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Identification of Halophilic Bacteria from Fish Sauce (Nam-Pla) in Thailand

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Identification of Halophilic Bacteria from Fish Sauce (Nam-Pla) in Thailand JOURNAL OF CULTURE COLLECTIONS Volume 6, 2008-2009, pp. 69-75 IDENTIFICATION OF HALOPHILIC BACTERIA FROM FISH SAUCE (NAM-PLA) IN THAILAND Somboon Tanasupawat1*, Sirilak Namwong1, Takuji Kudo2 and Takashi Itoh2 1Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; 2Japan Collection of Microorganisms, RIKEN BioResource Center, Wako-shi, Saitama 351-0198, Japan *Corresponding author, e-mail: [email protected] Summary Four strains of Gram-negative, rod-shaped, moderately halophilic bacteria, Group A, and eleven strains of strictly aerobic, extremely halophilic rods (10 strains, Group B) and coccoid (1 strain, Group C) were isolated from fish sauce fermentation (nam-pla) in Thailand. The 16S rRNA gene sequence analyses of the representative strains indicated that DS26-2 (Group A), HDS2-5 (Group B), and HRF6 (Group C), were closely related to Chromohalobacter salexigens KCTC 12941T, Halobacteirum salinarum JCM 8978T, and Halococcus saccharolyticus JCM 8878T with 99.3, 99.9, and 99.0 % similarity, respectively. Group A strains were identified as C. salexigens, Group B as H. salinarum, and Group C strain was H. saccharolyticus based on their DNA-DNA relatedness. Group A strains grew in 3–25 % (w/v) NaCl. Ubiquinone with nine isoprene units (Q-9) was a major component. The DNA G+C contents ranged from 63.1 to 64.2 mol %. Group B and Group C strains grew optimally in the presen- ce of 25-30 % NaCl. The tested strains of Group B contained major menaquinone with eight isoprene units (MK-8). DNA G+C contents ranged from 63.3 to 64.7 mol %. Group C strain had MK-8(H2) as a predominant menaquinone. The DNA G+C con- tent was 63.2 mol %. Key words: Archaea, Chromohalobacter salexigens, fish sauce, halophilic bacteria, Halobacterium salinarum, Halococcus saccharolyticus Introduction The moderately and extremely halophilic coccus and Halobacterium require at least bacteria occur to thrive in the salted environ- 1.5 M NaCl (optimal growth at 20–25 %, w/v ment, i.e. in salt lakes, soda lakes, saltern, cru- NaCl) for growth [11, 39]. de solar salts and proteinaceous products with In Thailand, Fish sauce contains high con- the capacity to balance the osmotic pressure of centration of salt (25–30 %, w/v NaCl), thus the environment and resist the denaturing ef- microorganisms found during fish sauce pro- fects of salts [5, 8, 14]. The moderately halo- duction are generally classified as halophilic philic bacteria i.e., Lentibacillus, Filobacillus, bacteria [16]. The strains of Lentibacillus sali- Tetragenococcus, Chromohalobacter are able campi, L. jurispiscarius [19], L. halophilus [30], to grow over a wide range of salt concentra- Filobacillus sp. RF2-5 [10], Halobacillus sp. tions and grow optimally in media containing SR5-3 [20], Piscibacillus salipiscarius [31], Tet- between 3 and 15 % (w/v) NaCl [1, 2, 19]. On ragenococcus halophilus and T. muriaticus [34], the other hand, the halophilic archaea i.e., Halo- Halobacterium salinarum [35], and Halococ- 69 cus thailandensis [21] were isolated from fer- teria isolated from fish sauce based on their mented fish sauce and related fish products. phenotypic and chemotaxonomic characteris- This work deals with the identification of tics including DNA-DNA relatedness and phy- the moderately and extremely halophilic bac- logenetic properties. Materials and Methods Source of samples and Isolation. Fish bed previously [12]. Polar lipids were determi- sauce samples (nam-pla) were collected from ned according to the method of Minnikin [17]. the factories (Sindhu Samuth Fish Sauce Fac- The quantitative analysis of cellular fatty acids tory (Squid Brand) LTD. (A), Samutprakarn, was employed with the Microbial Identification Thai Fish Sauce Factory (Squid Brand) Co., System (MIDI Inc.) [25]. DNAs were isolated LTD. (B), Samutsongkram, and Rayong Fish and purified according to the method of Saito Sauce Industry Co., Ltd. (C), Rayong Province, and Miura [23]. The DNA G+C content was de- in Thailand. The halophilic bacteria were isola- termined by the method of Tamaoka and Ko- ted by using spread-plate technique on agar magata [28] using reversed-phase HPLC. plates of JCM medium No. 169 and incubated DNA-DNA hybridization was conducted as at 37 °C for 1-2 weeks. described by Ezaki et al. [6] and detected as Identification methods. Morphological, reported by Tanasupawat et. al. [29]. The 16S cultural, physiological and biochemical charac- rRNA gene sequence of the moderately isolate teristics were determined as described by Bar- was determined as described previously [26], row and Feltham [3] and Namwong et al. [19, 21]; whereas the archaea strains were determined