Microbulbifer maritimus sp. nov., isolated from an intertidal sediment from the Yellow Sea, Korea

The genus Microbulbifer was proposed by González et al. (1997) for a novel rod-shaped and strictly aerobic marine bacterium that belonged to the Proteobacteria. A single species, Microbulbifer hydrolyticus, was included within this genus. The second species of the genus Microbulbifer was Microbulbifer salipaludis, which was proposed recently by Yoon et al. (2003b). Phylogenetic analyses based on 16S rDNA sequences have shown that the two Microbulbifer species fall within the γ-subclass of the Proteobacteria (González et al., 1997; Solano & Sanchez-Amat, 1999; Anzai et al., 2000; Yoon et al., 2003b). Chemical markers of this genus include the presence of a major amount of iso-C_15 : 0 and ubiquinone with eight isoprene units (Q-8) (González et al., 1997; Yoon et al., 2003b). The DNA G+C content of the genus is 58–59 mol% (González et al., 1997; Yoon et al., 2003b). Pigmentation of the colonies of these two Microbulbifer species is cream or greyish-yellow. Recently, Pseudomonas elongata was transferred to the genus Microbulbifer as Microbulbifer elongatus (Yoon et al., 2003a). The colour of M. elongatus was yellowish-brown, unlike the first two Microbulbifer species that were described. In the course of screening taxonomically useful micro-organisms that were present in an intertidal sediment from the Yellow Sea (Korea), a yellowish brown-coloured, slightly halophilic bacterial strain (TF-17^T) was isolated and characterized taxonomically. The result of 16S rDNA sequence comparison indicated that this isolate is related phylogenetically to members of the genus Microbulbifer. Accordingly, the aim of this work was to establish the exact taxonomic position of strain TF-17^T by polyphasic characterization, which included phenotypic properties, detailed phylogenetic analysis based on 16S rDNA sequences and genomic relatedness.

Strain TF-17^T was isolated by the dilution-plating technique on marine agar 2216 (MA; Difco). Cell biomass of strain TF-17^T for respiratory lipoquinone analysis and for DNA extraction was obtained from cultures in marine broth 2216 (MB; Difco) at 37 °C. For fatty acid methyl ester analysis, cell mass of strain TF-17^T was obtained from agar plates after incubation for 3 days at 37 °C on MA. Cell morphology was examined by light microscopy and transmission electron microscopy (TEM). Flagellum type was examined by TEM using cells from an exponentially growing culture. Gram-reaction was determined by using a Gram Stain kit (bioMérieux) according to the manufacturer's instructions. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber with anaerobically prepared MA. Growth in the absence of NaCl was investigated in trypticase soy broth to which NaCl was not added. Growth at various NaCl concentrations (0·5–15 %) was investigated in MB or in trypticase soy broth. Growth at various temperatures (4–55 °C) was measured on MA. Catalase activity was determined by bubble production in 3 % (v/v) H₂O₂ solution. Oxidase activity was determined by oxidation of 1 % p-aminodimethylaniline oxalate. Hydrolysis of gelatin and aesculin and nitrate reduction were determined as described by Lanyi (1987), with the modification that artificial sea water was used. Artificial sea water contained [(l distilled water)⁻¹]: 23·6 g NaCl; 0·64 g KCl; 4·53 g MgCl₂.6H₂O; 5·94 g MgSO₄.7H₂O; 1·3 g CaCl₂.2H₂O (Levring, 1946). Hydrolysis of casein and starch was determined as described by Cowan & Steel (1965). Hydrolysis of hypoxanthine, tyrosine, xanthine and Tween 80 was tested on MA plates by using substrate concentrations that were described by Cowan & Steel (1965). Hydrolysis of chitin (Sigma) and birchwood xylan (Sigma) was determined on solid and in liquid marine salts basal medium (Baumann & Baumann, 1981) that contained 0·5 % chitin or xylan as the sole carbon source, respectively. M. hydrolyticus DSM 11525^T was used as positive control for hydrolysis tests of chitin and xylan. Additional enzyme activity was determined by using the API ZYM system (bioMérieux). Acid production from carbohydrates was determined as described by Leifson (1963). Utilization of various substrates for growth was determined as described by Yurkov et al. (1994).

