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Int J Syst Evol Microbiol 56 (2006), 2185-2188; DOI  10.1099/ijs.0.64330-0
© 2006 International Union of Microbiological Societies

Glaciecola nitratireducens sp. nov., isolated from seawater

Keun Sik Baik1, Yoon-Dong Park2, Chi Nam Seong1, Eun Mi Kim1,3, Kyung Sook Bae4 and Jongsik Chun2

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


   ABSTRACT
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A marine bacterial strain, FR1064T, was isolated from a surface seawater sample collected off Jeju Island, South Korea. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the Gammaproteobacteria and was related to the genus Glaciecola with 97.6 % sequence similarity to Glaciecola pallidula, its nearest phylogenetic neighbour. DNA–DNA relatedness between strain FR1064T and G. pallidula ACAM 615T was 55 %. Cells of the novel isolate were Gram-negative, aerobic, rod-shaped, motile and halophilic, with an optimum sea salts concentration of 4–7 %. The major fatty acids were straight-chain saturated (C16 : 0), summed feature 3 and monounsaturated fatty acid C18 : 1. The DNA G+C content was 44 mol%. Several phenotypic characteristics differentiated the novel isolate from all previously described members of the genus Glaciecola. The polyphasic data obtained in this study clearly demonstrate that strain FR1064T represents a novel species of the genus Glaciecola. The name Glaciecola nitratireducens sp. nov. is therefore proposed, with strain FR1064T (=KCTC 12276T=JCM 12485T) as the type strain.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain FR1064T is AY787042.


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The genus Glaciecola was originally created to accommodate aerobic, psychrophilic, halophilic bacteria and initially comprised two species, Glaciecola punicea and Glaciecola pallidula. These species were isolated from sea-ice diatom assemblage samples collected from coastal areas of eastern Antarctica (Bowman et al., 1998Go). 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., 2003Go; Van Trappen et al., 2004Go). 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, 1940Go), 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, 1995Go). 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, 1967Go), maximum-likelihood (Felsenstein, 1981Go), maximum-parsimony (Fitch, 1971Go) and neighbour-joining (Saitou & Nei, 1987Go) methods. Evolutionary distance matrices were generated according to Jukes & Cantor (1969)Go. The resultant tree topologies were evaluated in bootstrap analyses (Felsenstein, 1985Go) of the neighbour-joining methods based on 1000 resamplings. The alignment and phylogenetic analyses were carried out using the jPHYDIT program (Jeon et al., 2005Go; http://chunlab.snu.ac.kr/jphydit) and PAUP 4.0 (Swofford, 1998Go) as described previously (Kim et al., 2005Go; Yi & Chun, 2006Go).

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, 1994Go) 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. 1Go) 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.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing relationships between strain FR1064T and members of the class Gammaproteobacteria. Bootstrap values, expressed as percentages of 1000 replications, are given at the nodes. Solid circles indicate that the corresponding nodes were also recovered in Fitch–Margoliash, maximum-likelihood and maximum-parsimony trees. Helicobacter pylori ATCC 43504T was used as an outgroup. Bar, 0.1 nucleotide substitutions per position.

 
The taxonomic relationship between strain FR1064T and G. pallidula ACAM 615T was further examined using DNA–DNA hybridization. Genomic relatedness was determined using a membrane filter technique (Seldin & Dubnau, 1985Go) 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., 1987Go).

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, 1941Go; 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, 1956Go), 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 1Go.


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  		Table 1. Phenotypic characteristics that differentiate strain FR1064T from its nearest phylogenetic neighbours

Species: 1, FR1064T; 2, Glaciecola polaris LMG 21857T; 3, G. mesophila LMG 21855T; 4, G. punicea ACAM 611T; 5, G. pallidula ACAM 615T. Data from Bowman et al. (1998)Go, Romanenko et al. (2003)Go, Van Trappen et al. (2004)Go and this study. +, Positive; –, negative; W, weakly positive; ND, not determined.

 
Cellular fatty acids of the novel isolate were analysed as methyl esters by GLC by the MIDI system according to the manufacturer's instructions. Fatty acid methyl esters were prepared from biomass grown on MA at 25 °C for 2 days. DNA G+C content (mol%) was determined by the thermal denaturation method of Marmur & Doty (1962)Go. The DNA G+C content for strain FR1064T was 44 mol% and the cellular fatty acid profile is given in Table 2Go.


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  		Table 2. Cellular fatty acid content (%) of strain FR1064T and Glaciecola species

Strains: 1, FR1064T; 2, G. polaris LMG 21857T; 3, G. mesophila LMG 21855T; 4, G. punicea ACAM 611T; 5, G. pallidula ACAM 615T. Data from Bowman et al. (1998)Go, Romanenko et al. (2003)Go, Trappen et al. (2004) and this study. Values are percentages of total fatty acids. ND, Not detected; tr, <1.0 %.

 
On the basis of phylogenetic analysis and DNA–DNA hybridization, it is evident that strain FR1064T should be assigned as representing a novel species of the genus Glaciecola. In addition, a number of physiological and chemotaxonomic characteristics clearly distinguish the novel isolate from other phylogenetically related species (Tables 1 and 2GoGo). 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, {alpha}-chymotrypsin, beta-glucuronidase, {alpha}-glucosidase, N-acetyl-beta-glucosaminidase, beta-glucosidase, {alpha}-mannosidase or {alpha}-fucosidase. Other physiological and biochemical characteristics are given in Table 1Go. Major fatty acids are summed feature 3 (38.2 %), C16 : 0 (20.5 %), C18 : 1{omega}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.


   ACKNOWLEDGEMENTS
 
We thank Dr John Bowman (Australian Food Safety Centre of Excellence) for the gift of the type strain of Glaciecola pallidula. This work was supported, in part, by Korea Ministry of Science and Technology under National Research Laboratory Program (M10500000110-05J0000-11010) and 21C Frontier Microbial Genomics and Applications Center Program (MG05-0101-2-0).


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Jeon, Y.-S., Chung, H., Park, S., Hur, I., Lee, J.-H. & Chun, J. (2005). jPHYDIT: a JAVA-based integrated environment for molecular phylogeny of ribosomal RNA sequences. Bioinformatics 21, 3171–3173.[Abstract/Free Full Text]

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