J. Gen. Appl. Microbiol., 53, 185–189 (2007)

Full Paper

Janibacter corallicola sp. nov., isolated from in Palau

Akiko Kageyama,1 Yoko Takahashi,1, * Mina Yasumoto-Hirose,2, † Hiroaki Kasai,2 Yoshikazu Shizuri,2 and Satoshi O¯ mura1, 3

1 Kitasato Institute for Life Sciences, Kitasato University, Minato-ku, Tokyo 108–8641, Japan 2 Marine Biotechnology Institute, Kamaishi, Iwate 026–0001, Japan 3 The Kitasato Institute, Minato-ku, Tokyo 108–8641, Japan

(Received February 6, 2007; Accepted March 20, 2007)

A novel Janibacter is described on the basis of phenotypic, chemotaxonomic and geno- typic data. Two bacterial strains were isolated in Palau, which were both Gram-positive, catalase- positive with meso-diaminopimelic acid as the diagnostic diamino acid of the peptido- glycan. The major menaquinone was MK-8(H4). Mycolic acids were not detected. The G C con- tent of the DNA was 70–71 mol%. Comparative 16S rDNA studies of the two isolated strains revealed that they both belonged to the genus Janibacter. DNA-DNA relatedness data revealed that 04PA2-Co5-61T and 02PA-Ca-009 belong to the same species, a new species of the genus Janibacter. From these results, Janibacter corallicola sp. nov. is proposed, with the type strain 04PA2-Co5-61T (=MBIC 08265T, DSM 18906T).

Key Words—; Janibacter corallicola sp. nov.; 16S rRNA;

Introduction Based on phenotypic and chemotaxonomic proper- ties, two strains we isolated from Palau were well The genus Janibacter was proposed by Martin et al. matched to the description of the genus Janibacter. (1997) with the type species Janibacter limosus. The The phenotypic and phylogenetic characteristics of genus Janibacter belongs to the family Intrasporan- these strains, coupled with genomic DNA-DNA relat- giaceae in the order Actinomycetales. There are cur- edness data, suggest that these strains belong to a rently four species in the genus Janibacter: Janibacter novel species, Janibacter corallicola sp. nov. limosus (Martin et al., 1997), Janibacter terrae (Yoon et al., 2000), Janibacter melonis (Yoon et al., 2004) Materials and Methods and Janibacter anophelis (Kämpfer et al., 2006). Jani- bacter brevis, originally described by Imamura et al. Bacterial strains and isolation. Strain 04PA2-Co5- (2000), has since been recognized as a heterotypic 61T was isolated from hard coral ( gemmifera) synonym of J. terrae (Lang et al., 2003). collected at the depth of 3–5 m in Angauru Coral Gar- den in Palau (GPS location: N 6°5333.0, E 134° * Address reprint requests to: Dr. Yoko Takahashi, Kitasato In- 821.4) in September 2004. The coral was cut into stitute for Life Sciences, Kitasato University, 5–9–1 Shirokane, small pieces (1 cm3), ground with a mortar and pestle, Minato-ku, Tokyo 108–8641, Japan. and transferred into 5 ml of sterile seawater. The Phone: 81–3–5791–6133 Fax: 81–3–5791–6133 homogenate was diluted to 1/100 with sterile seawater, E-mail: [email protected]. ac.jp then the bacterium was isolated from this suspension † Present address: Okinawa Prefecture Collaboration of Re- TM gional Entities for the Advancement of Technological Excellence, on 1/10 MA Ca medium [3.74 g of Difco Marine JST. Okinawa Health Biotechnology Research Development Broth 2216 (BD), 750 ml of filtrated seawater, and

Center, 12–75 Suzaki, Uruma, Okinawa 904–2234, Japan. 250 ml of distilled water with 1% CaCO3 containing 186 KAGEYAMA et al. Vol. 53

