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1 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2 National Center for Genetic Engineering and Biotechnology, Pathumthani 12120, Thailand
3 Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Correspondence
Somboon Tanasupawat
Somboon.T{at}chula.ac.th
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to accommodate red, extremely halophilic cocci that thrive in hypersaline environments, and the generic name was implemented by Kocur & Hodgkiss (1973) with the proposal of a single species, Halococcus morrhuae. In addition to Hcc. morrhuae, the genus Halococcus currently comprises five other recognized species, Halococcus saccharolyticus, Hcc. salifodinae, Hcc. dombrowskii, Hcc. hamelinensis and Hcc. qingdaonensis (Montero et al., 1989; Denner et al., 1994; Stan-Lotter et al., 2002; Goh et al., 2006; Wang et al., 2007). Fish sauce (nam-pla) fermentation in Thailand has also provided isolation sources of novel halophilic archaea and bacteria, for example Halobacterium salinarum, Halobacillus thailandensis, Tetragenococcus halophilus, Tetragenococcus muriaticus, Lentibacillus salicampi, Lentibacillus juripiscarius and Lentibacillus halophilus (Chaiyanan et al., 1999; Tanasupawat & Komagata, 2001; Thongthai et al., 1992; Thongsanit et al., 2002; Namwong et al., 2005; Tanasupawat et al., 2006). The present study describes the isolation of extremely halophilic archaeal cocci isolated from fish sauce fermentation and their characterization based on phenotypic, chemotaxonomic, DNA–DNA relatedness and 16S rRNA gene sequencing data. The halophilic archaeal strains were isolated from fish sauce samples (nam-pla) collected from factories in Thailand during the early, middle and late stages of the fermentation process by using a spread-plate technique on JCM medium 169 agar plates [consisting of (per litre): 250 g NaCl, 7.5 g Casamino acids, 10 g yeast extract, 2 g KCl, 3 g trisodium citrate, 20 g MgSO4.7H2O, 0.05 g FeSO4.4H2O, 0.2 g MnSO4.4H2O, 20 g agar, pH 7.2] incubated at 37 °C for 1–2 weeks. Unless otherwise stated, the test strains were grown in liquid or on agar medium of JCM medium 169.
Cell morphology of the isolates was observed by light microscopy and scanning electron microscopy for cells grown on agar plates at 37 °C for 7 days. Tests for general physiological and biochemical characteristics were performed as specified by Oren et al. (1997). Tests for catalase and oxidase activities, indole production, nitrate reduction and hydrolysis of casein, gelatin, arginine and Tween 80 were performed as described by Barrow & Feltham (1993). Carbon utilization and acid production from carbohydrates were determined in modified Leifson medium (Leifson, 1963) supplemented with 0.01 % yeast extract and 20 % (w/v) NaCl. Casitone was omitted for the carbon utilization tests and Tris/HCl was omitted for the acid production tests. Anaerobic growth was observed in standard growth medium with 0.5 % nitrate or arginine by using a Gaspak (BBL) anaerobic jar. The temperature range for growth was examined by incubating cultures on agar plates at 20, 28, 37, 45 and 50 °C. Effects of NaCl concentration for growth were tested in medium containing 0–30 % (w/v) NaCl. At lower NaCl concentrations (0–2 %), MgSO4.7H2O, KCl and trisodium citrate were omitted from the test medium. Similarly, the requirement for Mg2+ was tested in JCM 169 medium by omitting MgSO4.7H2O but supplementing with 2 % (w/v) Na2SO4 and 0–10 % (w/v) MgCl2. Growth was determined by measuring culture turbidity at 660 nm. The antibiotic susceptibility of the strains was tested as described by Stan-Lotter et al. (2002). Menaquinones were analysed as described by Komagata & Suzuki (1987). Polar lipids were determined according to the method of Minnikin et al. (1984).
DNA was isolated and purified according to the method of Saito & Miura (1963). The DNA G+C content was determined as described by Tamaoka & Komagata (1984) by using reversed-phase HPLC. DNA–DNA hybridization tests were performed as described by Ezaki et al. (1989) and levels of relatedness were determined according to Tanasupawat et al. (2000). The almost-complete 16S rRNA gene sequence of a representative strain, designated HDB5-2T, was amplified by PCR with primers D30F (5'-ATTCCGGTTCATCCTGC, positions 6–22 according to the Escherichia coli numbering system) and D56R (5'-GYTACCTTGTTACGACTT, positions 1492–1509). The amplified DNA fragment was separated by agarose gel electrophoresis and recovered by using a GenElute Minus EtBr spin column (Sigma). The sequence was determined by using the BigDye Terminator cycle sequencing ready reaction kit (version 3.0; Applied Biosystems) in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) with the following primers: D30F, D33R (5'-TCGCGCCTGCGCCCCGT, positions 344–360), D34R (5'-GGTCTCGCTCGTTGCCTG, positions 1096–1113), D56R, B99R (5'-GTGTTACCGCGGCTGCTG, positions 519–536), B36R (5'-GGACTACCAGGGTATCTA, positions 789–806) and X10R (5'-ACGGGCGGTGTGTRC, positions 1392–1406). The phylogenetic tree was constructed as described by Thompson et al. (1994), Saitou & Nei (1987), Kumar et al. (2001) and Felsenstein (1985).
