artificial sea water (ASW; Lyman & Fleming, 1940), spread onto a plate containing marine agar 2216 (MA; Difco) and incubated at 25 °C for 3 weeks. The isolate was routinely cultured on MA and maintained as a glycerol suspension (20 % v/v) at –80 °C.
Bacterial DNA preparation, PCR amplification and sequencing of the 16S rRNA gene were carried out as described previously (Chun & Goodfellow, 1995). The resultant sequence of strain FR1199T was aligned manually against sequences obtained from GenBank. Phylogenetic trees were inferred from the regions available for all sequences (positions 43–1438; Escherichia coli numbering system) using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resultant neighbour-joining tree topology was evaluated by bootstrap analyses (Felsenstein, 1985) based on 1000 resamplings. Alignment and phylogenetic analyses were carried out using the jPHYDIT program (http://chunlab.snu.ac.kr/jphydit/; Jeon et al., 2005) and PAUP 4.0 (Swofford, 1998) as described previously (Chun et al., 2000).
Preliminary sequence comparison with 16S rRNA gene sequences held in GenBank indicated that our isolate was closely related to the genus Pseudoalteromonas. The newly determined sequence was then aligned manually against representatives of the genus Pseudoalteromonas using information on bacterial 16S rRNA secondary structures. Strain FR1199T showed low levels of 16S rRNA gene sequence similarity to other pseudoalteromonads. The highest similarity was found with Pseudoalteromonas rubra ATCC 29570T (97·4 %), followed by Pseudoalteromonas mariniglutinosa NCIMB 1770T (97·2 %), Pseudoalteromonas luteoviolacea NCIMB 1893T (96·7 %) and Pseudoalteromonas piscicida ATCC 15057T (96·6 %). Our isolate formed an independent phyletic line in the Pseudoalteromonas clade in the comprehensive phylogenetic analysis (Fig. 1), to which two species (P. mariniglutinosa and P. luteoviolacea) showing >97 % 16S rRNA similarities were not phylogenetically related. It is clear from the phylogenetic analysis that the tidal flat isolate FR1199T represents a novel species in the genus Pseudoalteromonas.
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; 15 g Bacto agar, 5 g Bacto peptone, 1 g yeast extract, 0·1 g ferric citrate l–1 distilled water) prepared on an ASW base, supplemented with appropriate amounts of NaCl.
Growth at various temperatures was examined on MA at 4–50 °C. Growth under anaerobic conditions was checked in an anaerobic chamber [1 % (v/v) CO2, 10 % (v/v) H2, 80 % (v/v) N2; Sheldon Manufacturing] using anaerobically prepared MA. Catalase and oxidase activities were determined using 3 % (v/v) hydrogen peroxide and Kovacs reagent (Kovacs, 1956), respectively. Other biochemical tests were performed using API 20NE and API 20E kits (bioMérieux). Enzyme activities were tested using an API ZYM kit (bioMérieux) following the manufacturer's instructions. Strips were inoculated with a heavy bacterial suspension in ASW or AUX medium (bioMérieux) supplemented with 2 % (w/v) sea salts. The results of biochemical and physiological tests are given in Table 1 and the species description. Our isolate can be readily differentiated from other phylogenetically related species by several phenotypic properties as shown in Table 1.
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. The major cellular fatty acids of the strain FR1199T were C16 : 0 (27·6 ± 0·4 %), C16 : 17c and/or iso-C15 : 0 2-OH (21·7 ± 1·2 %) (summed feature 3) and C18 : 17c (23·3 ± 0·5 %). In addition, C12 : 0 (1·1 ± 0·1 %), C10 : 0 3-OH (4·0 ± 0·3 %), C12 : 0 3-OH (3·4 ± 0·2 %), iso-C16 : 0 3-OH (2·8 ± 0·6 %), C18 : 06c (2·2 ± 0·2 %), C17 : 0 (1·6 ± 0·2 %), C17 : 18c (1·0 ± 0·1 %) and C18 : 16c (2·9 ± 0·1 %) were also detected. The DNA G+C content of strain FR1199T was 39 mol%.
The formation of a distinctive phyletic line within the genus Pseudoalteromonas indicates that strain FR1199T can be assigned as a novel species in this genus. In addition, a number of physiological and chemotaxonomic characters clearly distinguished our isolate from other phylogenetically related species (Table 1
). Therefore, strain FR1199T should be classified as a member of a novel species within the genus Pseudoalteromonas, for which the name Pseudoalteromonas byunsanensis sp. nov. is proposed.
Description of Pseudoalteromonas byunsanensis sp. nov.
Pseudoalteromonas byunsanensis (by.un.san.en'sis. N.L. fem. adj. byunsanensis named after Byunsan, South Korea, the geographical origin of the type strain).
Cells are rod-shaped, 0·5–0·8 µm in diameter and 1·5–2·0 µm in length, Gram-negative and motile. Strictly aerobic. Cells do not form endospores. Oxidase- and catalase-positive. Requires sea water for growth. Growth occurs in media with 0·5–5 % (w/v) NaCl, with the optimum at 1·5–2 %. Temperature for growth ranges from 10 to 40 °C, with the optimum at 25–30 °C. The pH for growth ranges from 5·0 to 10·0, with the optimum at pH 8·0. Colonies are colourless at pH 5·0 but violet-coloured at pH 6–10. Colonies are usually 2–3 mm in diameter within 3 days at 25 °C. Does not reduce nitrate to nitrite. Produces gelatinase, cytochrome oxidase and aesculin dihydrolase, but not arginine dihydrolase, 
-galactosidase, lysine decarboxylase, ornithine decarboxylase, urease, H2S, tryptophan deaminase, indole or acetone. Produces alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, 
-glucosidase and acid phosphatase, but not lipase (C14), cystine arylamide, valine arylamidase, -chymotrypsin, -galactosidase, -galactosidase, -glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -mannosidase, -fucosidase, lysine decarboxylase, ornithine decarboxylase or tryptophan deaminase. Utilizes the following substrates as sole carbon and energy sources: glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose and tetramethyl-p-phenylenediamine dichloride. Does not utilize the following substrates: gluconate, caprate, malate, malonate, citrate, phenylacetate and p-nitrophenyl -galactopyranoside. Other phenotypic characteristics are given in Table 1. The major cellular fatty acids are C16 : 0 (27·6 ± 0·4 %), C16 : 17c and/or iso-C15 : 0 2-OH (21·7 ± 1·2 %) and C18 : 17c (23·3 ± 0·5 %). The DNA G+C content is 39 mol%.
The type strain, FR1199T (=JCM 12483T=KCTC 12274T), was isolated from Korean tidal flat sediment.
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ACKNOWLEDGEMENTS |
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