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1 Department of Biology, College of Natural Sciences, Sunchon National University, Sunchon 540-742, Republic of Korea
2 Department of Dental Hygiene, Kwangyang Health College, Kwangyang 545-703, Republic of Korea
3 School of Biological Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
4 Laboratory of Insect Resources, Korea Research Institute of Bioscience and Biotechnology, Yusung PO Box 115, Taejon 305-600, Republic of Korea
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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-Proteobacteria. Sequence similarity between strain FR1050T and the type strain of Hahella chejuensis was 94·7 %. Cells were Gram-negative, aerobic, rod-shaped, motile and halophilic; optimum growth occurred at sea salt concentrations of 4–6 %. The major fatty acids were C18 : 1
9c (39·0 %) and C16 : 0 (18·1 %). The DNA G+C content was 44 mol%. The polyphasic data obtained showed that strain FR1050T is affiliated to the genus Hahella but represents a novel species for which the name Hahella ganghwensis sp. nov. is proposed. The type strain is FR1050T (=KCTC 12277T=JCM 12486T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain FR1050T is AY676463.
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to accommodate a red-pigmented marine bacterial strain that formed a distinct phyletic line within the -Proteobacteria with low (<90 %) 16S rRNA gene sequence similarity to other recognized bacterial species. The only species of the genus, Hahella chejuensis, was isolated from a marine sediment sample from Korea (Marado, Cheju) and produced abundant extracellular polysaccharides. During the course of a study on marine microbial diversity, a Hahella-like strain, designated FR1050T, was isolated from a Korean sediment sample and was the subject of a taxonomic investigation. Based on its polyphasic properties, strain FR1050T is considered to represent a novel species, for which the name Hahella ganghwensis sp. nov. is proposed.
A marine sediment sample was collected from the getbol (Korean tidal flat) of Ganghwa Island, Korea (37° 35' 31·9'' N 126° 27' 24·5'' E). The sample was diluted with sterilized artificial sea water (ASW; Lyman & Fleming, 1940), spread onto a plate that contained 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 %, w/v) at –80 °C. H. chejuensis KCTC 2396T cultured on MA at 30 °C was used as a reference strain.
16S rRNA gene sequence analysis of strain FR1050T was performed using universal primers (Lane, 1991) as described by Chun & Goodfellow (1995), and an almost complete sequence was obtained (1454 bp). Phylogenetic analyses were performed using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1993), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resulting 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 (available at http://chunlab.snu.ac.kr/jphydit/) and PAUP 4.0 (Swofford, 1998) as described by Chun et al. (2000). Preliminary sequence comparison against the 16S rRNA gene sequences held in GenBank indicated that the getbol isolate belonged to the -Proteobacteria. The resultant sequence was then aligned manually based on 16S rRNA gene sequence secondary structure (Gutell, 1994) with representative sequences of the -Proteobacteria obtained from GenBank. Only unambiguously aligned nucleotide positions (1381 bp) were used to construct phylogenetic trees. On the basis of 16S rRNA gene sequence similarity values, the closest relatives were H. chejuensis KCTC 2396T (94·7 %), Zooshikella ganghwensis JC2044T (90·1 %) and Microbulbifer hydrolyticus DSM 11525T (90·7 %). No other recognized bacterial species showed more than 90 % 16S rRNA gene sequence similarity. This close relationship between strain FR1050T and H. chejuensis KCTC 2396T was also evident in the phylogenetic trees (Fig. 1). Strain FR1050T and H. chejuensis formed a monophyletic clade with 100 % bootstrap support and this grouping was recovered in all the phylogenetic trees employed in this study. Z. ganghwensis JC2044T was recovered as a sister group to the Hahella clade containing strain FR1050T in the neighbour-joining, Fitch–Margoliash and maximum-likelihood trees. However, the branching patterns of the other genera varied depending on the tree-building methods employed and were supported by relatively low bootstrap values. It is evident from phylogenetic analysis that strain FR1050T is affiliated to the genus Hahella with a novel species status.
