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. adjusted to an Optical Density (OD) 600 of 0.3 in in- Strain TPS4-2T was Gram-negative, aerobic, non- oculating fluid buffer supplemented with 1 M NaCl. The endospore-forming, in slightly curved rods and motile microplates were inoculated with 150 µl cell suspen- by means of one polar flagellum (Fig. 3). Colonies sion per well for each strain and incubated at 30°C. were round with entire edges, smooth, convex, opaque The results were read visually after incubation for 1, 2, and pale yellow (0.9×2.5 mm in diameter). Phenotyp- 3 and 4 days. Additional enzyme activities were re- ic characteristics are listed in the species description corded after 24-h incubation on API ZYM strips (bio- and in Table 2. Mérieux). Antibiotic susceptibility was determined ac- The cell biomass was produced on JCM medium cording to the conventional Kirby-Bauer method no. 377 agar plates at 37°C for 48 h for chemotaxo- (Bauer et al., 1966). Plates were incubated at 37°C for nomic characterization. Quinones were analyzed us- 48 h and the inhibition zone was interpreted according ing reversed-phase HPLC as described by Komagata

Table 2. Differential characteristics of Idiomarina piscisalsi strain TPS4-2T and closely related type strains.

Characteristic 1 2 3 4 5 Cell shape SCR Rods Curved rods SCR SCR Pigmentation Opaque/ Pale yellow Clear/ Pale yellow Cream Cream Cream NaCl range (%, w/v) 3‒20 1‒10 0‒25 3‒20 1‒20 Optimum growth in NaCl (%) 10‒15 3‒5 5 5 10 Maximum temperature (°C) 45 30 45 45 40 pH range 6‒9 6‒9 5‒9 5‒9 6‒10 Arginine hydrolysis + - + - + Esculin hydrolysis - - + + + Gelatin hydrolysis + - + + + Tween 80 hydrolysis + - + + + Utilization of (Biolog GN2) L-Arabinose + - + + + Pyruvic acid methyl ester + - - - - β-Hydroxybutyric acid + - - - - γ-Hydroxybutyric acid - + - - - α-Keto butyric acid + + + + - α-Keto valeric acid + - - + - Propionic acid + + + - - Succinic acid - - + - - L-Alaninamide + + - + - L-Asparagine - + - + + L-Aspartic acid - - - - + L-Glutamic acid - + + - + L-Leucine + - - - - L-Proline + - - - - L-Serine + + - + - Susceptibility to : Kanamycin - + - - - Penicillin G + - - + + Sulphonamide - - + - - Vancomycin - - - + + DNA G+C content (mol%) 47.0 48.6 48.0 48.2 45d Source of isolation Fermented fish Sea watera Baltic seab Hypersalinec Hypersalined

Abbreviations: 1, I. piscisalsi TPS4-2T; 2, I. zobellii DSM 15924T; 3, I. baltica DSM 15154T; 4, I. fontislapidosi DSM 16139T; 5, I. seosinensis KCTC 12296T. All phenotypic data were determined in this study. +, Positive reaction/growth; w, weakly positive; -, negative reaction/no growth; SCR, slightly curved rods. aData from Ivanova et al. (2000). bData from Brettar et al. (2003). cData from Martínez-Cánovas et al. (2004). dData from Choi and Cho (2005). 2013 Idiomarina piscisalsi sp. nov. 389

Table 3. Cellular fatty acid composition of Idiomarina piscisalsi strain TPS4-2T and closely related type strains.

