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Int J Syst Evol Microbiol 57 (2007), 2129-2131; DOI  10.1099/ijs.0.65120-0
© 2007 International Union of Microbiological Societies

Natronococcus jeotgali sp. nov., a halophilic archaeon isolated from shrimp jeotgal, a traditional fermented seafood from Korea

Seong Woon Roh1,2, Young-Do Nam1,2, Ho-Won Chang2, Youlboong Sung2, Kyoung-Ho Kim2, Ho-Jae Lee2, Hee-Mock Oh2 and Jin-Woo Bae1,2,3

1 University of Science & Technology, 52 Eoeun-dong, Daejeon 305-333, Korea
2 Biological Resource Center, KRIBB, Daejeon 305-806, Korea
3 Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea

Correspondence
Jin-Woo Bae
baejw{at}kribb.re.kr


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A novel halophilic archaeon (strain B1T) belonging to the genus Natronococcus was isolated from shrimp jeotgal, a traditional fermented food from Korea. Colonies of this strain were orange–red and cells were non-motile cocci that stained Gram-variable. Strain B1T grew in 7.5–30.0 % (w/v) NaCl and at 21–50 °C and pH 7.0–9.5, with optimal growth occurring in 23–25 % (w/v) NaCl and at 37–45 °C and pH 7.5. Strain B1T was most closely related to the type strain of Natronococcus occultus, with which it shared 97.91 % 16S rRNA gene sequence similarity. Within the phylogenetic tree, this novel strain shared a branching point with N. occultus and occupied a phylogenetic position that was distinct from the main Natronococcus branch. The degree of DNA–DNA hybridization with the type strain of N. occultus, the most closely related species phylogenetically, was 16.4 %. On the basis of these results, it is concluded that strain B1T represents a novel species of the genus Natronococcus, for which the name Natronococcus jeotgali is proposed. The type strain is B1T (=KCTC 4018T=DSM 18795T=JCM 14583T=CECT 7216T).

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain B1T is EF077631.


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Shrimp jeotgal, a traditional fermented food from Korea, is made by combining fresh, tiny shrimps with rock salt and stock and fermenting the mixture for several months. Throughout the fermentation period, this food acquires its own distinct taste and it is often used as an additive to improve the taste of other foods. To date, studies on the microflora of jeotgal have shown that the majority of its micro-organisms are Gram-positive, endospore-forming bacilli (Yoon et al., 2001Go), but no archaeon has been isolated from jeotgal. To further our understanding of archaea, jeotgal was analysed for novel strains of this domain and one strain belonging to the order Halobacteriales was isolated.

The genus Natronococcus, belonging to the order Halobacteriales, was first proposed by Tindall et al. (1984)Go. Currently, this genus contains only two species: Natronococcus occultus (Tindall et al., 1984Go) and Natronococcus amylolyticus (Kanai et al., 1995Go). In this study, a novel strain was isolated from shrimp jeotgal by using a dilution plating technique on medium for halophilic archaea. This strain, which was found to belong to the genus Natronococcus on the basis of its 16S rRNA gene sequence, is described herein. Accordingly, the main objective of the present work was to elucidate the taxonomic position of this strain, designated B1T, through phenotypic, genetic and chemotaxonomic analyses. N. occultus SP4T (=DSM 3396T) and N. amylolyticus Ah-36T (=DSM 10524T) served as the reference strains.