Fifteen extremely halophilic, Gram-negative cocci were isolated from various stages of the fish sauce fermentation process. Cells of these isolates were non-motile, 0.8–1.2 µm in diameter, occurring singly, in pairs or in tetrads (Fig. 1) during both exponential and stationary phases of growth. In addition, the cells did not lyse when suspended in distilled water for 1–2 h. They formed small, red-pigmented colonies (1–2 mm in diameter) on agar plates. The isolates grew in medium containing a high concentration of NaCl [at least 15 % (w/v) and optimally 20–30 % (w/v)]. Their growth temperature range was 15–45 °C (optimum growth at 37 °C) and pH range was 6–10 (optimum growth at pH 6–8). The physiological and biochemical properties of strain HDB5-2T are given in the species description below and in Table 1. A thin-layer chromatogram of the polar lipid fraction from four representative strains, HDS4-1, HDB5-2T, HDS7-4 and HIS10-2, revealed C20C20 and C20C25 diether lipids of phosphatidylglycerol, phosphatidylglycerol methylphosphate, a sulfated diglycosyl diether (S-DGA-1) and unidentified glycolipids, by comparing the profiles with those of Hcc. dombrowskii JCM 12289T and Hcc. morrhuae JCM 8876T. Phosphatidylglycerol sulfate was not detected. The novel strains had MK-8(H2) as the major menaquinone component. The DNA G+C contents of the novel strains were 60.0–61.8 mol% (Table 2).
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; Grant, 2001). A phylogenetic tree showing the position of strain HDB5-2T in the genus Halococcus was reconstructed as shown in Fig. 2. Strain HDB5-2T was closely related to Hcc. morrhuae JCM 8876T, Hcc. qingdaonensis JCM 13587T and Hcc. dombrowskii JCM 12289T, with 16S rRNA gene sequence similarities of 98.2–98.7 %, whereas it was more distantly related to Hcc. hamelinensis JCM 12892T, Hcc. saccharolyticus JCM 8878T and Hcc. salifodinae JCM 9578T, with sequence similarities of 93.7–94.1 %. DNA–DNA hybridization experiments revealed that the 15 novel strains constituted a homogeneous genetic group with more than 78.9 % DNA–DNA relatedness (Wayne et al., 1987). However, the novel strains showed low levels of DNA–DNA relatedness (<50 %) with Hcc. morrhuae JCM 8876T and Hcc. dombrowskii JCM 12289T (Table 2
). In addition, the 15 strains could be differentiated from recognized species of the genus Halococcus based on intolerance to high concentrations of MgCl2, growth at 45 °C, acid production from carbohydrates and utilization of various carbon sources (Table 1).
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Description of Halococcus thailandensis sp. nov.
Halococcus thailandensis (thai.lan.den'sis. N.L. masc. adj. thailandensis pertaining to Thailand, where the first strains were isolated).
Cells are Gram-negative, non-motile, strictly aerobic cocci, 0.8–1.2 µm in diameter, occurring singly, in pairs, tetrads or sarcina packets. Colonies are small, red-pigmented and circular with entire margins (1–2 mm in diameter after 1 week of incubation at 37 °C) when grown on complex medium of neutral pH. No lysis in distilled water. Grows aerobically but not anaerobically even in the presence of nitrate or arginine. Grows between 15 and 45 °C (optimally at 37 °C) and between pH 6 and 10 (optimally at pH 6–8). Extremely halophilic; requires at least 15 % (w/v) NaCl for growth and grows optimally at 20–30 % (w/v) NaCl. Does not require MgCl2 for growth. Catalase-, oxidase- and urease-positive. Nitrate is reduced but gas is not formed. Does not hydrolyse arginine, casein, gelatin, Tween 80 or starch. Produces acids from L-arabinose, cellobiose, D-glucose, lactose, D-mannitol, melibiose, sucrose and trehalose. Utilizes L-arabinose, D-fructose, D-glucose, D-xylose and glutamic acid, but not D-galactose or serine as the sole energy source. The type strain is susceptible to bacitracin, novobiocin and rifampicin but resistant to chloramphenicol, tetracycline, ampicillin, gentamicin, kanamycin, nalidixic acid and streptomycin. The DNA G+C content is 60.2–61.8 mol%. Possesses C20C20 and C20C25 diether core lipids. MK-8(H2) is the predominant menaquinone component. Possesses phosphatidylglycerol and phosphatidylglycerol methylphosphate as major polar lipid components. S-DGD-1, a sulfated mannosylglucosylglycerol diether, and unidentified glycolipids are also present.
The type strain, HDB5-2T (=BCC 20213T =JCM 13552T =PCU 278T), was isolated from fish sauce fermentation in Thailand.
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ACKNOWLEDGEMENTS |
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78 %, based on 1000 replications, are shown at nodes. Bar, 2 substitutions per 100 nucleotide positions.