Coraliomargarita akajimensis gen. nov., sp. nov., a novel member of the phylum ‘Verrucomicrobia’ isolated from seawater in Japan

Molecular phylogenetic approaches based on 16S rRNA gene sequence analysis have revealed that members of the phylum ‘Verrucomicrobia’ (Hedlund et al., 1997; Hugenholtz et al., 1998), a phylum represented by very few cultivated strains, are a globally distributed, abundant group of bacteria that are increasingly recognized as being of environmental significance (Sangwan et al., 2005). The phylum ‘Verrucomicrobia’ has been informally classified into subdivisions 1–5 by Hugenholtz et al. (1998), and has also been classified into subdivisions 1–6 by Vandekerckhove et al. (2000). At the time of writing, only three of these subdivisions are recognized by Bergey's Manual of Systematic Bacteriology (Garrity & Holt, 2001), all three corresponding to the rank of family: Verrucomicrobiaceae (subdivision 1), Opitutaceae (subdivision 4) and ‘Xiphinematobacteriaceae’ (subdivision 2) (Sangwan et al., 2005). Subdivision 4 comprises just a few recognized representatives, such as Opitutus terrae PB90-1^T and related strains (Janssen et al., 1997; Chin et al., 1999, 2001), which have been isolated from soil environments, and the marine bacteria ‘Fucophilus fucoidanolyticus’ SI-1234 (Sakai et al., 2003) and Alterococcus agarolyticus ADT3^T (Shieh & Jean, 1998), which were isolated from thermal springs. A. agarolyticus was misclassified as a member of the class Gammaproteobacteria (Sangwan et al., 2004).

A specimen of the hard coral Galaxea fascicularis L. was collected from 3–5 m below the surface of the sea at Majanohama, Akajima, Japan, in March 2004 together with the seawater around the coral, and these were kept in a 500-ml plastic bottle for 2–5 h. Strain 04OKA010-24^T was isolated from the seawater in the sample bottle. The seawater was diluted 1 : 10 with filtered, autoclaved seawater and used for isolation on 1 : 10 diluted marine agar [3.74 g marine broth 2216 (Difco), 750 ml filtered seawater, 250 ml distilled water, 15 g agar]. The phylogenetic position of strain 04OKA010-24^T was investigated by using a polyphasic taxonomic approach including 16S rRNA gene sequence analysis, together with physiological, biochemical and chemotaxonomic analyses. Based on these data, it is proposed that the isolate represents a novel species of a new genus in the phylum ‘Verrucomicrobia’.

The temperature range and pH range for growth were determined by incubating the isolate in marine agar 2216 (MA; Difco). The following buffers were used for pH tests in MA: MES (pH 5.5), ACES (pH 6.5 and 7.0), TAPSO (pH 7.6), TAPS (pH 8.5) and CHES (pH 9.0 and 9.5). Gram staining was performed as described by Murray et al. (1994). Cell morphology was observed by using light microscopy (BX60; Olympus) and transmission electron microscopy (TEM). For TEM observation, cells were mounted on Formvar-coated copper grids and negatively stained with 2 % (w/v) aqueous uranyl acetate. Grids were observed in an H-7000 transmission electron microscope (Hitachi) operated at 75 kV. In the course of TEM, cells of various sizes were observed. Cells were coccoid and generally 0.5–1.2 μm in diameter. In the stationary growth phase, smaller cells (0.5–0.6 μm in diameter) were predominantly observed. The cells were non-motile and no flagella were seen by electron microscopy (Fig. 1⇓). Growth under anaerobic conditions was determined after 2 weeks incubation in an AnaeroPack (Mitsubishi Gas Chemical Co., Inc.) on MA. Catalase activity was determined by bubble formation in a 3 % H₂O₂ solution. Oxidase activity was determined by use of cytochrome oxidase paper (Eiken Chemical Co., Ltd). API 20E and API 50CH strips (bioMérieux) were used to determine the physiological and biochemical characteristics of strain 04OKA010-24^T. These were read after incubation at 30 °C for 48 and 72 h, respectively. Determination of the respiratory quinone system and cellular fatty acid composition was carried out as described by Katsuta et al. (2005). DNA was prepared by using Genomic tips (Qiagen) from cells grown in DSM medium no. 607, and the DNA base composition was determined by using the HPLC method of Mesbah et al. (1989). Cell walls were prepared by the methods described by Schleifer & Kandler (1972), and amino acids in an acid hydrolysate of the cell walls were identified by TLC (Harper & Davis, 1979) and by HPLC, as their phenylthiocarbamoyl derivatives, with a model LC-10AD HPLC apparatus (Shimazu) equipped with a Wakopak WS-PTC column (Wako Pure Chemical Industries) (Yokota et al., 1993). An approximately 1500 bp fragment of the 16S rRNA gene was amplified from the extracted DNA by using bacterial universal primers 27F and 1492R (Weisburg et al., 1991) specific to the 16S rRNA gene. To ascertain the phylogenetic position of the new isolate, the 16S rRNA gene sequence of strain 04OKA010-24^T was compared with sequences obtained from GenBank (National Center for Biotechnology Information; ). Multiple alignments of the sequences were performed by using clustal_x (version 1.83) (Thompson et al., 1997). Alignment gaps and ambiguous bases were not taken into consideration when 1213 bases of the 16S rRNA gene nucleotides were compared. Phylogenetic relationships were analysed by using the same software. Distances were calculated by using the two-parameter model of Kimura (1980). Clustering with the neighbour-joining method (Saitou & Nei, 1987) was determined by using bootstrap values based on 1000 replications (Felsenstein, 1985). Sequence similarity values were calculated via mega3 (Kumar et al., 2004).