1.5% agar]. Colonies were picked after incubation at lar fatty acids were extracted from lyophilized cells, 25°C for about 1–3 weeks. Strain 02PA-Ca-009 was methylated and analyzed using a Shimadzu QP5050A isolated from polyurethane foam (PUF) blocks supple- gas chromatograph/mass spectrometer. Fatty acid mented with a seawater agar medium [filtered seawa- methyl ester (FAME) peaks were identified by retention ter containing 1.5% agar] which were fixed for 3 days time comparisons against authentic standards (BAME in the coral reef area at a depth of 5–6 m in the sea off Mix; Supelco). Further identification, including verifica- Palau (GPS location: N 7°204.5, E 134°2947.5) in tion of the double bond position, was performed using December 2002 (Yasumoto-Hirose et al., 2006). The the pyrrolidide derivatization method (Andersson and recovered PUF block was cut into small pieces Holman, 1974). (1 cm3), and homogenized with 5 ml of sterile seawater GC content of DNA and DNA-DNA hybridization. using a glass rod. The homogenate was diluted to DNA was isolated as described by Saito and Miura 1/1,000 with sterile seawater and then the bacterium (1963). The DNA base composition was estimated by was isolated from this mixture on MACa medium HPLC (Tamaoka and Komagata, 1984). DNA-DNA re- [37.4 g of DifcoTM Marine Broth 2216 (BD), 1 L of dis- latedness was determined by using the method of tilled water with 1% CaCO3 containing 1.5% agar]. Ezaki et al. (1989), using photobiotin and a microplate Colonies were picked after incubation at 25°C for format. about 1–3 weeks. Biomass for biochemical and chemo- 16S rDNA sequencing and phylogenetic analysis. taxonomic studies was prepared by culturing the DNA was prepared using InstaGene matrix (Bio-Rad, strains in tryptic soy broth (DifcoTM) at 27°C and har- CA, USA). 16S rDNA was amplified by PCR using vesting the cells using centrifugation. a forward primer corresponding to positions 8–27 Morphological and biochemical tests. Morphologi- and a reverse primer corresponding to 1492–1510 cal observations were carried out using a scanning (Escherichia coli numbering system; Weisburg et al., electron microscope (model JSM-5600; JEOL) and 1991) and sequenced using an automatic sequence cultures grown on GPM agar (1.0% glucose, 0.5% analyzer (Applied Biosystems 3730 DNA Analyzer; Ap- peptone, 0.5% meat extract, 0.3% NaCl and 1.2% plied Biosystems, CA, USA) with the BigDye® Termina- agar, pH 7.0) at 27°C for 3 days. For scanning electron tor v3.1 Cycle Sequencing Kit (Applied Biosystems). microscope inspections, the cultures were fixed in 4% Species related to the new strains were identified by osmium tetroxide vapor in situ at room temperature for performing sequence database searches using BLAST 18 h, and then dried at room temperature. Utilization of (Altschul et al., 1990). Sequence data for related various sole carbon sources was assessed using Prid- species were retrieved from GenBank. Phylogenetic ham-Gottlieb agar medium (Nihon Pharmaceutical analysis was performed using CLUSTAL W software Co., Ltd.) (Pridham and Gottlieb, 1948). NaCl toler- (Thompson et al., 1994). Nucleotide substitution rates ance, and pH and temperature ranges for growth were (Knuc values) were calculated (Kimura and Ohta, 1972) determined on 1/5 nutrient agar. The two isolates were and phylogenetic trees were constructed by using the characterized biochemically using API ZYM in accor- neighbor-joining method (Saitou and Nei, 1987). Se- dance with the manufacturer’s instructions. quence similarity values were determined by visual Chemotaxonomic tests. Purified cell walls were comparison and manual calculation. obtained using the method of Kawamoto et al. (1981). One milligram of purified cell wall was hydrolyzed at Results and Discussion 100°C with 1 ml of 6 M HCl for 16 h. The residue was dissolved in 100 ml of water and was used for amino In the course of an isolation program to obtain new acid analysis by thin layer chromatography (TLC). The microorganisms to produce useful bioactive com- presence of mycolic acid was examined using the TLC pounds, we have attempted to determine the taxo- method of Tomiyasu (1982). Menaquinones were ex- nomic positions of many isolates from marine environ- tracted and purified using the method of Collins et al. ments. Strains 04PA2-Co5-61T and 02PA-Ca-009 were (1977), and then analyzed by high performance liquid isolated from marine samples from Palau. Both strains chromatography (HPLC) (model 802-SC; Jasco) on a were Gram-positive, catalase positive cocci. chromatograph equipped with a Capcell Pak C18 col- Almost complete 16S rDNA gene sequences were umn (Shiseido Co., Ltd.) (Tamaoka et al., 1983). Cellu- determined for the two isolated strains. Subsequent 2007 Janibacter corallicola sp. nov. 187

Table 1. Fatty acid composition (%) of the isolated strains and Janibacter species.

