Photobacterium aplysiae sp. nov., a lipolytic marine bacterium isolated from eggs of the sea hare Aplysia kurodai

A bacterium, named GMD509^T, showing lipolytic activity was isolated from the eggs of the sea hare Aplysia kurodai collected at Mogiyeo (depth, 12 m), an uninhabited small island in the South Sea of Korea. The strain is Gram-negative, motile, facultatively anaerobic, mesophilic and weakly halophilic. Optimal growth of strain GMD509^T occurs in the presence of 3·0 % (w/v) NaCl and at pH 8 and 25 °C. The whole-cell fatty acid profile of the isolate includes C16 : 1, C16 : 0 and C18 : 1 as major fatty acids and its DNA G+C content is 45 mol%. Phylogenetic analyses of 16S rRNA gene sequences place this bacterium in the γ-Proteobacteria, within the genus Photobacterium. The 16S rRNA gene sequence of strain GMD509^T is most similar to those of Photobacterium frigidiphilum (97·8 %), Photobacterium profundum (97·5 %) and Photobacterium indicum (97·4 %). DNADNA relatedness levels between the isolate and its closest known phylogenetic relatives, P. frigidiphilum and P. indicum, are 25·3 and 13·7 %, respectively. Strain GMD509^T therefore represents a novel species, for which the name Photobacterium aplysiae sp. nov. is proposed, with the type strain GMD509^T (=KCTC 12383^T=JCM 12948^T).

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Published online ahead of print on 10 June 2005 as DOI 10.1099/ijs.0.63765-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GMD509^T is AY781193.

A transmission electron micrograph of a negatively stained cell of strain GMD509^T is available as supplementary material in IJSEM Online.

The genus Photobacterium, with Photobacterium phosphoreum as the type species, belongs to the γ-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 β-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 GMD509^T, isolated from eggs of the sea hare Aplysia kurodai. Based on this polyphasic evidence, it is proposed that GMD509^T 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 (10¹, 10² and 10³) 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 GMD509^T 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 0·50·8 µm in width by 1·04·0 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 3·04·0 mm in diameter. Strain GMD509^T can grow between 10 and 31 °C, at pH 49 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 GMD509^T are similar to those reported for other Photobacterium species (Nogi et al., 1998). The phenotype of strain GMD509^T, including characters for differentiating it from all other all Photobacterium species, is indicated in Table 1. The most clear differentiating characters between strain GMD509^T 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.

Table 1. Differential phenotypic characteristics of strain GMD509T and related Photobacterium species Taxa: 1, GMD509T; 2, P. frigidiphilum KCTC 12384T [data from Seo et al. (2005)]; 3, P. indicum NBRC 14233T [data from the present study and Xie & Yokota (2004)]; 4, P. profundum JCM 10084T [data from Nogi et al. (1998) and Seo et al. (2005)]; 5, P. lipolyticum KCTC 10562BPT [data from Yoon et al. (2005)]; 6, P. angustum ATCC 25915T [data from Nogi et al. (1998) and Seo et al. (2005)]; 7, P. phosphoreum ATCC 11040T [data from Nogi et al. (1998) and Seo et al. (2005)]; 8, P. iliopiscarium ATCC 51760T [data from Onarheim et al. (1994)]; 9, P. damselae subsp. damselae JCM 8968 [data from Nogi et al. (1998)]; 10, P. damselae subsp. piscicida NCIMB 25918 [data from Gauthier et al. (1995)]; 11. P. leiognathi ATCC 25521T [data from Nogi et al. (1998)]; 12, P. rosenbergii LMG 22223T [data from Thompson et al. (2005)]. +, Positive; , negative; V, variable; W, weak; ND, not determined. All species were positive for utilization of α-D-glucose. All species were negative for Gram stain, nitrite reduction to nitrogen and utilization of L-arabinose and α-D-lactose.

The genomic DNA G+C content was determined by using the HPLC method as described by Mesbah et al. (1989). Unmethylated lambda phage DNA (Sigma) was used as the calibration reference. The DNA G+C content of strain GMD509^T was 45 mol%, which is within the accepted range for the genus Photobacterium (3848 mol %).

The 16S rRNA gene was analysed as described previously (Seo et al., 2005). The 16S rRNA gene sequence (1504 bp) of strain GMD509^T 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 GMD509^T 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 %.

(31K): Fig. 1. Neighbour-joining tree showing the phylogenetic positions of strain GMD509T and related taxa, based on 16S rRNA gene sequences. Numbers at nodes represent the levels of bootstrap support based on neighbour-joining analyses of 1000 resampled datasets; only values >70 % are given. Solid circles represent clades that were also recovered in the maximum-parsimony and maximum-likelihood trees. Bar, 0·01 substitutions per site.

DNADNA hybridization was performed according to the method of Ezaki et al. (1989). 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, 0·50·8 µm in width and 1·04·0 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 49, with an optimum at pH 8. Obligate requirement for NaCl for growth; growth occurs at concentrations of 15 % (w/v) and is optimal at 3 % (w/v). Catalase, oxidase, arginine dihydrolase, β-galactosidase, α-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 β-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, α-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, GMD509^T (=KCTC 12383^T=JCM 12948^T), 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.