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Int J Syst Evol Microbiol 55 (2005), 379-383; DOI  10.1099/ijs.0.63365-0
© 2005 International Union of Microbiological Societies

Idiomarina seosinensis sp. nov., isolated from hypersaline water of a solar saltern in Korea

Dong Han Choi and Byung Cheol Cho

Research Institute of Oceanography and School of Earth and Environmental Sciences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea

Correspondence
Byung Cheol Cho
bccho{at}plaza.snu.ac.kr


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A halophilic {gamma}-proteobacterium, designated CL-SP19T, was isolated from hypersaline water from a solar saltern located in Seosin, Korea. Analysis of the 16S rRNA gene sequence revealed an affiliation with the genus Idiomarina. The sequence similarities between CL-SP19T and type strains of the genus Idiomarina ranged from 95·9 to 96·9 %. Cells were straight or slightly curved rods and were motile by means of a single polar flagellum. The major fatty acids were C15 : 0 iso (17·1 %) and C17 : 0 iso (15·2 %). Three fatty acids, C19 : 0{omega}8c cyclo (3·5 %), C14 : 1{omega}5c (1·4 %) and C18 : 3{omega}6c (1·2 %), were found in minor quantities, but uniquely in CL-SP19T among Idiomarina species. The DNA G+C content was 45·0 mol%. On the basis of its physiology, fatty acid composition and 16S rRNA gene sequence, strain CL-SP19T could be assigned to the genus Idiomarina but distinguished from the recognized species of the genus. Strain CL-SP19T, therefore, represents a novel species, for which the name Idiomarina seosinensis sp. nov. is proposed, with CL-SP19T (=KCTC 12296T=JCM 12526T) as the type strain.

Published online ahead of print on 17 September 2004 as DOI 10.1099/ijs.0.63365-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CL-SP19T is AY635468.

Additional phenotypic data are available as supplementary material in IJSEM Online.


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The genus Idiomarina was first described by Ivanova et al. (2000)Go. Six species, Idiomarina abyssalis (type species) and Idiomarina zobellii (Ivanova et al., 2000Go), Idiomarina baltica (Brettar et al., 2003Go), Idiomarina loihiensis (Donachie et al., 2003Go), Idiomarina fontislapidosi and Idiomarina ramblicola (Martínez-Cánovas et al., 2004Go), have been isolated from sea water, i.e. from deep waters of the north-western Pacific Ocean, from surface water of the central Baltic Sea, from hydrothermal fluids from a submarine volcano in Hawaii and from hypersaline habitats in Spain, respectively.

A hypersaline water sample (318 practical salinity units) from a solar saltern in Seosin, Korea, was spread on a plate containing marine agar 2216 (MA; Difco); the plate was incubated at 30 °C for 2 weeks. Strain CL-SP19T, appearing as a slightly yellowish colony, was isolated from the plate and subsequently purified four times on MA at 30 °C. The strain was maintained both on MA at 4 °C and in marine broth (MB; Difco) supplemented with 30 % (v/v) glycerol at –80 °C.

The 16S rRNA gene was amplified from a single colony by a PCR with Taq DNA polymerase (Bioneer) and primers 27F and 1492R (Lane, 1991Go). The PCR product was purified by using the AccuPrep PCR purification kit (Bioneer) and cloned using the pCR2.1 TOPO TA cloning kit (Invitrogen). Sequencing of the 16S rRNA gene was performed with an Applied Biosystems automatic sequencer (ABI3730XL) at Macrogen (Korea). The sequence of strain CL-SP19T was compared with 16S rRNA gene sequences available in GenBank, using a BLAST (Altschul et al., 1990Go) search. The sequence of strain CL-SP19T was manually aligned with six species of the genus Idiomarina and type species of genera belonging to the family Alteromonadaceae obtained from the GenBank and Ribosomal Database Project (Cole et al., 2003Go) databases using known 16S rRNA secondary structure information. Phylogenetic trees were obtained by using neighbour-joining (Saitou & Nei, 1987Go), maximum-parsimony (Fitch, 1971Go) and maximum-likelihood (Felsenstein, 1981Go) methods. An evolutionary distance matrix for the neighbour-joining method was generated according to the model of Jukes & Cantor (1969)Go. The robustness of tree topologies was assessed by bootstrap analyses based on 1000 replications for neighbour-joining and maximum-parsimony methods and 100 replications for the maximum-likelihood method. Alignment and phylogenetic analyses were carried out using the PHYDIT program (version 3.2, available at http://plaza.snu.ac.kr/~jchun/phydit/) and PAUP*, version 4.0 (Swofford, 1998Go). Likelihood parameters were estimated by the hierarchical ratio tests in MODELTEST, version 3.04 (Posada & Crandall, 1998Go). The almost-complete 16S rRNA gene sequence of strain CL-SP19T (1463 bp) was obtained. Sequence similarity indicated that the closest relatives of strain CL-SP19T were I. fontislapidosi (96·9 %), I. zobellii (96·8 %), I. abyssalis (96·8 %), I. baltica (96·4 %), I. loihiensis (96·3 %) and I. ramblicola (95·9 %). Phylogenetic analyses based on the 16S rRNA gene sequence showed that strain CL-SP19T formed a robust cluster with species of the genus Idiomarina (Fig. 1Go). Thus, it is clear that our isolate belongs to the genus Idiomarina. However, low similarities (95·9–96·9 %) between the 16S rRNA gene sequence of the novel isolate and those of previously described species of the genus Idiomarina indicated that strain CL-SP19T represents a novel species in the genus (Stackebrandt & Goebel, 1994Go; Rosselló-Mora & Amann, 2001Go).



