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1 Department of Biology, College of Natural Sciences, Sunchon National University, Sunchon 540-742, Republic of Korea
2 School of Biological Sciences and Institute of Microbiology, Seoul National University, NS70, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
3 Department of Dental Hygiene, Kwangyang Health College, Kwangyang 545-703, Republic of Korea
4 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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). Recently, two further species of the genus, namely Glaciecola mesophila and Glaciecola polaris, have been described, isolated from marine invertebrate specimens and Arctic Ocean seawater, respectively (Romanenko et al., 2003; Van Trappen et al., 2004). In the course of our study on marine microbial diversity, a Glaciecola-like strain, designated strain FR1064T, was isolated from surface seawater and was the subject of a comprehensive taxonomic investigation. In this study, the polyphasic taxonomic properties of strain FR1064T are presented.
Strain FR1064T was isolated from a coastal surface seawater sample collected off Jeju Island, Republic of Korea. The sample was diluted with sterilized artificial seawater (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 %, w/v) at –80 °C. G. pallidula ACAM 615T, cultured on MA at 15 °C, was used as a reference strain.
Bacterial DNA preparation, PCR amplification and sequencing of the 16S rRNA gene were carried out as described previously (Chun & Goodfellow, 1995). The resulting gene sequence of strain FR1064T was aligned manually against sequences obtained from GenBank. Phylogenetic trees were inferred from the regions available for all sequences (positions 28–1433; Escherichia coli numbering system) using the Fitch–Margoliash (Fitch & Margoliash, 1967), maximum-likelihood (Felsenstein, 1981), maximum-parsimony (Fitch, 1971) and neighbour-joining (Saitou & Nei, 1987) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969). The resultant tree topologies were evaluated in bootstrap analyses (Felsenstein, 1985) of the neighbour-joining methods based on 1000 resamplings. The alignment and phylogenetic analyses were carried out using the jPHYDIT program (Jeon et al., 2005; http://chunlab.snu.ac.kr/jphydit) and PAUP 4.0 (Swofford, 1998) as described previously (Kim et al., 2005; Yi & Chun, 2006).
Preliminary sequence comparisons with the 16S rRNA gene sequences held in GenBank indicated that the novel isolate belonged to the class Gammaproteobacteria. The resultant sequence was then aligned manually based on 16S rRNA secondary structure (Gutell, 1994) with representative sequences of the Gammaproteobacteria obtained from GenBank. Strain FR1064T showed the highest 16S rRNA gene sequence similarity to G. pallidula ACAM 615T (97.6 %), followed by G. polaris LMG 21857T (95.4 %), G. mesophila LMG 21855T (95.2 %) and G. punicea ACAM 611T (94.2 %). To elucidate the phylogenetic relationship between the novel isolate and the other species of the genus Glaciecola, phylogenetic trees were constructed using four different tree-making algorithms. The neighbour-joining tree (Fig. 1) showed that strain FR1064T formed a monophyletic clade with G. pallidula ACAM 615T with 100 % bootstrap support. This relationship was confirmed by all other tree-inferring methods used in this study.
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) using a DIG High Prime DNA labelling and detection starter kit II (Roche). When DNAs were used individually as labelled DNA probes for reciprocal hybridization experiments and conducted in duplicate, the DNA–DNA relatedness value between strain FR1064T and G. pallidula ACAM 615T was 55 %. It is clear from 16S rRNA gene sequence and DNA–DNA hybridization data that strain FR1064T represents a novel species in the genus Glaciecola (Wayne et al., 1987).
For phenotypic characterization, strain FR1064T was grown on MA at 25 °C unless otherwise specified. Cellular morphology was observed by differential interference microscopy (Axioskop 40; Zeiss) and scanning electron microscopy (JSM-6400; JEOL) using cells grown at 25 °C for 3 days. Motility was examined using wet mounts. Growth under anaerobic conditions was checked in an anaerobic chamber (CO2/H2/N2, 10 : 10 : 80; Sheldon Manufacturing). The pH range (pH 4–12) for growth was determined using MA. The requirement for sea salts (0–11 %, Sigma) for growth was tested using synthetic ZoBell medium (ZoBell, 1941; 15 g Bacto agar, 5 g Bacto peptone, 1 g yeast extract, 0.1 g ferric citrate in 1 l distilled water). Growth at various temperatures was examined on MA at 4–50 °C. Biochemical tests were performed using the API 20E, API 20NE and API ZYM kits (bioMérieux). Strips were inoculated with a heavy bacterial suspension in ASW or AUX medium (bioMérieux) supplemented with 2 % (w/v) sea salts. Catalase and oxidase activities were determined using 3 % (v/v) hydrogen peroxide and Kovacs' reagent (Kovacs, 1956), respectively. The novel isolate is halophilic as it required 2–9 % (w/v) artificial sea salts for growth (optimum 4–7 %). The results of biochemical and physiological tests are given in the species description and Table 1.
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. The DNA G+C content for strain FR1064T was 44 mol% and the cellular fatty acid profile is given in Table 2.
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). Therefore, we propose that strain FR1064T be classified as a representative of a novel species within the genus Glaciecola, for which the name Glaciecola nitratireducens sp. nov. is proposed.
Description of Glaciecola nitratireducens sp. nov.
Glaciecola nitratireducens (ni.tra.ti.re.du'cens. N.L. n. nitras nitrate; L. part. adj. reducens converting to a different state; N.L. part. adj. nitratireducens reducing nitrate).
Gram-negative, aerobic and halophilic. Catalase-positive. Colonies on MA are circular, convex with an entire margin, slightly cream-coloured and approximately 1.5 mm in diameter after 5 days at 25 °C. Cells are motile, oval- or rod-shaped and 0.5–0.6x1.0–1.5 µm. Spores are not formed. Does not grow without sea salts. Growth occurs in 2–9 % (w/v) sea salts (optimum of 4–7 %). Growth occurs at pH 6–9 (optimum pH of 8) and at temperatures between 15 and 30 °C (optimum of 25 °C). Does not utilize citrate, caprate, adipate or phenylacetate. Produces cytochrome oxidase, but not ornithine decarboxylase, arginine dihydrolase, lysine decarboxylase, urease, indole, acetoin, H2S or tryptophan deaminase. Negative in tests for the fermentation of inositol, sorbitol, rhamnose, melibiose and amygdalin. Produces alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and trypsin, but not lipase (C14), valine arylamidase, cystine arylamidase, 
-chymotrypsin, 
-glucuronidase, -glucosidase, N-acetyl--glucosaminidase, -glucosidase, -mannosidase or -fucosidase. Other physiological and biochemical characteristics are given in Table 1. Major fatty acids are summed feature 3 (38.2 %), C16 : 0 (20.5 %), C18 : 1
7c (7.7 %) and C14 : 0 (7.0 %). The DNA G+C content of the type strain is 44 mol%.
The type strain, FR1064T (=KCTC 12276T=JCM 12485T), was isolated from seawater off Jeju Island, Republic of Korea.
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ACKNOWLEDGEMENTS |
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