Oren et al. [22]. Acid production from carbohy- as described by Namwong et al. [21]. The phy- drate was determined in the medium described logenetic tree based on 16S rRNA gene sequen- by Leifson [15] supplemented with 6.5 % for ces was constructed as described by Kumar the moderately and 16.5 % NaCl for the ex- et al. [13]; Felsenstein [7]; Saitou and Nei [24]; tremely strains. Determination of the antibiotic Thompson et al. [33]. susceptibility of halophilic archaea was tested The GenBank/ EMBL/DDBJ accession num- as described by Stan-Lotter et al. [27]. The bers for the 16S rRNA gene sequences of strain meso-diaminopimelic acid in the peptidoglycan DS26-2, HDS2-5, and HRF6 were AB193815, and menaquinones were analyzed as descri- AB284264 and AB284265, respectively. Results and Discussion A total of fifteen isolates were divided into se, D-fructose, D-galactose, D-ribose and D-xy- three groups, the moderately rod-shaped halo- lose but did not produce it from D-amydalin, D- philic bacteria, Group A (4 isolates) and the ex- cellobiose (some weakly), glycerol, inulin, myo- tremely rod-shaped halophilic bacteria, Group inositol, lactose, D-maltose, D-mannitol, D-man- B (10 isolates) and a sphere-shaped bacterium, nose, D-melibiose, D-melezitose, raffinose, Group C (1 isolate, HRF6) based on their phe- rhamnose, salicin, sucrose, D-sorbitol and D- notypic and chemotaxonomic characteristics, trehalose (Table 1). Group A isolates contain- DNA-DNA relatedness including 16S rRNA ge- ed meso-diaminopimelic acid as the diagnostic ne sequence analyses (Fig. 1, 2; Tables 1, 2). diamino acid in the cell wall peptidoglycan. Group A contained isolates KS11-1, DS26-2, Strain DS26-2 contained fatty acids of C10:0 KS87-5, and PB12. All were Gram-negative, (5.0 %), C12:0 (7.5 %), C12:0 3OH (12.2 %), C14:0 cream-pigmented, non-spore-forming rods. (0.6 %), C16:0 (32.0 %), Cyclo C17:0 (11.9 %), Cells measured 0.7-1.0 µm in width and 2.0- C18:1ω7c (3.2 %), C18:0 (1.1 %), cyclo C19:0 ω8c 5.0 µm in length and occurred singly or in pairs (23.2 %) and trace (less than 0.5 %) of C10:0 3OH, on JCM. No. 169 agar plates supplemented methyl C18:1 ω7c and C20:1 ω7c and had ubiqui- with 10 % NaCl. Catalase, oxidase, urease, none with nine isoprene units (Q-9) as the pre- and nitrate reduction were positive but indole dominant quinone. The polar lipids of the repre- formation was negative. Group A isolates hyd- sentative strain, DS26-2 were phosphatidyl- rolysed arginine and starch but did not hydro- glycerol (PG), diphosphatidylglycerol (DPG), lyse gelatin, casein, Tween 80 and tyrosine. phosphatidyl ethanolamine (PE) and unidenti- They were capable to grow at temperature ran- fied glycolipids. The DNA G+ C contents of ging from 10 to 45 °C, at pH 5.0 to pH 9.0 and strains ranged from 63.1 to 63.5 mol %. On the in 3 to 25 %, w/v NaCl (optimally at 10 % NaCl). basis of the 16S rRNA gene sequence analy- They produced acid from L-arabinose, D-gluco- ses, strain DS26-2 was placed within the radia- 70 tion of the cluster comprising the members of related among themselves (100-102.9 %) and the family Halomonadaceae and the genus they exhibited high levels of DNA-DNA related- Chromohalobacter (Fig. 1) and was closely ness with C. salexigens KCTC 12941T (97.4- related to C. salsexigens KCTC 12941T (99.3 % 105.6 %) as shown in Table 2. Therefore, the sequence similarity). A DNA–DNA hybridiza- group A isolates were identified as C. salexi- tion experiment revealed that they were closely gens [1, 2, 36, 38]. Table 1. Differential characteristics of strains in Chromohalobacter, Halobacterium and Halococcus. Strains* Characteristics 1 2 3 4 5 6 Cell form Rods Rods Rods Rods Cocci Cocci Pigmentation Cream Cream Red Red Red Red NaCl range (%) 3-25 0.9-25 15-30 15-30 15-30 15-30 pH range 5-9 5-9 5-10 5-10 5-10 5-10 Nitrate reduction + + - - - - Hydrolysis of Arginine + + - - - - Casein - - d + + + Gelatin - - d + + + Acid from L-Arabinose + + - - - - D-Cellobiose d w - - - - D-Fructose + + D-Galactose + + - - - - D-Glucose + + - - - - *Strains: 1, 4 strains in Group A; 2, C. salexigens KCTC 12941T; 3, 10 strains in Group B; 4, H. salinarum JCM 8978T; 5, HRF6; 6, H. saccharolyticus JCM 8878T. Reaction: +, positive; d, some positive; w, weak positive; -, negative. JCM, Japan Collection of Microorganisms, RIKEN BioResource Center, Saitama, Japan; KCTC, Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. Group B contained isolates HDB1-1, HDB1- at pH 6.0 to 7.0 and the pH range for growth 11, HDB1-31, HDS2-5, HDB10-5, HIB20-2, was pH 5.0 to 10.0. At least 100 mM MgCl2 HIB60-1, HIS10-4, HIS30-1, and HIS50-2(1). was required for growth of strains in both All were Gram-negative, red-pigmented, non- Groups. The best growth was obtained at MgCl2 spore-forming rod-shaped archaea. Cells me- concentrations between 3 to 5 % (w/v).
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