International Journal of Systematic and Evolutionary Microbiology (2013), 63, 1805–1809 DOI 10.1099/ijs.0.043604-0

Pseudoalteromonas arabiensis sp. nov., a marine polysaccharide-producing bacterium

Hidetoshi Matsuyama,1 Hideki Minami,1 Hirokazu Kasahara,1 Yoshihisa Kato,2 Masafumi Murayama3 and Isao Yumoto4

Correspondence 1School of Biological Science and Engineering, Tokai University, Minamisawa, Minami-ku, Sapporo Hidetoshi Matsuyama 005-8601, Japan [email protected] 2School of Marine Science and Technology, Tokai University, Orido 3-20-1, Shimizu-ku, Shizuoka 424-8610, Japan 3Center for advanced Marine Core Research, Kochi University, B200 Nankoku, Kochi 783-0032, Japan 4Bioprocess Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517, Japan

A novel exopolysaccharide-producing bacterium, designated strain k53T, was isolated from sediment from the Arabia Sea, Indian Ocean. The strain was Gram-negative, motile, strictly + aerobic, oxidase-positive and catalase-positive, and required Na for growth. Its major isoprenoid quinone was ubiquinone-8 (Q-8), and its cellular fatty acid profile mainly consisted

of C16 : 1v7c,C16 : 0 and C18 : 1v7c. The DNA G+C content was 43 mol%. 16S rRNA gene sequence analysis suggested that strain k53T is a member of the genus . Strain k53T exhibited close phylogenetic affinity to Pseudoalteromonas lipolytica LMEB 39T (98.0% 16S rRNA gene sequence similarity) and Pseudoalteromonas donghaensis HJ51T (97.3 %).The DNA–DNA reassociation values between strain k53T and P. lipolytica JCM 15903T and P. donghaensis LMG 24469T were 17 % and 12 %, respectively. Owing to the significant differences in phenotypic and chemotaxonomic characteristics, and phylogenetic analysis based on the 16S rRNA gene sequence and DNA–DNA relatedness data, the isolate merits classification as a representative of a novel species, for which the name Pseudoalteromonas arabiensis is proposed. The type strain of this species is k53T (5JCM 17292T5NCIMB 14688T).

There have been many reports of marine micro-organisms the largest within the class .Inthis that produce exopolysaccharides (EPSs). It remains possible study, we isolated a polysaccharide-producing bacterium that new polysaccharide-producing could be found ‘belonging to genus Pseudoalteromonas’ and the phenotypic in various habitats, and we have been trying to find them in and chemotaxonomic characteristics and results of phylo- sediment from the ocean bed (Matsuyama et al., 2006). Strain genetic analysis based on the 16S rRNA gene sequence of the k53T was isolated as a polysaccharide producer and was isolate showed that it merited classification as a novel considered to be a ‘Pseudoalteromonas-like’ strain. The genus Pseudoalteromonas species. originally described by Baumann et al. (1972) for Sediment samples were collected from the Arabia Sea at a marine aerobic, Gram-negative, non-fermentative, polarly depth of 3615 m (16u 459 0199 N, 69u 009 0299 E) using a flagellated bacteria, was later divided into two genera, multiple corer during the GEOTRACES cruise in 2009. Alteromonas and Pseudoalteromonas, on the basis of phylo- Strain k53T was isolated by selective enrichment. The genetic analysis (Gauthier et al., 1995). At the time of writing, sediment sample was inoculated in 10 ml mineral salts the genus Pseudoalteromonas comprised 38 species and two medium (KJ medium) containing (l21) 23.4 g NaCl, 5 g subspecies (http://www.bacterio.cict.fr/p/pseudoalteromonas. MgCl2, 1.1 g CaCl2 .2H2O, 0.7 g KCl, 0.2 g NaHCO3, html). Members of the genus Pseudoalteromonas have been 6.05 g Tris, 5 g peptone, 10 g glucose, 0.01 g K2HPO4, isolated from marine environments and the genus is one of 0.008 g Na2HPO4, 1.0 g (NH4)2SO4, 0.0016 g NH4NO3, 3.9 g Na2SO4, pH 7.8. This medium was incubated at 15uC Abbreviation: EPS, exopolysaccharide. with shaking at 150 r.p.m. After being incubated for several The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene days, a portion of the suspension was transferred into sequence of strain k53T is AB576636. 10 ml fresh medium and the medium was reincubated. A supplementary table is available with the online version of this paper. After three successive transfers, the suspension was plated