Percentage of total Fatty acida 1 2 3 4 5

C10:0 3OH tr tr 1.0 1.6 tr iso-C11:0 1.2 3.2 2.5 2.7 2.5 iso-C11:0 3-OH 3.1 3.5 2.7 2.5 3.8 iso-C13:0 3.1 1.9 1.2 1.1 tr iso-C13:0 3-OH 2.5 5.6 3.0 3.4 3.3 C15:0 tr tr 1.0 tr tr Fig. 3. Transmission electron micrograph of Idiomarina pi- iso-C15:0 39.8 42.1 45.4 32.7 32.9 T scisalsi strain TPS4-2 with single polar flagellum, grown on iso-C15:1 F 1.9 tr 1.6 1.3 2.2 JCM medium no. 377 at 37°C. C16:0 4.5 6.7 8.3 7.2 9.4 Bar, 0.5 µm. iso-C16:0 1.0 tr 1.1 tr tr C17:0 1.2 1.3 1.2 1.6 tr iso-C17:0 14.2 15.5 10.6 11.6 9.4 and Suzuki (1987). For quantitative analysis of cellular C17:0 cyclo 1.5 2.5 3.2 2.3 2.8 fatty acid composition, cell mass was harvested and C17:1 ω8c tr tr tr 1.2 tr fatty acid methyl esters (FAMEs) were prepared and iso-C17:1 ω9c 8.9 4.4 4.3 4.9 11.3 identified according to the instructions of the Microbial C18:1 ω7c tr tr tr 1.0 5.6 Identification System (MIDI) (Kämpfer and Kroppenst- 11-methyl C18:1 ω7c tr - 2.9 2.1 tr edt, 1996; Sasser, 1990). The DNA G+C content was C18:1 ω9c 3.2 2.7 1.2 6.5 tr determined by reversed-phase HPLC (Tamaoka and C18:0 1.2 1.3 1.1 2.2 1.1 C cyclo 8c tr tr tr tr 1.2 Komagata, 1984). 19:0 ω Summed feature 3b 3.3 1.4 3.0 7.8 5.6 Strain TPS4-2T contained Q-8 as the respiratory li- T poquinone and contained iso-C15:0(39.8%) and iso- Abbreviations: 1, I. piscisalsi TPS4-2 ; 2, I. zobellii DSM T T C17:0(14.2%) as the predominant cellular fatty acids. 15924 ; 3, I. baltica DSM 15154 ; 4, I. fontislapidosi DSM T T Strain TPS4-2T and the closely related type strains 16139 ; 5, I. seosinensis KCTC 12296 ; -, not detected. a showed similar cellular fatty acid profiles but the Values are percentage of total cellular fatty acids. All data were determined in this study under the same conditions and amount of some components were different as shown T same time. in Table 3. The DNA G+C content of strain TPS4-2 b Summed feature 3 contained C16:1ω7c and /or iso-C15:0 T was 47.0 mol%, while that of I. zobellii DSM 15924 2OH. tr, trace (＜1%). was 48.6 mol%. The differential characteristics of strain TPS-2 are polar flagellum. Growth occurs within the pH range shown in Table 2. On the basis of the phenotypic char- 6‒9 (optimally at pH 8), at 25‒45°C (optimally at acteristics, phylogenetic position, DNA G+C content, 30‒37°C) and in 3‒25% (w/v) NaCl (optimally in the level of DNA-DNA relatedness (Table 1) and the 10‒15%, w/v NaCl) but no growth in the absence of rep-PCR fingerprinting pattern as mentioned above NaCl. Positive for catalase, oxidase, hydrolysis of argi- (Fig. 2), the strain represents a novel species of the nine, casein, gelatin and Tween 80 but negative for ni- genus Idiomarina, for which the name Idiomarina pi- trate reduction, hydrolysis of esculin, starch and L-ty- scisalsi sp. nov is proposed. rosine; urease, H2S production, methyl-red and indole formation. In the Biolog GN2 test, the strain utilizes L- Description of Idiomarina piscisalsi sp. nov. arabinose, pyruvic acid methyl ester, acetic acid, Idiomarina piscisalsi (pis.ci.sal’si. L. n. piscis fish; L. β-hydroxybutyric acid, α-keto butyric acid, α-keto vale- adj. salsus salted, salt; N.L. gen. n. piscisalsi of salted ric acid, propionic acid, L-alaninamide, L-alanyl-gly- fish, from which the type strain was isolated). cine, glycyl-L-glutamic