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1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-0032, Japan
2 Marine Biotechnology Institute Co. Ltd, 3-75-1, Heita, Kamaishi, Iwate 026-0001, Japan
Correspondence
Jaewoo Yoon
aa57058{at}mail.ecc.u-tokyo.ac.jp
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ABSTRACT |
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9c and C18 : 0 were the major fatty acids. On the basis of these data, it was concluded that strain 04OKA010-24T represents a novel species in a new genus in subdivision 4 of the phylum ‘Verrucomicrobia’, for which the name Coraliomargarita akajimensis gen. nov., sp. nov. is proposed. The type strain of Coraliomargarita akajimensis is 04OKA010-24T (=MBIC06463T=IAM 15411T=KCTC 12865T).
Present address: Okinawa Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST. Okinawa Health Biotechnology Research Development Center, 12-75 Suzaki, Uruma Okinawa 904-2234, Japan. 
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; 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 PB90-1T 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 ADT3T (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 04OKA010-24T 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 04OKA010-24T 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 04OKA010-24T. 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 04OKA010-24T was compared with sequences obtained from GenBank (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov). 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).
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): cells lacked flagella and were non-motile (Fig. 1
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9c (23.5 %) and C18 : 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 04OKA010-24T 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 04OKA010-24T 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 04OKA010-24T 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.
Description of Coraliomargarita gen. nov.
Coraliomargarita (Co.ra'li.o.mar.ga.ri'ta. Gr. n. koralion coral; L. fem. n. margarita a pearl; N.L. fem. n. Coraliomargarita coral pearl, referring to a white-colony-forming, coccoid micro-organism isolated from seawater in a sample bottle of hard coral).
Cells are Gram-negative, obligately aerobic cocci. Cells lack flagella and are non-motile. No spores are formed. Catalase-negative, but oxidase-positive. Nitrate is not reduced. The major respiratory quinone is MK-7. The G+C content of the genomic DNA of the type strain of the type species is 53.9 mol%. Predominant cellular fatty acids are C14 : 0, C18 : 19c and C18 : 0. The type species is Coraliomargarita akajimensis.
Description of Coraliomargarita akajimensis sp. nov.
Coraliomargarita akajimensis (a.ka.ji.men'sis. N.L. fem. adj. akajimensis pertaining to Akajima, an island in Okinawa, from where the type strain was isolated).
Main characteristics are as given for the genus. In addition, cells are 0.5–1.2 µm in diameter. Neither cellular gliding movement nor swarming growth is observed. Colonies grown on half-strength R2A agar medium with 75 % artificial seawater are circular, convex and white. The optimum temperature for growth is 20–30 °C; no growth occurs at 4 or 45 °C. The pH range for growth is 7.0–9.0. NaCl is required for growth and can be tolerated at up to 5 % (w/v). Urea and DNA are hydrolysed but agar, casein, aesculin and gelatin are not. Reactions for acetoin, ONPG and tryptophan deaminase are positive, but reactions for citrate utilization, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, hydrogen sulfide and indole production are negative. Acid is produced from glycerol, galactose, fructose, mannose, mannitol, sorbitol, trehalose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol and 5-ketogluconate, but not from erythritol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, adonitol, methyl 
-D-xylopyranoside, glucose, sorbose, rhamnose, dulcitol, inositol, methyl 
-D-mannnopyranoside, methyl -D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, salicin, cellobiose, maltose, lactose, melibiose, sucrose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentiobiose, gluconate or 2-ketogluconate. The usual components of bacterial cell walls such as muramic acid and diaminopimelic acid are not detected. Major fatty acid components (>2.0 % of the total) are iso-C14 : 0 (8.2 %), C14 : 0 (24.2 %), anteiso-C15 : 0 (2.9 %), C16 : 0 (3.3 %), C18 : 19c (23.5 %), C18 : 0 (15.5 %), C19 : 0 (2.8 %) and C21 : 0 (6.9 %).
The type strain, 04OKA010-24T (=MBIC06463T=IAM 15411T=KCTC 12865T), was isolated from seawater in a sampling bottle of the hard coral Galaxea fascicularis L., collected at Majanohama, Akajima, Japan.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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