On the basis of 16S rRNA gene sequence analysis, strain 04OKA010-24^T was found to be related most closely to the as yet undescribed marine bacterium ‘Fucophilus fucoidanolyticus’ SI-1234 (94.8 % similarity). The most closely related type strains of species with validly published names were Alterococcus agarolyticus ADT3^T (87.6 % similarity) and Opitutus terrae PB90-1^T (83.9 %). Based on levels of 16S rRNA gene sequence divergence, strain 04OKA010-24^T is thus determined to be related to the genera Alterococcus and Opitutus, which are members of ‘Verrucomicrobia’ subdivision 4. These results indicate that strain 04OKA010-24^T belongs to the phylum ‘Verrucomicrobia’ within subdivision 4 (Fig. 2⇓).

Strain 04OKA010-24^T also showed distinct phenotypic features that discriminated it from recognized members of ‘Verrucomicrobia’ subdivision 4 (Table 1⇓): cells lacked flagella and were non-motile (Fig. 1⇑).

Biochemical and physiological characteristics of strain 04OKA010-24^T further supported the phylogenetic results. The major quinone system was menaquinone MK-7. The G+C content of the DNA of strain 04OKA010-24^T was 53.9 mol%. Major fatty acids of strain 04OKA010-24^T were C_14 : 0 (24.2 %), C_18 : 1ω9c (23.5 %) and C_18 : 0 (15.6 %). The cell wall was prepared by disrupting cells, followed by heating with 3 % SDS, washing and centrifugation. Amino acid analysis of the cell-wall hydrolysate indicated the absence of muramic acid and diaminopimelic acid, suggesting that the cell wall did not contain peptidoglycan.

Strain 04OKA010-24^T was obligately aerobic, was isolated from seawater and was able to tolerate 5 % NaCl; by contrast, the other members of ‘Verrucomicrobia’ subdivision 4, originating from rice paddy soil and hot springs, could only tolerate up to 1–3 % NaCl. Strain 04OKA010-24^T could also be distinguished on the basis of the following characteristics: catalase, oxidase, nitrate reduction, temperature range for growth and hydrolysis of DNA and urea. Some differential characteristics were noted from the API 50CH results, including acid production from galactose, mannitol and mannose.

Based on the results of the phylogenetic analysis and its biochemical and physiological properties, strain 04OKA010-24^T was considered to represent a novel species in a new genus belonging to subdivision 4 of the phylum ‘Verrucomicrobia’. We propose the name Coraliomargarita akajimensis gen. nov., sp. nov. for this organism.