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

Home , Chromohalobacter, Halobacterium noricense

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 presence 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 content 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). asured 0.7-1.0 µm in width and 2.0-3.0 µm in Group B strains proved to be positive for length and occurred singly or in pairs. Colonies catalase and oxidase. Some strains hydroly- were circular and convex on JCM. No. 169 agar sed casein and gelatin but did not hydrolyse plates. arginine and tween 80. Growth on single car- Group C, HRF6 was red-pigmented and bon sources was not observed. No acids were sphere-shaped. The cells measured 0.8-1.0 µm produced from L-arabinose, D-cellobiose, D- in size and appeared singly or in tetrads. The glucose, lactose, D-mannitol, D-melibiose, raf- isolates in both two Groups grew optimally in finose, sucrose, and D-trehalose (Table 1). The the presence of 20–30 % (w/v) NaCl. Growth strains were susceptible to bacitracin (10 units), did not occur in the presence of less than 15 % novobiocin (5 µg) and rifampicin (30 µg) but NaCl. Cells of Group B strains lysed in distilled resistant to ampicillin (10 µg), chloramphenicol water, but Group C strain did not. They grew (30 µg), gentamicin (10 µg), kanamycin (30 µg), optimally at 37 °C and no growth was obser- nalidixic acid (30 µg), tetracycline (30 µg), and ved at 10 or at 50°C. Optimal growth occurred streptomycin (10 µg).

71 The representative of Group B strain, HDS2- analyses, the representative strains of Group 5 contained menaquinones of MK-8 (95.8 %) B, HDS2-5 and Group C, HRF6, were repre- and MK-8 (H2) (4.2 %). The major polar lipids sented the genera Halobacterium and Halo- presented were phytanyl-sesterterpanyl moie- coccus, respectively (Fig. 2). HDS2-5 was clo- T ties (C20C25) of phosphatidylglycerol (PG), phos- sely related to the H. salinarum JCM 8978 phatidylglycerol phosphate (PGP) and phos- with 99.9 % similarity whereas HRF6 corres- phoglycerol sulfate (PGS). Glycolipids were ponded to Halococcus saccharolyticus JCM S-TGD-1, and S-TeGD. Group B strains con- 8878Twith 99.3 % similarity. The DNA G+C tained two polar lipids, the glycerol diether ana- contents of tested strains in Group B ranged logs (C20C25 and C20C20) of PG and PGP. from 63.3 to 64.7 mol % and HRF6 was 63.2 On the basis of 16S rRNA gene sequence mol %, respectively (Table 2).

Table 2. DNA G+C contents and DNA-DNA relatedness of strains in Chromohalobacter, Halobacterium and Halococcus.

DNA G+C Factory/ % Homology with labeled Strains PCU content Fermentation strains (mol %) Time (days) Group A KCTC 12941T DS26-2 KS11-1 283 63.4 A/11 106.1 102.9 DS26-2 284 63.2 B/26 97.4 100.0 KS87-5 285 63.5 A/87 102.3 101.1 PB12 286 63.1 A/360 105.6 100.6 C. salexigens KCTC 12941T 64.2 100.0 85.6 Group B JCM 8978T JCM 8876T HIB20-2 287 64.7 A/20 94.6 13.9 HDB1-1 288 64.2 B/30 101.1 15.9 HDB1-11 289 63.3 B/30 98.5 1.6 HDB1-31 290 63.4 B/30 92.3 4.7 HIS30-1 291 63.7 A/30 94.6 4.8 HIS50-2(1) 292 63.8 A/50 97.3 16.7 HIB60-1 293 ND A/60 92.9 8.9 HDS2-5 294 63.7 B/60 89.5 3 HDB10-5 295 ND B/300 98.8 2.7 HIS10-4 296 ND A/300 87.3 2.5 H. salinarum JCM 8978T 66.4a 100 8.8 H. morrhuae JCM 8876T 3.2 100 Group C HRF6 JCM 8878T HRF6 297 63.2 C/180 100 79.9 H. saccharolyticus JCM 8878T 59.5 or 64b 88.5 100 H. morrhuae JCM 8876T 57.8c 8.3 6.5

ND, Not determined. aData from Gutiérrez et al. [9]. bData from Montero et al. [18] and Ventosa et al. [37]. cData from Bohácek et al. [4]. PCU, Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.

72 75 Chromohalobacter canadensis ATCC 43984T (AJ295143) Pseudomonas beijerinckii ATCC 19372T (AB021386) 99 Chromohalobacter sarecenensis LV4T (AY373448) 100 Chromohalobacter marismortui ATCC 17056T (X87219) Chromohalobacter israelensis ATCC 43985T (AJ295144) Chromohalobacter salexigens DSM 3043T (AJ295146) 100 DS26-2 (AB193815) Halomonas muralis LMG 20969T (AJ320530) 95 T Halomonas desiderata FB2 (X92417) 65 Halomonas boliviensis LC1T (AY245449) Deleya pacifica DSM 4742T (L42616) Halomonas eurihalina ATCC 49336T (X87218) 87 99 Halomonas salina F8-11T (AJ295145) Deleya marina ATCC 25374T (M933544) 78 Zymobacter palmae T-109T (D14555) Pseudomonas aeruginosa ATCC 27853T (AY268175) 100 Pseudomonas oleovorans IAM 1508T (D84018)

0.01

Fig. 1. Phylogenetic tree showing the relationships between strain DS26-2T and related taxa based on 16S rRNA gene sequence. The branching pattern was generated by neighbor-joining method. Bootstrap percentages above 65 %, based on 1000 replications are shown at the nodes. Bar, 0.01 substitutions per nucleotide position.