Downloaded from www.microbiologyresearch.org by 043604 G 2013 IUMS Printed in Great Britain 1805 IP: 54.70.40.11 On: Sun, 09 Jun 2019 21:06:58 H. Matsuyama and others onto Marine agar 2216 (Difco) to isolate pure cultures. The Table 1. Phenotypic characteristics of strain k53T and closely isolates were checked for their ability to produce EPSs by related type strains of species of the genus Pseudoalteromonas adding three volumes of ethanol to the supernatant of broth Taxa: 1, strain k53T;2,P. donghaensis LMG 24469T;3,P. lipolytica JCM (Matsuyama et al., 2003). Of the strains isolated, k53T,which 15903T 4, P. haloplanktis JCM 20767T. All data were taken from this study showed good EPS production, was selected for further study. except where indicated. All strains were positive for production of catalase, In addition to strain K53T, Pseudoalteromonas donghaensis oxidase, reduction of nitrates to nitrites, alkaline phosphatase, esterase (C4), LMG 24469T (Oh et al., 2011), Pseudoalteromonas lipolytica esterase lipase (C8), valine arylamidase, leucine arylamidase, acid JCM 15903T (Xu et al., 2010) and Pseudoalteromonas T phosphatase, and naphthol-AS-BI-phosphohydrolase, and hydrolysis of haloplanktis JCM 20767 were used as reference strains for aesculin and gelatin, acid production from sucrose and maltose, phenotypic characterization and fatty acid composition. T assimilation of a-D-glucose, maltose, D-mannose, sucrose and succinic Pseudoalteromonas haloplanktis JCM 20767 is a type species acid. All strains were negative for Gram-staining, production of arginine of genus Pseudoalteromonas (Gauthier et al., 1995). These dihydrolase, cystine arylamidase, a-galactosidase, b-galactosidase, b-gluco- micro-organisms were cultured in marine broth 2216 with sidase and a-fucosidase, production of indole, assimilation of adonitol, L- u reciprocal shaking (150 r.p.m.) at 27 C until the early fucose, D-galactose, a-lactose, melibiose, raffinose, L-rhamnose, D-gluconic stationary phase of growth. acid, formic acid and D-serine. +, Positive; 2, negative; W, weakly positive For phenotypic characterization, marine broth 2216 was used as the basal medium. Acid production from Characteristic 1 2 3 4 carbohydrates was determined by the method of Leifson Pigmentation 22 2Melanin-like (1963). Growth at different temperatures (4, 5, 6, 10, 15, Growth at 15 % (w/v) NaCl 2 + 22 20, 25, 30, 35, 37 and 40uC) was tested using marine broth Growth at 37uC 2 ++ + 2216 (MB). The pH range for growth was determined by Production of EPS + 22 2 adding MES (pH 5.0–7.0), TAPS (pH 7.5–9.0) and CHES Production of: (pH 9.0–10.0) to MB at a concentration of 50 mM. NaCl Trypsin 2 ++ 2 requirement for growth was determined after 1 day of a-Glucosidase 2 ++ + cultivation at 27uC in the following medium: 0.1 % Lipase (C14) 22 W 2 peptone, 0.1 % yeast extract, 0.03 % KCl, 0.25 % a-Chymotrypsin 2 WW 2 ++ MgSO .7H O, 0.05 % CaSO .2H O, (0–20 %) NaCl. a-Mannosidase 2 2 4 2 4 2 ++ + Other physiological and biochemical characteristics were N-Acetyl-b-glucosaminidase 2 examined according to the methods described in Cowan & Acid production from: D-Glucose 2 ++ + Steel’s Manual (Barrow & Feltham, 1993). Phenotypic Trehalose 2 + 22 characterization was also performed for comparing char- D-Arabinose ++ 22 acteristics with the related species under the same Cellobiose ++ + 2 conditions, using the API ZYM and API 20NE kits D-Mannose ++ + 2 (bioMe´rieux), and the Biolog GN2 microwell plates, Assimilation of: according to the manufacturers’ instructions, but with Tween 40 2 ++ + minor modifications, i.e. cell suspensions for inoculation Tween 80 2 ++ + were prepared in seawater. N-Acetyl-D-galactosamine + 22 2 D-Arabinose ++ 22 Cultural properties, cell morphology, motility and the + T Gentiobiose 22 2 results of some physiological tests of strain k53 are a-Cyclodextrin ++ + 2 described in Table 1 and the species description. Cells were D-Sorbitol + 22 2 motile. Growth occurs in media with 0.5–10 % (w/v) NaCl, Trehalose 2 + 2 + with the optimum at 2–3 %. Temperature range for growth L-Serine ++ + 2 # is 6–35 C, optimum 25uC. The pH range for growth is 5.5– Dextrin ++ + 2 T 9.5, optimum pH 7–8. Strain k53 can be readily differ- Propionic acid ++ 2 + entiated from the phylogenetically closely related species P. N-Acetyl-D-glucosamine ++ + 2 T T lipolytica JCM 15903 and P. donghaensis LMG 24469 and L-Aspartic acid 2 + 22 from the type species of the genus Pseudoalteromonas (P. Cellobiose ++ + 2 haloplanktis JCM 20767T) by several phenotypic properties Acetic acid 2 + 2 + as shown in Table 1. Strain k53T produce EPS in KJ medium, Citric acid + 22 + however, neither P. lipolytica JCM 15903T, P. donghaensis L-Alanine + W 22 ++ LMG 24469T nor P. haloplanktis JCM 20767T produce EPS L-Leucine 2 2 ++ in KJ medium. L-Proline 2 2 DNA G+C content (mol%)* 43 41.8 42.3 42–44 For the identification of isoprenoid quinones, they were extracted from freeze-dried cells by the method of *Data for P. donghaensis LMG 24469T, P. lipolytica JCM 15903T and Nishijima et al. (1997) and analysed by HPLC (Waters P. haloplanktis JCM 20767T were taken from Oh et al. (2011), Xu et al. 600 series). Ubiquinone-8 (97.3 %), ubiquinone-7 (2.3 %) (2010) and Bowman & McMeekin (2005), respectively.