Strain B1T was isolated from shrimp jeotgal on a complex medium containing (g l–1): Casamino acids (Difco) (5), yeast extract (Difco) (5), MgCl2 . 6H2O (20), KCl (2), Tris (12), CaCl2 . 2H2O (0.2) and NaCl (200). The medium also contained antibiotics (penicillin G, erythromycin and cycloheximide all at 100 µg ml–1) that are known to inhibit bacteria and eukarya, but not archaea (Purdy et al., 2004Go). The pH was adjusted to 7.4 and incubation was conducted at 37 °C for 5–7 days. A pure culture was obtained by repeated re-streaking on agar plates. The methods used for phenotypic tests are in accordance with the proposed minimal standards for the description of new taxa in the order Halobacteriales (Oren et al., 1997Go). Optimal conditions for growth were determined in media containing 0–30 % (w/v) NaCl and the pH range for growth was assayed in media of pH 5.0–11.0 at intervals of 0.5 pH units. Oxidase activity was determined using an oxidase reagent (bioMérieux). Enzyme testing was carried out for 19 hydrolytic enzymes for each strain using the microenzyme API ZYM system (bioMérieux). Total lipids were extracted by the modified method of Xin et al. (2000)Go.

Chromosomal DNA was extracted and purified as described by Sambrook et al. (1989)Go. The 16S rRNA gene was amplified by PCR using two universal primers: forward primer 21F (5'-TTCCGGTTGATCCTGCCGGA-3') and reverse primer 1492R (5'-GGYTACCTTGTTACGACTT-3'). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed according to the methods described by Yoon et al. (2003)Go. DNA–DNA hybridization was performed by the fluorometric method of Ezaki et al. (1989)Go. The 16S rRNA gene sequence of the novel isolate was aligned with 10 reference sequences from the NCBI database (Fig. 1Go) by using the multiple sequence alignment program CLUSTAL_X (1.8) (Thompson et al., 1997Go). Phylogenetic relationships between representatives of the genus Natronococcus were determined using the MEGA version 2.1 software program. Distance matrices were determined by following the assumptions described by Kimura (1980)Go. These matrices were used to elaborate dendrograms by using the neighbour-joining method (Saitou & Nei, 1987Go). Bootstrap analysis investigating the stability of the trees was performed by obtaining a consensus tree based on 1000 randomly generated trees.


Figure 1
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  		Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain B1T with respect to other species of the genus Natronococcus and related species. Numbers at nodes indicate bootstrap values (1000 replications). Bar, 1 bp substitution per 100 nt.

 
The results of biochemical and physiological tests are given in Table 1Go and the species description. Strain B1T could be readily differentiated from other closely related species on the basis of phenotypic properties, as shown in Table 1Go. Polar lipid analysis indicated that strain B1T contained phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester.


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  		Table 1. Characteristics that differentiate Natronococcus jeotgali sp. nov. from closely related Natronococcus species

Species: 1, N. jeotgali sp. nov.; 2, N. occultus; 3, N. amylolyticus.

 
A BLAST search of 16S rRNA gene sequences in the NCBI database and construction of a phylogenetic tree using 16S rRNA gene sequences from members of the genus Natronococcus and related species revealed that strain B1T fell within the cluster of Natronococcus species (Fig. 1Go). Strain B1T showed 97.91 % 16S rRNA gene sequence similarity to the type strain of N. occultus and 96.46 % similarity to the type strain of N. amylolyticus. DNA–DNA hybridization studies were then performed to determine the genomic relationship between strain B1T, N. occultus SP4T and N. amylolyticus AH-36T. The mean DNA–DNA relatedness value between strain B1T and N. occultus SP4T was 16.4 % and that between strain B1T and N. amylolyticus AH-36T was 12.2 %. Other differences between strain B1T and these two reference strains are shown in Table 1Go.

Thus, on the basis of phenotypic, genetic and chemotaxonomic comparisons to previously described taxa, strain B1T is the type strain of a novel species of the genus Natronococcus, for which the name Natronococcus jeotgali sp. nov. is proposed.

Description of Natronococcus jeotgali sp. nov.
Natronococcus jeotgali (je.ot.ga'li. N.L. gen. n. jeotgali of jeotgal, a traditional Korean fermented seafood).

