Asanoa iriomotensis sp. nov., isolated from mangrove soil

The genus Asanoa was proposed by Lee & Hah (2002) to accommodate actinomycetes with meso-diaminopimelic acid (A₂pm) and 3-OH A₂pm in the cell wall, MK-10(H₆, H₈) as the major menaquinone and fatty acid type 2d and which belong to the family Micromonosporaceae by their 16S rRNA gene sequences. The genus was established with Asanoa ferruginea (basonym Catellatospora ferruginea Asano and Kawamoto 1986) and Asanoa ishikariensis (formerly ‘Catellatospora ishikariense’) and was distinguished from the genus Catellatospora by members of the latter genus displaying major menaquinone MK-10(H₄) or MK-9(H₄, H₆) and fatty acid type 3b (Lee & Hah, 2002).

Mangroves are salt-adapted plants found along many of the world's tropical and subtropical coastlines (Dodd, 2000). In Japan, mangroves develop along estuaries in the subtropical zone, where sea water and river water mix. During a study of the diversity of actinomycetes that inhabit the mangrove zone, strain TT 97-02^T was isolated from soil around the roots of the mangrove Bruguiera gymnorrhiza. Strain TT 97-02^T formed spore chains borne on the tip of short sporophores arising directly from the agar surface. The isolate formed a monophyletic cluster with the members of the genus Asanoa and had fatty acid type 2d and major menaquinone MK-10(H₆, H₈). This isolate represents a novel species of Asanoa, for which we propose the name Asanoa iriomotensis.

Strain TT 97-02^T was isolated on humic acid-vitamin (HV) agar (Hayakawa & Nonomura, 1987) by the yeast extract/SDS method (Hayakawa & Nonomura, 1989) from soil around the roots of B. gymnorrhiza growing along the Shiira River in Iriomote Island, Okinawa Prefecture, Japan. Freeze-dried cells for chemotaxonomic analyses were grown in yeast extract/glucose broth (10 g yeast extract and 10 g d-glucose per litre distilled water, pH 7·0) on a rotary shaker at 28 °C. A. ferruginea NBRC 14496^T and A. ishikariensis NBRC 14551^T were used as reference strains.

Cultural and physiological characteristics were determined as described previously (Gordon et al., 1974; Seino et al., 1985; Shirling & Gottlieb, 1966; Yokota et al., 1993). Morphology was observed by scanning electron microscopy as described previously (Tamura et al., 2000). Analyses of whole-cell sugar patterns, cell-wall amino acids, menaquinones, cellular fatty acids, isomers of A₂pm, acyl type of peptidoglycan, mycolic acid and DNA base composition were performed as described previously (Tamura et al., 1994). The microplate hybridization method developed by Ezaki et al. (1988, 1989) was applied with minor modifications to determine DNA relatedness (Tamura et al., 1999). PCR amplification and sequencing of the 16S rRNA gene were performed as described previously (Tamura & Hatano, 2001) with a model ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) according to the manufacturer's protocol. Phylogenetic analysis of 16S rRNA gene sequences was performed as described previously (Tamura & Hatano, 2001).

The isolate was a Gram-positive, non-acid-fast and aerobic organism with branched hyphae. A non-fragmenting substrate mycelium was formed. A 14-day-old culture grown on HV agar showed spore chains borne on the tip of short sporophores arising directly from the agar surface (see supplementary figure in IJSEM Online). Motile spores were not observed.

The isolate exhibited good growth on yeast extract-malt extract agar (ISP medium 2), moderate growth on oatmeal agar (ISP medium 3), poor growth on inorganic salts-starch agar (ISP medium 4), glycerol-asparagine agar (ISP medium 5) and peptone-yeast extract-iron agar (ISP medium 6) and almost no growth on tyrosine agar (ISP medium 7). The isolate formed spore chains on water agar and HV agar, but not on yeast extract-malt extract agar, oatmeal agar, inorganic salts-starch agar, glycerol-asparagine agar, peptone-yeast extract iron agar or tyrosine agar.

Strain TT 97-02^T hydrolysed starch, but did not reduce nitrate. It grew at 20 and 30 °C, but not at 15 or 37 °C. The isolate utilized (−)-d-mannitol, (+)-d-melibiose, (+)-d-maltose, (+)-l-rhamnose, methyl α-d-glucoside, (+)-d-raffinose, (+)-d-galactose, (+)-d-mannose and glucose and grew weakly on (+)-l-arabinose and (+)-d-xylose, but did not utilize l-erythritol, adonitol, (+)-d-lactose, l-inositol, d-sorbitol or dulcitol as sole carbon sources. The isolate did not grow on 4 % NaCl. Hydrolysis of aesculin, decomposition of urea, utilization of sodium succinate, sodium oxalate, sodium malate and sodium citrate and growth on MacConkey agar were negative.

The cell walls contained meso-A₂pm, d-glutamic acid, glycine and l-alanine, indicating that the peptidoglycan is type A1γ according to Schleifer & Kandler (1972). The isolate contained xylose, mannose, galactose and glucose as whole-cell sugars, but arabinose was not detected. The major menaquinones were MK-10(H₆) and MK-10(H₈). The major cellular fatty acids were anteiso-C_15 : 0 (22·3 %), anteiso-C_17 : 0 (19·6 %), iso-C_15 : 0 (18·0 %), C_17 : 0 (13·6 %) and iso-C_16 : 0 (12·2 %), and small amounts of iso-C_17 : 0, C_16 : 0, C_18 : 0, C_15 : 0 and iso-C_14 : 0 were also detected. Mycolic acids were absent. Phosphatidylethanolamine was detected, but phospholipids including unidentified glucosamine and phosphatidylcholine were not detected, corresponding to phospholipid type II of Lechevalier et al. (1981). The acyl type of cell-wall polysaccharides was acetyl. The G+C content of the genomic DNA was 69 mol%.

The 16S rRNA gene sequence was determined for strain TT 97-02^T (positions 28–1525 according to the Escherichia coli numbering system; Brosius et al., 1978). Positions at which secondary structures varied between strains and positions at which the sequence was not determined in some reference organisms were excluded from the analysis. The phylogenetic tree obtained by applying the neighbour-joining method to K_nuc values is depicted in Fig. 1⇓. 16S rRNA gene sequence analysis revealed that the isolate fell in the cluster of the family Micromonosporaceae and formed a monophyletic cluster with members of the genus Asanoa. The isolate was closely related to A. ferruginea and A. ishikariensis (96·9–97·1 % similarity). In a DNA–DNA hybridization study, strain TT 97-02^T exhibited levels of DNA–DNA relatedness of 15–28 % with A. ferruginea NBRC 14496^T and A. ishikariensis NBRC 14551^T.

The previously known members of the genus Asanoa show poor sporulation that occurs only on tap water agar and glycerol/calcium malate agar (Asano & Kawamoto, 1986; Lee & Hah, 2002). Sporulation of strain TT 97-02^T was not observed on richly nutritious media but developed well on water agar and HV agar. Chemotaxonomic characteristics and phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate is a member of the genus Asanoa. The isolate could be distinguished from the two known species, A. ferruginea and A. ishikariensis, by its low relatedness in DNA–DNA hybridization, its cellular fatty acid profile and the utilization of some carbon sources (Table 1⇓). On the basis of the data presented above, we propose that strain TT 97-02^T be classified in a novel species, Asanoa iriomotensis sp. nov.