T 100 Halobacterium noricense A1 (AJ548827) Halobacterium salinarum DSM 3754T (AJ496185) 100 HDS2-5 (AB284264) T 100 Halococcus salifodinae Blp (AB004877) HRF6 (AB284265) 100 99 T Halococcus saccharolyticus JCM 8878 (AB004876) Halococcus morrhuae JCM 8876T (D11106) Haloferax denitrificans strain S1T (D14128) T 91 Halobiforma haloterrestris strain 135 (AF333760) Natronococcus occultus JCM 88 59T (Z28378) 100 Natrialba aegyptiaca 40T (AF251941) 94 Haloterrigena turkmenica 4kT (AB004878) Natronobacterium pharaonis JCM 8858T (D87971)

Methanobacterium beijingense 8-2T (AY350742) 0.02

Fig. 2. Phylogenetic tree showing the relationships between strain HDS2-5 , HRF6, and related taxa based on 16S rRNA gene sequence. The branching pattern was generated by neighbor-joining method. Bootstrap percentages above 91%, based on 1000 replications are shown at the nodes. Bar, 0.02 substitutions per nucleotide position

73 Group B strains were closely related among JCM 8876T. Therefore, a sphere-shaped HRF6 themselves with 90-102.9 % of DNA-DNA rela- (Group C) was identified as H. saccharolyticus tedness, suggesting that they belong to the sa- [18, 27]. me species. On the other hand, all strains show- In fish sauce and related products, Lenti- ed the high levels of DNA-DNA relatedness to bacillus jurispiscarius, Filobacillus sp., Haloba- H. salinarum JCM 8978T (87.3 - 101.1 %) as cillus sp., T. halophilus, T. muriaticus, H. Salina- shown in Table 2. The representative strain, rum, H. piscisalsi, and Natrinema gari [10, 19, HDS2-5 contained C20C25 derivative of several 20, 29, 32, 34, 35, 39] were reported. In this kinds of polar lipid such as glycolipids (S-TGD-1 study, the moderately halophilic bacteria, C. sa- and S-TEGD) that are characteristics and dia- lexigens and the extremely halophilic archaea, gnostic for the genus Halobacterium [8]. There- H. salinarum and H. saccharolyticus strains were fore, Group B strains were identified as Halo- commonly distributed and played some roles in bacterium salinarum [38]. many stages of fish sauce fermentation. HRF6 contained two major polar lipids, the Acknowledgements. We are very grateful glycerol diether analogs (2-O-sesterterpanyl-3- to Sindhu Samuth Fish Sauce Factory (Squid O-phytanyl-sn-glicerol, C20C25 and 2, 3-di-O-phy- Brand) LTD., Samutprakarn, Thai Fish Sauce tanyl-sn-glycerol, C20C20) of phosphatidylglyce- Factory (Squid Brand) Co., LTD., Samutsongk- rol (PG), phosphatidylglycerol-methylphospha- ram and Rayong Fish Sauce Industry Co., Ltd., te (PGP-Me) and MK-8(H2) (89.4 %), and MK-8 Rayong Province, for providing the samples. (10.6 %) that were different from the strain of This study was in part supported by the Thai- Group B [8]. HRF6 exhibited high level of DNA- land Research Fund for a 2001 Royal Golden DNA relatedness (88.5 %) with H. saccharo- Jubilee Scholarship as a research grant to S. N. lyticus JCM 8878T but had low level of DNA- and by Ratchadapiseksomphot Endowment DNA relatedness (8.3 %) with H. morrhuae Fund, Chulalongkorn University (2002).

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ИДЕНТИФИКАЦИЯ НА ХАЛОФИЛНИ БАКТЕРИИ ОТ РИБЕН СОС (NAM-PLA) В ТАЙЛАНД

Сомбоон Танасупават*, Сирилак Намвонг, Такуджи Кудо, Такаши Итох

Резюме От ферментирал рибен сос (nam-pla) в Тайланд са изолирани четири щама Грам-отрицателни бактерии, с пръчководна форма, умерено хало- филни (Група А) и единадесет щама на облигатно аеробни, крайно хало- филни бактерии, от които 10 щама пръчковидни (Група В) и един кокоиден щам (Група С). Секвенционният анализ на 16S рРНК на представителни щамове, обозначени като DS26-2 (Група A), HDS2-5 (Група B) и HRF6 (Група C), показва, че те са тясно родствени на Chromohalobacter salexigens KCTC 12941T, Halobacteirum salinarum JCM 8978T и Halococcus saccharolyticus JCM 8878T, съответно със сходство 99.3, 99.9 и 99.0 %. На база на ДНК-ДНК род- ството щамовете от Група А се идентифицират като C. salexigens, от Група В като H. salinarum, а от Група С като H. saccharolyticus. Щамовете от Група А растат в 3–25 % (w/v) NaCl. Главният компонент е убихинонът с девет изопренови единици (Q-9), а съдържанието на ДНК G+C варира от 63.1 до 64.2 mol %. Щамовете от Група В и Група С растат оптимално в присъствието на 25-30 % NaCl. Изследваните щамове от Група В съдържат основно менахинон с девет изопренови единици (MK-8). Съдържанието им а ДНК G+C е от 63.3 to 64.7 mol %. Щамът от Група С има MK-8(H2) като прео- бладаващ менахинон, а съдържанието на ДНК G+C е 63.2 mol %.