Downloaded from www.microbiologyresearch.org by 1806 International Journal of Systematic and Evolutionary Microbiology 63 IP: 54.70.40.11 On: Sun, 09 Jun 2019 21:06:58 Pseudoalteromonas arabiensis sp. nov. and ubiquinone-9 (0.5 %) are identified as the major The level of DNA–DNA relatedness was determined quinones of strain k53T. For the fatty acid analysis, strain fluorometrically by the method of Ezaki et al. (1989) using k53T and reference strains were grown in marine broth 2216 photobiotin-labelled DNA probes and black microplates. (Difco) at 27uC for 24 h. Whole-cell fatty acids were According to sequence similarities and phylogenetic analysis analysed according to the methods of Yumoto et al. data based on the 16S rRNA gene sequence, strains that (2001); they were extracted from 100 mg freeze-dried cells exhibited greater than 97 % similarity with strain k53T were esterified by acid methanolysis and analysed by GC (model used for the estimation of DNA–DNA relatedness. The T GC 353; GL Sciences) using a 0.25 mm (i.d.) 6 100 m, DNA–DNA relatedness values of strain k53 with P. T T 0.2 mm film SP-2560 column (Supelco). Fatty acids were lipolytica JCM 15903 and P. donghaensis LMG 24469 identified by comparing them with fatty acid methyl esters (17 % and 12 %, respectively) were significantly below the purchased from Supelco and GL Sciences, and using GC/MS value of 70 % that is considered to be the threshold for the (model INCOS 50; Finnigan mat) connected a GLC (model delineation of species (Wayne et al., 1987). T 3400; Varian). The fatty acid composition of strain k53 is The formation of a distinctive phyletic line within the shown in Table S1 (available in IJSEM Online). These data genus Pseudoalteromonas indicates that strain k53T can be were obtained using the same growth conditions for all assigned as a novel species in this genus. In addition, a T strains. The cellular fatty acid profiles of K53 , P. lipolytica number of physiological and chemotaxonomic characters and P. donghaensis mainly consisted of C16 : 1v7c,C16 : 0 and clearly distinguished our isolate from other phylogeneti- T T C18 : 1v7c. The fatty acid profile of strain k53 clearly cally related species (Table 1). Therefore, strain k53 resembles those determined for other marine genera of the should be classified as a member of a novel species within Gammaproteobacteria,e.g.Alteromonas, Pseudoalteromonas the genus Pseudoalteromonas, for which the name and Glaciecola (Ivanova et al., 2000). Pseudoalteromonas arabiensis sp. nov. is proposed. Bacterial DNA was prepared according to the method of Marmur (1961). The DNA obtained was digested with Description of Pseudoalteromonas arabianensis nuclease P1 (Yamasa Shoyu) and resulting nucleotides were sp. nov. separated by HPLC (Tamaoka & Komagata, 1984). The Pseudoalteromonas arabiensis (a.ra.bi.en9sis. N.L. fem. adj. DNA G+C content of strain k53T was 43 mol%, which is arabiensis of or belonging to the Arabian Sea, where the consistent with that of the genus Pseudoalteromonas, 38– type strain was isolated). 