Chromosomal DNA was isolated and purified according to a method described previously (Yoon et al., 1996), except that ribonuclease T1 was used together with ribonuclease A. Respiratory lipoquinones were analysed as described by Komagata & Suzuki (1987), using reversed-phase HPLC. For quantitative analysis of cellular fatty acid compositions, a loop of cell mass was harvested and fatty acid methyl esters were prepared and identified by following the instructions of the Microbial Identification system (MIDI). DNA G+C content was determined by the method of Tamaoka & Komagata (1984). DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC.

16S rDNA was amplified by PCR using two universal primers (Yoon et al., 1998). The PCR product was purified with a QIAquick PCR purification kit (Qiagen). Sequencing of the purified 16S rDNA was performed by using an ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) as recommended by the manufacturer. The purified sequencing reaction mixtures were electrophoresed automatically on an Applied Biosystems model 377 automatic DNA sequencer. Alignment of sequences was carried out with clustal w software (Thompson et al., 1994). Gaps at the 5′ and 3′ ends of the alignment were omitted from further analysis. Phylogenetic trees were inferred by using three tree-making algorithms: the neighbour-joining (Saitou & Nei, 1987), maximum-likelihood (Felsenstein, 1981) and maximum-parsimony (Kluge & Farris, 1969) methods, implemented within the phylip package (Felsenstein, 1993). Evolutionary distance matrices for the neighbour-joining method were calculated according to Jukes & Cantor (1969) by using the program dnadist in the phylip package. Stability of relationships was assessed by a bootstrap analysis based on 1000 resamplings of the neighbour-joining dataset, using the programs seqboot, dnadist, neighbor and consense of the phylip package. DNA–DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989), using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample; the highest and lowest values obtained for each sample were excluded and the remaining three values were used to calculate similarity values. DNA–DNA relatedness values quoted are the means of these three values.

Cells of strain TF-17^T were rods that measured approximately 0·3–0·5 μm in width and 3·0–6·0 μm in length after cultivation for 3 days at 37 °C on MA. Strain TF-17^T had no flagella. Colonies on MA were yellowish-brown in colour, smooth, irregular, slightly raised and 2·0–4·0 mm in diameter after 3 days incubation at 37 °C on MA. Strain TF-17^T grew optimally at 37 °C, pH 6·5–7·5 and in the presence of 2–4 % (w/v) NaCl. Strain TF-17^T did not grow under anaerobic conditions on MA. Phenotypic properties of strain TF-17^T are shown in Table 1⇓ or are given in the species description (see below). As shown in Table 1⇓, there are differences between TF-17^T and the other Microbulbifer species in some phenotypic characteristics.

The predominant respiratory lipoquinone found in strain TF-17^T was unsaturated ubiquinone with eight isoprene units (Q-8), at a peak area ratio of approximately 95 %. Strain TF-17^T had a cellular fatty acid profile that contained major amounts of straight-chain, branched and hydroxy fatty acids; it contained iso-C_15 : 0 as the major fatty acid and also significant amounts of iso-C_11 : 0 3-OH and iso-C_17 : 1ω9c (Table 2⇓). This profile was similar to those of the type strains of M. hydrolyticus, M. salipaludis and M. elongatus, although there are differences in the proportions of some fatty acids, such as iso-C_11 : 0, iso-C_11 : 0 3-OH and C_18 : 1ω7c. The DNA G+C content of strain TF-17^T was 59·9 mol%.