Cells are non-motile cocci with a diameter of 1–2 µm and occur in irregular clusters. The Gram reaction is mixed: some cells stain positive and others are negative. Colonies are orange–red, circular and 0.5–1.0 mm in diameter after 5–7 days of growth at 37 °C. Cell lysis does not occur in distilled water. Growth occurs in 7.5–30.0 % (w/v) NaCl, at 21–50 °C and pH 7.0–9.5. Optimal growth conditions are 23–25 % (w/v) NaCl, 37–45 °C, pH 7.5. Strictly aerobic, catalase-positive, oxidase-negative and reduces nitrate to nitrite. The following substrates can be utilized as sole carbon and energy sources: sucrose, fructose, glucose, acetate and lactose. Citrate is not utilized. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase and naphthol-AS-BI-phosphohydrolase activities (API ZYM system). Cystine arylamidase, {alpha}-galactosidase, beta-glucuronidase, beta-glucosidase, {alpha}-glucosidase, N-acetyl-beta-glucosaminidase, {alpha}-mannosidase and {alpha}-fucosidase activities are not observed. The polar lipid fraction consists of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester. Resistant to the following antibiotics (µg ml–1): bacitracin (25), penicillin (50), ampicillin (50), chloramphenicol (50) and erythromycin (50). Sensitive to the following antibiotics (µg ml–1): novobiocin (25), bacitracin (50), anisomycin (25) and aphidicolin (25).

The type strain, B1T (=KCTC 4018T=DSM 18795T=JCM 14583T=CECT 7216T), was isolated from shrimp jeotgal, a traditional fermented food in Korea.


   ACKNOWLEDGEMENTS
 
The authors are supported by the KRIBB Research Initiative Program and Environmental Biotechnology National Core Research Center (KOSEF: R15-2003-012-02002-0) from the Ministry of Science and Technology (MOST) of the Republic of Korea. We thank Dr J. P. Euzéby for doing background research when naming strain B1T.


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Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[Abstract/Free Full Text]

Kanai, H., Kobayashi, T., Aono, R. & Kudo, T. (1995). Natronococcus amylolyticus sp. nov., a haloalkaliphilic archaeon. Int J Syst Bacteriol 45, 762–766.[Abstract/Free Full Text]

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]

Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriol 47, 233–238.[Abstract/Free Full Text]

Purdy, K. J., Cresswell-Maynard, T. D., Nedwell, D. B., McGenity, T. J., Grant, W. D., Timmis, K. N. & Embley, T. M. (2004). Isolation of haloarchaea that grow at low salinities. Environ Microbiol 6, 591–595.[CrossRef][Medline]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[Abstract/Free Full Text]

Tindall, B. J., Ross, H. N. M. & Grant, W. D. (1984). Natronobacterium gen. nov. and Natronococcus gen. nov., two new genera of haloalkaliphilic archaebacteria. Syst Appl Microbiol 5, 41–57.

Xin, H., Itoh, T., Zhou, P., Suzuki, K., Kamekura, M. & Nakase, T. (2000). Natrinema versiforme sp. nov., an extremely halophilic archaeon from Aibi salt lake, Xinjiang, China. Int J Syst Evol Microbiol 50, 1297–1303.[Abstract]

Yoon, J. H., Kang, S. S., Lee, K. C., Kho, Y. H., Choi, S. H., Kang, K. H. & Park, Y. H. (2001). Bacillus jeotgali sp. nov., isolated from jeotgal, Korean traditional fermented seafood. Int J Syst Evol Microbiol 51, 1087–1092.[Abstract]

Yoon, J. H., Kang, K. H. & Park, Y. H. (2003). Halobacillus salinus sp. nov., isolated from a salt lake on the coast of the East Sea in Korea. Int J Syst Evol Microbiol 53, 687–693.[Abstract/Free Full Text]




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S. W. Roh, Y.-D. Nam, H.-W. Chang, K.-H. Kim, Y. Sung, M.-S. Kim, H.-M. Oh, and J.-W. Bae
Haloterrigena jeotgali sp. nov., an extremely halophilic archaeon from salt-fermented food
Int J Syst Evol Microbiol, September 1, 2009; 59(9): 2359 - 2363.
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