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Idiomarina Piscisalsi Sp. Nov., from Fermented Fish (Pla-Ra) in Thailand

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Idiomarina Piscisalsi Sp. Nov., from Fermented Fish (Pla-Ra) in Thailand J. Gen. Appl. Microbiol., 59, 385‒391 (2013) Short Communication Idiomarina piscisalsi sp. nov., from fermented fish pla-ra( ) in Thailand Jaruwan Sitdhipol,1,† Wonnop Visessanguan,2 Soottawat Benjakul,3 Pattaraporn Yukphan,4 and Somboon Tanasupawat1,* 1 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Food Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand 3 Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 4 BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand (Received August 17, 2012; Accepted June 18, 2013) Key Words—fermented fish; Idiomarina piscisalsi; γ-Proteobacteria; 16S rRNA gene The family Idiomarinaceae, class Gammaproteobac- Korea (Choi and Cho, 2005), Idiomarina homiensis teria, was proposed by Ivanova et al. (2004) according from seashore sand in Korea (Kwon et al., 2006), Idi- to the phylogenetic relationships of marine Alteromon- omarina salinarum from a marine solar saltern in Korea as-like bacteria. The family formerly contained two (Yoon et al., 2007), Idiomarina insulisalsae sp. nov., genera, Idiomarina and Pseudidiomarina, which were isolated from the soil of a sea salt evaporation pond, proposed by Ivanova et al. (2000) and Jean et al. Idiomarina marina, Idiomarina maritima and Idiomarina (2006), respectively. At the time of writing, the genus donghaiensis from the sea, Idiomarina sediminum Idiomarina comprises nineteen species, Idiomarina from sediment, Idiomarina tainanensis from Tainan, abyssalis and Idiomarina zobellii from the deep sea Taiwan and Idiomarina taiwanensis from Taiwan (Ivanova et al., 2000), Idiomarina baltica from surface (Taborda et al., 2009), Idiomarina xiamensis from sur- water of the central Baltic Sea (Brettar et al., 2003), face sea water around the Xiamen island, Idiomarina Idiomarina loihiensis from a submarine volcano aestuarii (Wang et al., 2011), Idiomarina maris from (Donachie et al., 2003), Idiomarina fontislapidosi and sediment of the South China Sea (Zhang et al., 2012) Idiomarina ramblicola from inland hypersaline habitats and Idiomarina aquimaris isolated from reef-building in Spain (Martínez-Cánovas et al., 2004), Idiomarina coral Isopora palifera (Chen et al., 2012). During our seosinensis from hypersaline water of a solar saltern in investigation on moderately halophilic bacteria in Thai fermented fish (pla-ra) which contain 7.8‒17.9% NaCl * Corresponding author: Dr. Somboon Tanasupawat, Depart- (w/v), the strain TPS4-2T was isolated. In this paper, we ment of Biochemistry and Microbiology, Faculty of Pharmaceu- propose the name Idiomarina piscisalsi sp. nov. for tical Sciences, Chulalongkorn University, Bangkok 10330, Thai- strain TPS4-2T. land. Strain TPS4-2T (=NBRC 108617T=PCU 325T=TISTR E-mail: [email protected] T †Bioscience Department, Thailand Institute of Scientific and 2054 ) was isolated from pla-ra which was collected Technological Research (TISTR), Pathumthani 12120, Thailand. from a market in Suphanburi Province, Thailand. The The GenBank/EMBL/DDBJ accession number for the 16S fermented fish sample was aseptically minced and di- rRNA sequence gene of strain TPS4-2T is AB619724. luted in 10% NaCl solution. Then, the sample was 386 SITDHIPOL et al. Vol. 59 spread on JCM medium no. 377 agar plates (per liter): 1980), maximum-parsimony (Fitch, 1971) and maxi- 100 g NaCl, 5 g casamino acids, 5 g yeast extract, 1 g mum-likelihood (Felsenstein, 1981) methods in the glutamic acid, 2 g KCl, 3 g trisodium citrate, 20 g program MEGA 5 (Tamura et al., 2011). The confi- MgSO4･7H2O, 36 mg FeCl2･4H2O, 0.36 mg MnCl2･ dence values of branches of the phylogenetic trees 4H2O, and 20 g agar; pH 7.2; and incubated at 37°C were determined using bootstrap analyses (Felsen- for 48 h. The colonies were picked up and purified. stein, 1985) based on 1,000 resamplings. The DNA- The type strains, I. zobellii DSM 15924T, I. seosinensis DNA hybridization was performed as described by KCTC 12296T, I. baltica DSM 15154T and I. fontislapi- Ezaki et al. (1989). dosi DSM 16139T were used as reference strains. Comparison of the 16S rRNA gene sequence of Chromosomal DNA was extracted and purified from strain TPS4-2T with those of other members of the fam- cells grown on JCM medium no. 377 agar plates ac- ily Idiomarinaceae indicated that it was placed in the cording to the method of Saito and Miura (1963). The genus Idiomarina and was closely related to I. zobellii 16S rRNA gene of the isolate was amplified, purified, DSM 15924T(98.7%), I. baltica DSM 15154T(96.9%), I. and sequenced as described by Namwong et al. fontislapidosi DSM 16139T(96.6%), I. seosinensis (2005). The 16S rRNA gene sequence was aligned KCTC 12296T(96.4%) (Fig. 1). Strain TPS4-2T exhibited with selected