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; 15 g Bacto agar, 5 g Bacto peptone, 1 g yeast extract, 0·1 g ferric citrate in 1000 ml distilled water). Sea salts were not added to the synthetic medium when the growth range for NaCl concentration was tested. The getbol isolate was halophilic, requiring media containing 1–10 % (w/v) artificial sea salts for growth (optimum 4–6 %), and was unable to grow on ZoBell medium containing 0–10 % (w/v) NaCl alone. Growth at various temperatures was examined on MA at 4–50 °C. Growth under anaerobic conditions was checked in an anaerobic chamber (1 % CO2, 10 % H2, 80 % N2; Sheldon Manufacturing) using anaerobically prepared MA, trypticase soy agar (Difco; supplemented with 1 % sea salts) and nutrient agar (Difco; supplemented with 1 % sea salts). Biochemical tests were performed using the API 20NE, API 20E and API ZYM kits (bioMérieux). Strips were inoculated with a heavy bacterial suspension in ASW or AUX medium (bioMérieux) supplemented with 2 % sea salts. Catalase and oxidase activities were determined using 3 % (v/v) hydrogen peroxide and Kovacs reagent (Kovacs, 1956), respectively. Results from these biochemical and physiological tests are given in the species description and in Table 1. In contrast to the result given by Lee et al. (2001), the type strain of H. chejuensis, together with strain FR1050T, showed no growth under anaerobic conditions when the media were prepared anaerobically. The reported anaerobic growth of H. chejuensis may have resulted from residual oxygen in the media tested by Lee et al. (2001). Based on the result obtained here, an emended description for the genus Hahella is given below.
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. The DNA G+C ratio of strain FR1050T was 44 mol% and the cellular fatty acid profile is given in Table 2. Although the culture conditions for growth of strain FR1050T and H. chejuensis KCTC 2396T were identical, their fatty acid compositions were very different, especially for C18 : 17c, C18 : 19c, C17 : 0 10-methyl and a mixture of iso-C16 : 0 2OH and/or C16 : 17c.
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). Based on the polyphasic evidence presented here, the getbol isolate merits novel species in the genus Hahella. The name Hahella ganghwensis sp. nov. is therefore proposed for strain FR1050T.
Description of Hahella ganghwensis sp. nov.
Hahella ganghwensis (gang.hwen'sis. N.L. fem. adj. ganghwensis pertaining to Ganghwa Island, Republic of Korea, the geographical origin of the type strain of the species).
Gram-negative, oxidase- and catalase-positive, aerobic and halophilic. Colonies on MA are circular, smooth, convex with entire margin, slightly cream-coloured and approximately 1 mm in diameter after 5 days at 30 °C. Produces slightly brown pigment after 5 days on MA at 40 °C. Cells are motile rods, 0·4–0·5x1·0–1·5 µm in size. Spores are not formed. Growth occurs in 1–10 % (w/v) sea salts (optimum 4–6 %). Does not grow without sea salts. Growth occurs at pH 5–10 (optimum 7–8) and at 15–40 °C (optimum 35 °C). Utilizes N-acetylglucosamine, but not amygdalin, gluconate, caprate or adipate. Other physiological and biochemical characteristics are given in Table 1. Major fatty acids are C18 : 19c (39·0 %) and C16 : 0 (18·1 %); the complete fatty acid profile is given in Table 2. The DNA G+C content is 44 mol%.
The type strain, FR1050T (=KCTC 12277T=JCM 12486T), was isolated from sediment of getbol, the Korean tidal flat.
Emended description of the genus Hahella
The description of the genus Hahella remains that given by Lee et al. (2001), with the following modifications. Aerobes. Reduction of nitrate to nitrite is variable. Require sea salts or NaCl for growth.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chun, J., Bae, K. S., Moon, E. Y., Jung, S. O., Lee, H. K. & Kim, S. J. (2000). Nocardiopsis kunsanensis sp. nov., a moderately halophilic actinomycete isolated from a saltern. Int J Syst Evol Microbiol 50, 1909–1913.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef]
Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package), version 3.5c. Distributed by the author. Department of Genetics, University of Washington, Seattle, USA.
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[CrossRef]
Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic trees: a method based on mutation distances as estimated from cytochrome c sequences is of general applicability. Science 155, 279–284.
Gutell, R. R. (1994). Collection of small subunit (16S- and 16S-like) ribosomal RNA structure: 1994. Nucleic Acids Res 22, 3502–3507.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
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Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115–175. Edited by M. Goodfellow. Chichester: Wiley.
Lee, H. K., Chun, J., Moon, E. Y., Ko, S. H., Lee, D. S., Lee, H. S. & Bae, K. S. (2001). Hahella chejuensis gen. nov., sp. nov., an extracellular-polysaccharide-producing marine bacterium. Int J Syst Evol Microbiol 51, 661–666.[Abstract]
Lyman, J. & Fleming, R. H. (1940). Composition of sea water. J Mar Res 3, 134–146.
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Zobell, C. E. (1941). Studies on marine bacteria. I. The cultural requirements of heterotrophic aerobes. J Mar Res 4, 42–75.
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