48 mol% (Bowman & McMeekin, 2005). Aerobic, Gram-negative, oxidase- and catalase-positive, non- The 16S rRNA gene was amplified by PCR using primers 9F pigmented, non-endospore-forming, motile, rod-shaped (GAGTTTGATCCTGGCTCAG) and 1510R (GGCTACCT- cells, 1.0–1.5 mm long and 0.6–0.7 mm wide. On Marine TGTTACGA). The resulting PCR product was purified with agar, they form smooth, convex, circular and entire colonies. QIAquick PCR purification kit (Qiagen) and sequenced Grows in media with 0.5–10 % (w/v) NaCl, optimum at 2– directly by the dideoxynucleotide chain-termination method 3 %. Grows at 6–35#C, optimum at 25#C, and pH 5.5–9.5, using a DNA sequencer (PRISM 3100; Applied Biosystems) optimum at pH 7–8. Produces EPS. Hydrolyses aesculin, with a Big Dye termination RR mix version 3 . 1 (Applied gelatin, casein and starch, but not urea. Reduces nitrates to Biosystems) according to the manufacturer’s instructions. nitrite but not to nitrogen (API). Does not produce indole Multiple alignments of the sequences were performed using (API). Produces alkaline phosphatase, esterase (C4), esterase the program CLUSTAL W program (Thompson et al., 1994). A lipase (C8), leucine arylamidase, valine arylamidase, acid phylogenetic tree was constructed by the neighbour-joining phosphatase, naphthol-AS-BI-phosphohydrolase and N- (Saitou & Nei, 1987), maximum-likelihood (Guindon & acetyl-b-glucosaminidase, but not lipase (C14), cystine Gascuel, 2003) and maximum-parsimony (Felsenstein, 1981) arylamidase, trypsin, chymotrypsin, a-galactosidase, b-galac- method in MEGA 5 (Tamura et al., 2011). For neighbour- tosidase, a-glucosidase, b-glucosidase, a-mannosidase or a-fucosidase. Utilizes a-cyclodextrin, dextrin, N-acetyl-D- joining analysis, the distance between sequences (Knuc value) was calculated using Kimura’s two-parameter model galactosamine, N-acetyl-D-glucosamine, L-arabinose, gentio- (Kimura, 1980). The similarity between sequences was biose, D-glucose, maltose, D-mannose, D-sorbitol, sucrose, calculated using the GENETYX computer program (Software cis-aconitic acid, citric acid, propionic acid, succinic acid, L- Development). The 16S rRNA gene sequence (1501 bp) of alanine, L-alanyl glycine, L-glutamic acid, glycyl L-aspartic strain k53T was obtained and analysed and the similarity with acid, glycyl L-glutamic acid, L-serine and L-threonine, but not previously reported strains was determined. Phylogenetic glycogen, Tween 40, Tween 80, D-fructose, D-galactose, myo- analysis of the 16S rRNA gene sequence showed that strain inositol, melibiose, raffinose, L-rhamnose, trehalose, formic K53T was a member of the genus Pseudoalteromonas.The acid or acetic acid (Biolog). Acid is produced from sucrose, neighbour-joining (Fig. 1) and maximum-likelihood trees maltose, cellobiose, D-arabinose and D-mannose, but not T from D-glucose or D-sorbitol. The cellular fatty acid profiles revealed that strain k53 clustered with P. lipolytica LMEB mainly consist of C v7c,C and C v7c. The major 39T (Xu et al., 2010) and P. donghaensis HJ 51T (Oh et al., 16 : 1 16 : 0 18 : 1 quinone is ubiquinone-8. 2011). Strain K53T was most similar to P. lipolytica LMEB 39T (98.0 % 16S rRNA gene sequence similarity) and P. The type strain is k53T (5JCM 17292T5NCIMB 14688T), donghaensis HJ 51T (97.3 %). isolated from sediment in the Arabian Sea in the Indian