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  		Fig. 1. Neighbour-joining tree showing the relationship between strain CL-SP19T and members of Idiomarina and other related genera belonging to the Alteromonadaceae. Only those bootstrap values above 70 % are shown (1000 resamplings) at the branching points. Solid circles indicate that the corresponding nodes are also recovered in maximum-likelihood and maximum-parsimony trees. Burkholderia cepacia (M22518) was used as an outgroup (not shown). Bar, 0·1 nucleotide substitution per site.

 
Subsequently, morphological and physiological tests were also performed. Gram-staining was performed as described by Smibert & Krieg (1994)Go. Cell morphology and motility were examined by using phase-contrast microscopy (BX60 microscope; Olympus) with cells grown at 30 °C in MB. The presence of a flagellum was observed using a scanning electron microscope (JSM-6700, FE-SEM; JEOL) (Fig. 2Go). Anaerobic growth was checked on MA using the GasPak anaerobic system (BBL). Catalase and oxidase activities were determined according to the protocols described by Smibert & Krieg (1994)Go, while gelatinase, amylase, DNase and nitrate reductase activities were examined as described by Hansen & Sørheim (1991)Go.



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  		Fig. 2. Scanning electron micrograph of negatively stained cells of strain CL-SP19T.

 
The temperature range for growth was determined on the basis of colony formation on MA plates that were incubated at 4–46 °C. Optimum temperature (a range of 25–40 °C) and pH range (a range of pH 5–10) for growth were determined by observing changes in OD600 values over time in MB. The final pH was adjusted using NaOH and HCl solutions. The sea salt concentrations allowing growth of CL-SP19T were determined using synthetic ZoBell broth [Bacto peptone (Difco), 5 g; yeast extract, 1 g; ferric citrate, 0·1 g; distilled water, 1 litre] with various concentrations of sea salt (0, 0·5, 1, 3, 5, 7, 10, 15, 20 and 25 %, w/v). Indole production, acid production from glucose, arginine dihydrolase, urease, hydrolysis of aesculin, and gelatinase and {beta}-galactosidase were tested using the API 20NE kit (bioMérieux) according to the manufacturer's instructions, except that cell suspensions were prepared using artificial sea water (NaCl, 24 g; MgCl2, 5·1 g; Na2SO4, 4 g; CaCl2, 1·1 g; KCl, 0·7 g; NaHCO3, 0·2 g; KBr, 0·1 g; H3BO3, 0·027 g; SrCl2, 0·024 g; NaF, 0·003 g; distilled water, 1 litre; Yi & Chun, 2004Go) as the suspension medium. Other enzyme activities were also assayed, using the API ZYM kit (bioMérieux) and artificial sea water as the suspension medium. The utilization of single carbon sources was investigated using Biolog GN plates and using artificial sea water for cell suspension; the results were read visually after incubation for 1, 2, 3 and 5 days. For the analyses of API 20NE, API ZYM and Biolog, I. loihiensis L2-TRT, I. baltica OS145T, I. abyssalis KMM 227T and I. zobellii KMM 231T were employed as reference strains. The results of morphological, biochemical and physiological tests are given in Table 1Go and Table 2Go and in the species description. The DNA G+C content was determined by HPLC analysis (Tamaoka & Komagata, 1984Go) at the Korean Culture Centre of Microorganisms. The results are shown in Table 1Go and in the species description.


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  		Table 1. Selected phenotypic characteristics of Idiomarina seosinensis sp. nov. and other Idiomarina species

Strains: 1, I. seosinensis CL-SP19T (this study); 2, I. abyssalis KMM 227T (Ivanova et al., 2000Go); 3, I. zobellii KMM 231T (Ivanova et al., 2000Go); 4, I. baltica OS145T (Brettar et al., 2003Go); 5, I. loihiensis L2-TRT (Donachie et al., 2003Go); 6, I. fontislapidosi F23T (Martínez-Cánovas et al., 2004Go); 7, I. ramblicola R22T (Martínez-Cánovas et al., 2004Go). ND, No data available; +, positive response; –, negative response. All strains are Gram-negative.