sequences obtained from the GenBank/ low DNA-DNA relatedness with I. zobellii DSM EMBL/DDBJ databases by using the CLUSTAL W pro- 15924T(47.7%), I. baltica DSM 15154T(24.7%), I. fontis- gramme version 1.8 (Thompson et al., 1994). The lapidosi DSM 16139T(7.4%) and I. seosinensis KCTC alignment was manually verified and adjusted prior to 12296T(14.1%). I. zobellii DSM 15924T exhibited recip- the construction of a phylogenetic tree. The phyloge- rocally low DNA-DNA relatedness (51.4%) to strain netic tree was constructed by using neighbor-joining TPS4-2T (Table 1). The DNA-DNA relatedness of strain (Saitou and Nei, 1987) with genetic distances com- TPS4-2T with its closest phylogenetic neighbors was puted by using Kimura’s 2-parameter model (Kimura, well below the 70% cut-off point recommended for the Fig. 1. Neighbor-joining tree based on 16S ribosomal RNA gene se- quences showing relationship between Idiomarina piscisalsi strain TPS4-2T and closely related type strains. Only bootstrap values above 50% (percentages of 1,000 replications) are indicated. ●, indicates branches of the maximum-parsimony tree; *, indi- cates branches of the maximum-likelihood tree; Bar, 0.02 substitutions per nucleotide position. 2013 Idiomarina piscisalsi sp. nov. 387 Table 1. DNA-DNA relatedness of Idiomarina piscisalsi strain TPS4-2 T and closely related type strains. DNA-DNA relatedness (%) with labeled strainsa Strain TPS4-2T DSM 15924T I. piscisalsi TPS4-2T 100±0.02 51.4±0.01 I. zobellii DSM 15924T 47.7±0.01 100±0.04 I. fontislapidosi DSM 16139T 7.4±0.02 11.6±0.01 I. baltica DSM 15154T 24.7±0.01 27.2±0.03 I. seosinensis KCTC 12296T 14.1±0.02 16.8±0.06 aValues are expressed as the means of three determinations. Fig. 2. Dendrogram and agarose gel electrophoresis of (GTG)5 -PCR ge- nomic fingerprint of Idiomarina piscisalsi strainTPS4-2T and closely related type strains. assignment of the strains to the same genomic spe- DSM 16139T, were more closely related, with a Dice cies (Wayne et al., 1987). Based on the above DNA- coefficient of 70.6%, while strain TPS4-2T was sepa- DNA relatedness data, strain TPS4-2T warrants a sepa- rated from them with a 61.1% Dice coefficient (Fig. 2). rate species of the genus Idiomarina. Cell shape, size and cell arrangement were exam- Repetitive sequencing based on polymerase chain ined on JCM medium no. 377 agar at 37°C for 48 h. reaction (rep-PCR) fingerprinting as described by Ver- Hucker-Conn modification was used for Gram staining salovic et al. (1994) was carried out with (GTG)5 primer (Hucker and Conn, 1923). Spore formation was exam- (5′-GTGGTGGTGGTGGTG-3′) (Gevers et al., 2001) ined on Gram-stained specimens. Flagella were exam- and the PCR and electrophoresis conditions were ined as described by Forbes (1981) and observed by modified from Chokesajjawatee et al. (2008). The re- transmission electron microscopy. Effects of growth at actions were performed in a Thermo cycler (DYAD various NaCl concentrations (0, 0.5 and 1‒25%, w/v), ALD 1244, MJ Research, Inc., Waltham, MA). The gel initial pH (4‒9, with intervals of 1) and temperatures (4, image was captured by using a Typhoon 9410 image 10, 25, 30, 37, 45 and 50°C) was investigated in JCM scanner (Amersham Biosciences, Little Chalfont, UK). medium no. 377 broth (Namwong et al., 2005). Cata- The resulting fingerprints were analyzed by using a lase, oxidase and esculin hydrolysis, H2S production, pattern analysis software package, Gel ComparII ver- methyl-red and indole formation and nitrate reduction sion 4.5 (Applied Maths BVBA, Sint-Martens-Latem, were determined as described by Barrow and Feltham Belgium). An UPGMA dendrogram based on Dice’s (1993). Hydrolysis of casein, gelatin, starch and Tween coefficient calculated from similarities of the DNA fin- 80, tyrosine, deoxyribonuclease and urease activity gerprint patterns was used to reveal genetic relation- were determined as described by Namwong et al. ship between strains. The Idiomarina strains compared (2005). Arginine decarboxylase was tested by using were separated into two clusters at a 48.5% Dice coef- the medium reported by Thornley (1960). All tests ficient (Fig. 2). The first cluster comprised two strains, were carried out in the medium supplemented with I. baltica DSM 1514T and I. seosinensis KCTC 12296T 10% NaCl, except for the investigation of effects of with the Dice coefficient of 54.1%. The second group growth at various NaCl concentrations. The utilization comprised three strains, I. zobellii DSM 15924T, I. fon- of carbon sources was investigated using Biolog GN2 tislapidosi DSM 16139T, and TPS4-2T. The first two plates (Choi and Cho, 2005). Cell preparation of the strains, I. zobellii DSM 15924T and I. fontislapidosi tested strains for Biolog GN2 plates were grown in log 388 SITDHIPOL et al. Vol. 59 phase at 30°C and the density of cell suspension was to the manufacturer’s manual.
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