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T 73 Pseudoalteromonas atlantica IAM 12927 (X82134) T 64 Pseudoalteromonas espejiana NCIMB 2127 (X82143) 54 T Pseudoalteromonas agarivorans KNN 255 (AJ417594) T 77 Pseudoalteromonas paragorgicola KMM 3548 (AY040229) T . Pseudoalteromonas carrageenovora IAM 12662 (X82136) T 77 Pseudoalteromonas tetraodonis IAM 14160 (X82139) 57. NCIMB 2128T (X82140) Pseudoalteromonas undina T Pseudoalteromonas haloplanktis ATCC 14393 (X67024) 0.005 100 . . CECT 4664T (X98336) 86 . Pseudoalteromonas antarctica T 99 Pseudoalteromonas translucida KMM 520 (AY040230) T Pseudoalteromonas aliena KMM 3562 (AY387858) T 99. Pseudoalteromonas mariniglutinosa KMM 3635 (AJ507251) T . Pseudoalteromonas prydzensis MB8-11 (U85855) 65 T 59 . Pseudoalteromonas arabiensis k53 (AB576636) HJ 51T 69 . Pseudoalteromonas donghaensis (FJ754319) T 78 67 Pseudoalteromonas lipolytica LME B39 (FJ404721) Pseudoalteromonas ruthenica KMM 300T (AF316891) T .Pseudoalteromonas aurantia ATCC 33046 (X82135) T 100 Pseudoalteromonas citrea NCIMB 1889 (X82137) T . Pseudoalteromonas phenolica O-BC30 (AF332880) T . Pseudoalteromonas luteoviolacea NCIMB 1893 (X82144) 86 T 83 . Pseudoalteromonas rubra ATCC 29570 (X82147) T 91 Pseudoalteromonas peptidolytica F12-50-A1 (AF007286) T Pseudoalteromonas byunsanensis FR1199 (DQ011289) T Pseudoalteromonas tunicata D2 (Z25522)

Fig. 1. Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic relationships of strain k53T within the genus Pseudoalteromonas. Bootstrap values (.50 %) based on 1000 replicates are shown at branch nodes. Filled squares indicate that the corresponding nodes were also recovered with the maximum-likelihood method. Bar, 0.005 substitutions per nucleotide position.

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