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Marine Biotechnology Research Centre, Korea Ocean Research & Development Institute, PO Box 29, Ansan 425-600, Republic of Korea
Correspondence
Sang-Jin Kim
s-jkim{at}kordi.re.kr
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ABSTRACT |
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-Proteobacteria, within the genus Photobacterium. The 16S rRNA gene sequence of strain GMD509T is most similar to those of Photobacterium frigidiphilum (97·8 %), Photobacterium profundum (97·5 %) and Photobacterium indicum (97·4 %). DNA–DNA relatedness levels between the isolate and its closest known phylogenetic relatives, P. frigidiphilum and P. indicum, are 25·3 and 13·7 %, respectively. Strain GMD509T therefore represents a novel species, for which the name Photobacterium aplysiae sp. nov. is proposed, with the type strain GMD509T (=KCTC 12383T=JCM 12948T).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GMD509T is AY781193.
A transmission electron micrograph of a negatively stained cell of strain GMD509T is available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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-subclass of the Proteobacteria and was first proposed by Beijerinck (1889)
. Members of this genus are Gram-negative, straight, plump rods with one to three polar flagella (although some are not motile), and they are facultatively anaerobic and isolated from the marine environment. They cannot utilize the exogenous monomer 
-hydroxybutyrate. Presently, the genus includes 11 species with validly published names: Photobacterium leiognathi (Boisvert et al., 1967), P. phosphoreum (Reichelt & Baumann, 1973), Photobacterium fischeri (Beijerinck, 1889; Reichelt & Baumann, 1973), Photobacterium angustum (Reichelt et al., 1976), Photobacterium damselae subsp. damselae (Smith et al., 1991), Photobacterium damselae subsp. piscicida (Gauthier et al., 1995), Photobacterium iliopiscarium (Onarheim et al., 1994), Photobacterium profundum (Nogi et al., 1998), Photobacterium indicum (Xie & Yokota, 2004), Photobacterium rosenbergii (Thompson et al., 2005), Photobacterium lipolyticum (Yoon et al., 2005) and Photobacterium frigidiphilum (Seo et al., 2005). Among them, P. profundum (Nogi et al., 1998) and P. frigidiphilum were isolated from deep-sea sediments and P. damselae subsp. damselae, P. iliopiscarium and P. leiognathi were isolated from marine animals, i.e. skin ulcers of damselfish (Smith et al., 1991), intestine of herring (Onarheim et al., 1994) and the light organ of teleostean fish (Boisvert et al., 1967), respectively. In this article, we describe the morphological, phenotypic, phylogenetic and genomic characteristics of strain GMD509T, isolated from eggs of the sea hare Aplysia kurodai. Based on this polyphasic evidence, it is proposed that GMD509T be assigned as the type strain of the novel species Photobacterium aplysiae sp. nov.
Eggs of the sea hare Aplysia kurodai were collected in August 2003 by diving at Mogiyeo (depth, 12 m; 34° 04' 35'' N, 127° 15' 18'' E), an uninhabited small island in the South Sea of Korea. Immediately after sampling, a 1 g sample of egg was ground in a mortar containing 3 ml aged sea water that was autoclaved at 121 °C for 20 min. An aliquot (100 µl) was diluted serially (10–1, 10–2 and 10–3) in sterile-filtered (0·2 µm) or autoclaved aged sea water. Diluted sample (100 µl) was spread onto plates of marine 2216E agar (MA; Difco) supplemented with 1 % (v/v) tributyrin (TBN). The plates were then incubated at 10 °C for 7 days. Individual colonies with a halo were isolated from the TBN plates. The process was repeated on MA until pure cultures were obtained. Strain GMD509T was selected as a lipase-producing bacterium and studied further.
Gram staining was performed and growth characteristics were determined as described previously (Bae et al., 2005) and growth potential under anaerobic conditions was tested as described by Sohn et al. (2004). Cell morphology and flagellum type were observed under a transmission electron microscope at x12 000, with cells grown for 2 days at 25 °C on MA (Seo et al., 2005). Susceptibility to vibriostatic agent 2,4-diamino-6,7-diisopropylpteridine (O/129; Sigma) was determined by the disc-diffusion method (Seo et al., 2005). The isolate is Gram-negative, rod-shaped, motile by means of a single polar flagellum, facultatively anaerobic, approximately 0·5–0·8 µm in width by 1·0–4·0 µm in length (see Supplementary Figure, available in IJSEM Online) and susceptible to vibriostatic agent O/129. Colonies grown for 2 days at 25 °C on MA are cream-coloured and opaque with a smooth surface, circular form and convex elevation, with entire margins, and 3·0–4·0 mm in diameter. Strain GMD509T can grow between 10 and 31 °C, at pH 4–9 and between 1 and 5 % NaCl (w/v). Optimal growth occurs at 25 °C, pH 8 and in the presence of 3 % (w/v) NaCl.
Results of the physiological characterization and chemotaxonomic analyses are given in the species description with methods as described previously (Seo et al., 2005), except that the concentration of NaCl was 3 % (w/v) instead of 2 % in the modified saline solution used as the bacterial suspension solution and cells were incubated in marine 2216E broth (MB; Difco) at 25 °C. The major fatty acids of strain GMD509T are similar to those reported for other Photobacterium species (Nogi et al., 1998). The phenotype of strain GMD509T, including characters for differentiating it from all other all Photobacterium species, is indicated in Table 1. The most clear differentiating characters between strain GMD509T and its closest phylogenetic neighbours, P. frigidiphilum, P. indicum and P. profundum, are indole production and cellobiose utilization, as well as optimal temperature for growth, catalase and oxidase activities and the utilization of N-acetyl-D-glucosamine, D-fructose, D-galactose, sucrose and D-mannose.