An almost-complete 16S rDNA sequence of 1490 nt (approximately 96 % of the Escherichia coli 16S rRNA gene sequence) was determined for strain TF-17^T. Strain TF-17^T was found to have highest 16S rDNA sequence similarity to members of the γ-subclass of the Proteobacteria and, in particular, to the genus Microbulbifer. In the phylogenetic tree based on the neighbour-joining algorithm, strain TF-17^T clustered with the type strains of M. hydrolyticus, M. salipaludis and M. elongatus (Fig. 1⇓). The relationship between strain TF-17^T and the clade comprising the type strains of the three Microbulbifer species was supported by bootstrap analysis at a confidence level of 100 %. Similar tree topologies were generated by the maximum-parsimony method (data not shown). Strain TF-17^T showed 16S rDNA sequence similarity levels of 95·1, 95·7 and 95·3 % to M. hydrolyticus ATCC 700072^T, M. salipaludis KCCM 41586^T and M. elongatus DSM 6810^T, respectively. Sequence similarities to all other taxa included in the phylogenetic analysis were <90·5 % (Fig. 1⇓). DNA–DNA hybridization was performed to determine genotypic relatedness between strain TF-17^T and the type strains of the three Microbulbifer species. Strain TF-17^T exhibited DNA–DNA relatedness levels of 11·5, 10·0 and 13·0 % to M. hydrolyticus DSM 11525^T, M. salipaludis KCCM 41586^T and M. elongatus DSM 6810^T, respectively.

The results of 16S rDNA sequence analysis revealed that strain TF-17^T has the closest phylogenetic affiliation to the genus Microbulbifer, confirmed by a high bootstrap resampling value. Colonies of strain TF-17^T are similar to those of M. elongatus in colour, whereas they are distinguishable from colonies of M. hydrolyticus and M. salipaludis in colour. The results obtained in chemotaxonomic analyses are most similar to the chemotaxonomic properties that are characteristic of the genus Microbulbifer (Yoon et al., 2003a, b). In particular, the predominant respiratory lipoquinone type and cellular fatty acid profile are useful for distinguishing strain TF-17^T and other members of the genus Microbulbifer from some other related genera. Strain TF-17^T and other members of the genus Microbulbifer contain Q-8 as the predominant respiratory lipoquinone (Yoon et al., 2003a, b), whereas members of the genus Marinobacter and authentic pseudomonads have been known to contain Q-9 as the predominant respiratory lipoquinone (Oyaizu & Komagata, 1983; Yumoto et al., 2001; Yoon et al., 2003a). Strain TF-17^T can be distinguished from members of the genus Alcanivorax by cellular fatty acid profiles: strain TF-17^T and other members of the genus Microbulbifer contain iso-C_15 : 0 as the major fatty acid, whereas members of the genus Alcanivorax contain C_18 : 1ω7c and C_16 : 0 as the major fatty acids (Fernández-Martínez et al., 2003). Therefore, both phylogenetic and chemotaxonomic results indicate clearly that strain TF-17^T belongs to the genus Microbulbifer.

Although strain TF-17^T is similar to M. elongatus in its morphological properties, it is differentiated from M. elongatus in some physiological properties (Table 1⇑). There are some noteworthy differences between strain TF-17^T and the other two Microbulbifer species in morphological, physiological and biochemical properties, including colour of colonies, maximum growth temperature, ability to hydrolyse some substrates and acid production from carbohydrates (Table 1⇑). Levels of 16S rDNA similarity between strain TF-17^T and the type strains of Microbulbifer species (95·1–95·7 %) are low enough to exclude the possibility of assigning strain TF-17^T to a previously described Microbulbifer species (Stackebrandt & Goebel, 1994). In addition, DNA–DNA relatedness levels justify that strain TF-17^T is separate from the three Microbulbifer species with validly published names (Wayne et al., 1987). Therefore, on the basis of phenotypic, chemotaxonomic and phylogenetic data and genotypic distinctiveness, strain TF-17^T should be placed in the genus Microbulbifer as the type strain of a novel species, for which we propose the name Microbulbifer maritimus sp. nov.