acid, L-leucine, L-proline and L- Cells are Gram-negative, aerobic, non-endospore- serine but not D-fructose, D-galactose, D-glucose, forming, slightly curved rods approximately 0.3‒0.4 µm glycerol, maltose, D-mannitol, D-mannose, ribose, su- in width and 1.2‒2 µm in length. Colonies are circular, crose, trehalose or D-xylose. In the API ZYM test, the convex, opaque and pale yellow. Motile by a single strain is positive for acid phosphatase and alkaline 390 SITDHIPOL et al. Vol. 59 phosphatase, esterase (C4), esterase lipase (C8), α- mination of clonality and relatedness of Vibrio cholerae iso- chymotrypsin, napthol-AS-BI-phosphohydrolase, leu- lates by genomic fingerprinting, using long range repetitive cine arylamidase, valine arylamidase and cystein aryl- element sequence-based PCR. Appl. Environ. Microbiol., 74, 5392‒ 5401. amidase but negative for trypsin, lipase (C14), Donachie, S. P., Hou, S., Gregory, T. S., Maloahoff, A., and Alam, α-glucosidase, β-glucosidase, N-acetyl-β- M. (2003) Idiomarina loihiensis sp. nov., a halophilic glucosaminidase, α-fucosidase, α-galactosidase, γ-Proteobacterium from the Lō’ihi submarine volcano, β-galactosidase, β-glucuronidase, and α- mannosi- Hawai’i. Int. J. Syst . Evol. Microbiol., 53, 1873‒ 1879. dase. Susceptible to carbenicillin (100 µg), penicillin Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric G (20 units), sulphonamide (300 µg), imipenem deoxyribonucleic acid-deoxyribonucleic acid hybridization (10 µg), erythromycin (15 µg), ampicilllin (10 µg), in microdilution wells as an alternative to membrane filter cephalothin (30 µg) and novobiocin (5 µg), but resis- hybridization in which radioisotopes are used to determine tant to kanamycin (30 µg), tobramycin (10 µg), vanco- genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol., 39, 224‒ 229. mycin (30 µg), streptomycin (10 µg) and gentamicin Felsenstein, J. (1981) Evolutionary trees from DNA sequences: (10 g). Q-8 is presented as the respiratory lipoqui- µ A maximum likelihood approach. J. Mol. Evol., 17, 368‒ none. Major cellular fatty acids are iso-C15:0 and 376. iso-C17:0. DNA G+C content of the type strain is Felsenstein, J. (1985) Confidence limits on phylogenies: An ap- 47.0 mol%. The type strain, TPS4-2T (=NBRC 108617T proach using the bootstrap. Evolution, 39, 783‒ 791. =PCU 325T=TISTR 2054T) was isolated from ferment- Fitch, W. M. (1971) Toward defining the course of evolution: ed fish (pla-ra) in Thailand. Minimum change for a specific tree topology. Syst. Zool., 20, 406‒ 416. Forbes, L. (1981) Rapid flagella stain. J. Clin. Microbiol., 13, Acknowledgments 807‒ 809. Gevers, D., Huys, G., and Swings, J. (2001) Applicability of rep- This work was financially supported by the Ministry of Sci- PCR fingerprinting for identification of Lactobacillus spe- ence and Technology and the Thailand Research Fund for fi- cies. FEMS Microbiol. Lett., 26, 18‒ 25. nancial support under the TRF Senior Research Scholar pro- Hucker, G. J. and Conn, H. J. (1923) Method of gram staining. gramme to S.B. Thanks are also due to the National Center for Technical Bulletin 93, New York State Agricultural Experi- Genetic Engineering and Biotechnology (BIOTEC) for providing ment Station, Ithaca, pp. 3‒ 37. laboratory equipment and experimental space. 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