1 23456

C15 : 0 —— — ——12.1

C17 : 0 11.3 14.5 16.4 — 17.8 13.4

C18 : 0 — 5.9 — — — —

C17 : 1 w8c 12.8 13.0 13.8 11.9 26.2 20.0

C18 : 1 w9c 7.8 9.4 — 13.3 17.5 7.6

iso-C15 : 0 —— — ——5.1

iso-C16 : 0 34.0 29.0 32.9 38.1 12.2 16.7

anteiso-C17 : 0 —— — 5.7 — —

10-methyl-C17 : 0 10.2 7.5 — 6.0 — —

1, Janibacter corallicola 04PA2-Co5-61T; 2, Janibacter coralli- cola 02PA-Ca-009; 3, Janibacter anophelis H2.16BT; 4, Jani- Fig. 1. Phylogenetic tree constructed on the basis of 16S bacter terrae DSM 13876T; 5, Janibacter limosus DSM 11140T; rDNA sequences using the neighbor-joining method. 6, Janibacter melonis CM2104T. Fatty acids constituting 5% of Numbers at branch points are bootstrap values (1,000 resam- the total are listed. Data for the type strains of J. anophelis, J. plings). The tree was unrooted and Ornithinicoccus hortensis terrae, J. limosus and J. melonis are from Kämpfer et al. (2006). was used as an outgroup. GenBank accession codes are given in parentheses. to same species, DNA-DNA hybridization relatedness was determined. The relatedness of the two isolates 16S rDNA-based phylogenetic analysis demonstrated was 92%, showing that the two isolates were the same that the strains belonged to the genus Janibacter. species. Figure 1 shows the relationship between the two iso- Based on the present results, we propose that the lated strains and their nearest phylogenetic relatives. two isolated strains represent one novel Janibacter Strains 04PA2-Co5-61T and 02PA-Ca-009 were closely species: Janibacter corallicola sp. nov. related to Janibacter anophelis CCUG 49715T with a high bootstrap value. Its percentage of the occurrence Description of Janibacter corallicola sp. nov. was 84% in 1,000 bootstrapped trees. The level of se- Janibacter corallicola (co.ral.li.co’la. L. n. corallum, quence similarity between the two isolates was 98.8%, coral; L. suff. -cola, inhabitant dweller; N.L. n. corrali- while that between the isolated strains and the other cola, coral-dweller). four Janibacter species ranged from 96.0% to 97.4%. Cells are coccus, varying in their cell size from 0.6 The DNA base composition of the two strains was to 1.1 mm (Fig. 2). Gram-positive, catalase positive, 70–71 mol% GC. The cell wall peptidoglycan of both aerobic. Colonies are pale yellow. Growth occurs be- isolates contained meso-diaminopimelic acid as the tween pH 5 and pH 9 (optimum growth occurs be- diagnostic diamino acid. The peptide subunit in both tween pH 6 and pH 8), and 16°C and 38°C (optimum strains consisted of alanine, glutamic acid, and meso- growth occurs between 23°C and 36°C). In 1/5 nutrient diaminopimelic acid. The major menaquinone was agar medium, NaCl is tolerated up to 7%. D-Fructose,

MK-8(H4). The major cellular fatty acid was iso-C16 : 0 D-galactose, D-glucose, maltose, D-mannose, raffinose (Table 1). and trehalose are assimilated, but L-arabinose, D-man- The chemotaxonomic and morphological character- nitol, sucrose and D-xylose are not (Fig. 2). Esterase istics (Tables 1 and 2) of these two isolated strains are (C4), esterase lipase (C8), leucine arylamidase, naph- consistent with their assignment to the genus Janibac- thol-AS-BI-phosphohydrolase, a-glucosidase, are de- ter (Martin et al., 1997). The range of phenotypic and tected by the API ZYM enzyme assay; alkaline phos- chemotaxonomic characteristics that distinguish the phatase, chymotrypsin, a-galactosidase, b-galactosi- isolated strains from the other four Janibacter species dase, b-glucuronidase, N-acetyl-b-glucosaminidase, a- is presented in Table 2. mannosidase and a-fucosidase are negative. Weak In order to assess whether these two strains belong reaction for lipase (C14), valine arylamidase, acid 188 KAGEYAMA et al. Vol. 53

Table 2. Differential characteristics of the two isolates and all species of the genus Janibacter.

Characteristic 1 2 3 4 5 6

Growth at 37°C w Color of colonies pale pale ND white, cream cream or cream yellow yellow or yellowish pale cream Morphology cocci cocci cocci, rods cocci or cocci, rods cocci short rods Utilization of (as sole carbon) D-Fructose D-Galactose ND ND ND Sucrose w

1, Janibacter corallicola 04PA2-Co5-61T; 2, Janibacter corallicola 02PA-Ca-009; 3, Janibacter anophelis H2.16BT; 4, Janibacter terrae DSM 13876T; 5, Janibacter limosus DSM 11140T; 6, Janibacter melonis CM2104T. Data for the type strains of J. anophelis, J. terrae, J. limosus and J. melonis are from Kämpfer et al. (2006) (growth at 37°C and utilization of sole carbon), or from Yoon et al. (2004) (color of colonies and morphology). Abbreviations: , positive; w, weakly positive; , negative; ND, no data.

Acknowledgments

We appreciate Dr. Hideki Yukihira in Palau International Coral Reef Center, for his help in collecting and identifying the hard coral. We are grateful to Dr. Jean P. Euzéby (Société de Bactériologie Systématique et Vétérinaire and Ecole Nationale Vétérinaire de Toulouse, France) for his help with the latiniza- tion of the species name. This study was supported in part by a Grant of the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and The JSPS Grant-in-Aid for Science Research Foundation, and New Energy and Industrial Technology Development Orga- nization (NEDO).

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