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. Unmethylated lambda phage DNA (Sigma) was used as the calibration reference. The DNA G+C content of strain GMD509T was 45 mol%, which is within the accepted range for the genus Photobacterium (38–48 mol %).
The 16S rRNA gene was analysed as described previously (Seo et al., 2005). The 16S rRNA gene sequence (1504 bp) of strain GMD509T was aligned manually with representative sequences of the genus Photobacterium and related taxa by using known 16S rRNA secondary-structure information. Phylogenetic trees were inferred by the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1993) and maximum-parsimony (Fitch, 1971) methods. Evolutionary-distance matrices (for the neighbour-joining method) were generated according to the model of Jukes & Cantor (1969). The trees were rooted by using Escherichia coli (GenBank accession no. X80725) as an outgroup. The PHYLIP package (Felsenstein, 1993) was used for all analyses. The resultant unrooted tree topology was evaluated by bootstrap analyses (1000 replicates; Felsenstein, 1985) using the neighbour-joining method.
Sequence-similarity analysis indicated that the closest relatives of strain GMD509T were P. frigidiphilum (97·8 %), P. profundum (97·5 %) and P. indicum (97·4 %) (Fig. 1). Sequence similarities to all other species included in the phylogenetic analyses were <97·0 %.
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. The resultant estimates of DNA relatedness between the isolate and the type strains of the closely related species P. frigidiphilum and P. indicum were 25·3 and 13·7 %, respectively, which is significantly lower than that accepted as the genotypic definition of a species (Wayne et al., 1987).
Description of Photobacterium aplysiae sp. nov.
Photobacterium aplysiae (ap.ly'si.ae. N.L. gen. fem. n. aplysiae of Aplysia, a zoological genus, referring to the isolation of the type strain from a sea hare, genus Aplysia).
Cells are rod-shaped, 0·5–0·8 µm in width and 1·0–4·0 µm in length, motile by means of a polar flagellum and Gram-negative. Cream-coloured, opaque, smooth, circular, convex colonies with entire margins are formed after 2 days on MA at 25 °C. Facultatively anaerobic. Susceptible to vibriostatic agent O/129. Mesophilic. Growth occurs between 10 and 31 °C, with an optimum at 25 °C. The pH range for growth is 4–9, with an optimum at pH 8. Obligate requirement for NaCl for growth; growth occurs at concentrations of 1–5 % (w/v) and is optimal at 3 % (w/v). Catalase, oxidase, arginine dihydrolase, -galactosidase, 
-glucosidase, alkaline phosphatase, esterase/lipase, lipase, leucine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities and hydrolysis of gelatin are positive. Reduction of nitrate to nitrite occurs and acid is produced from glucose. Does not utilize -hydroxybutyrate. The following carbon sources are utilized: dextrin, glycogen, N-acetyl-D-glucosamine, cellobiose, D-fructose, maltose, D-mannitol, D-mannose, sucrose, D-trehalose, DL-lactic acid, L-alanine, methyl pyruvate, glycyl L-aspartic acid, L-serine, L-threonine, L-alanyl glycine, glutamic acid, inosine, uridine, glycerol and glucose 6-phosphate. Tweens 40 and 80, D-galactose, D-psicose, D-gluconic acid, -ketoglutaric acid, succinic acid, glycyl L-glutamic acid and asparagine are utilized weakly. The major fatty acids are palmitoleic acid (C16 : 1), palmitic acid (C16 : 0) and oleic acid (C18 : 1). The DNA G+C content is 45 mol%.
The type strain, GMD509T (=KCTC 12383T=JCM 12948T), was isolated from the eggs of a sea hare (Aplysia kurodai) collected in August 2003 at Mogiyeo (depth, 12 m), an uninhabited small island in the South Sea of Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Beijerinck, M. W. (1889). Le Photobacterium luminosum. Bactérie lumineuse de la Mer du Nord. Arch Neerl Sci Exactes Nat 23, 401–427.
Boisvert, H., Chatelain, R. & Bassot, J.-M. (1967). Étude d'un Photobacterium isolé de l'organe lumineux de poissons Leiognathidae. Ann Inst Pasteur (Paris) 112, 520–524.
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Smith, S. K., Sutton, D. C., Fuerst, J. A. & Reichelt, J. L. (1991). Evaluation of the genus Listonella and reassignment of Listonella damsela (Love et al.) MacDonell and Colwell to the genus Photobacterium as Photobacterium damsela comb. nov. with an emended description. Int J Syst Bacteriol 41, 529–534.
Sohn, J. H., Lee, J.-H., Yi, H., Chun, J., Bae, K. S., Ahn, T.-Y. & Kim, S.-J. (2004). Kordia algicida gen. nov., sp. nov., an algicidal bacterium isolated from red tide. Int J Syst Evol Microbiol 54, 675–680.
Thompson, F. L., Thompson, C. C., Naser, S., Hoste, B., Vandemeulebroecke, K., Munn, C., Bourne, D. & Swings, J. (2005). Photobacterium rosenbergii sp. nov. and Enterovibrio coralii sp. nov., vibrios associated with coral bleaching. Int J Syst Evol Microbiol 55, 913–917.
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Xie, C.-H. & Yokota, A. (2004). Transfer of Hyphomicrobium indicum to the genus Photobacterium as Photobacterium indicum comb. nov. Int J Syst Evol Microbiol 54, 2113–2116.
Yoon, J.-H., Lee, J.-K., Kim, Y.-O. & Oh, T.-K. (2005). Photobacterium lipolyticum sp. nov., a bacterium with lipolytic activity isolated from the Yellow Sea in Korea. Int J Syst Evol Microbiol 55, 335–339.
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