 

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  		Table 2. Selected phenotypic characteristics that differentiate Idiomarina seosinensis sp. nov. from other Idiomarina species

Strains: 1, I. seosinensis CL-SP19T; 2, I. abyssalis KMM 227T; 3, I. zobellii KMM 231T; 4, I. baltica OS145T; 5, I. loihiensis L2-TRT. +, Positive response; –, negative response; W, weak response. Data for all strains in the table were obtained in this study. Additional phenotypic data are available as supplementary material in IJSEM Online.

 
The fatty acid methyl esters in whole cells grown at 30 °C for 2 days on MA were analysed by GC, according to the instructions of the Microbial Identification System (MIDI), at the Korean Culture Centre of Microorganisms. The profile for strain CL-SP19T shows a predominance of iso-branched fatty acids, which is characteristic of the genus Idiomarina (Table 3Go). However, in CL-SP19T, the proportion of 15 : 0 iso fatty acids (17·1 %) is obviously lower than those of other Idiomarina species (24·7–40·6 %), whilst the proportion of 17 : 0 iso fatty acids is slightly higher in strain CL-SP19T (15·2 %) than in Idiomarina species (8·8–12·9 %). Three fatty acids, C19 : 0{omega}8c cyclo (3·5 %), C14 : 1{omega}5c (1·4 %) and C18 : 3{omega}6c (1·2 %), were found in minor quantities, but uniquely in CL-SP19T among Idiomarina species. Therefore, the fatty acid pattern of strain CL-SP19T differs distinctly from those of previously described species of Idiomarina.


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  		Table 3. Fatty acid compositions of Idiomarina seosinensis sp. nov. and other Idiomarina species

Strains: 1, I. seosinensis CL-SP19T (this study); 2, I. abyssalis KMM 227T (Ivanova et al., 2000Go); 3, I. zobellii KMM 231T (Ivanova et al., 2000Go); 4, I. baltica OS145T (Brettar et al., 2003Go); 5, I. loihiensis L2-TRT (Donachie et al., 2003Go); 6, I. fontislapidosi F23T (Martínez-Cánovas et al., 2004Go); 7, I. ramblicola R22T (Martínez-Cánovas et al., 2004Go). –, Not detected or <1 %.

 
Phylogenetic analyses based on 16S rRNA gene sequences, fatty acid profiles and physiological features suggest that strain CL-SP19T represents a novel species of the genus Idiomarina, for which the name Idiomarina seosinensis sp. nov. is proposed.

Description of Idiomarina seosinensis sp. nov.
Idiomarina seosinensis (se.o.sin.en'sis. N.L. fem. adj. seosinensis referring to the Seosin region in Korea, where the type strain was found).

Cells are Gram-negative, strictly aerobic and straight or slightly curved rods that are approximately 0·3–0·6 µm wide and 1·0–1·9 µm long. Motile by means of a single polar flagellum. On MA solid medium, colonies are slightly yellowish, entire circular, convex, opaque and shiny. After 3 days on MA at 30 °C, colonies are approximately 2–3 mm in diameter. They grow within the temperature range 4–40 °C (optimum of 30–35 °C) and at pH values between 6 and 10. Growth occurs in sea salt concentrations of 1–20 % (w/v) (optimum of 5–10 %), whereas no growth occurs in media containing NaCl as the only salt. Positive for oxidase, catalase, DNase, gelatinase and nitrate reductase activities but negative for amylase activity. The major fatty acids are 15 : 0 iso (17·1 %) and 17 : 0 iso (15·2 %). Minor amounts of C19 : 0{omega}8c cyclo (3·5 %), C14 : 1{omega}5c (1·4 %) and C18 : 3{omega}6c (1·2 %) are also found. In API 20NE tests, cells are positive for nitrate reductase activity and hydrolysis of aesculin, but negative for indole production, acid production from glucose, arginine dihydrolase, urease, gelatinase and {beta}-galactosidase. In API ZYM tests, cells are positive for alkaline phosphatase, esterase (C4 and C8) and leucine arylamidase activities, but negative for valine arylamidase, cysteine arylamidase, trypsin, {alpha}-chymotrypsin and naphthol-phosphohydrolase activities. In Biolog tests, the following substrates are utilized: glycogen, Tween 40, Tween 80, {alpha}-ketobutyric acid, {alpha}-ketovaleric acid, succinic acid, L-alaninamide, L-alanine, L-alanylglycine, L-asparagine, L-aspartic acid, L-glutamic acid, glycyl-L-glutamic acid, L-proline and L-threonine. The DNA G+C content is 45·0 mol%.

The type strain is CL-SP19T (=KCTC 12296T=JCM 12526T), isolated from hypersaline water from a solar saltern located in Seosin, Korea.


   ACKNOWLEDGEMENTS
 
We thank Professor M. Alam for the gift of the type strain of I. loihiensis, and Dr J. Chun and Ms H. Lee for discussions and help during this work. This work was supported by the BK21 project of the Korean Government.


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