International Journal of Systematic and Evolutionary Microbiology (2001), 51, 559–563 Printed in Great Britain Asaia siamensis sp. nov., an acetic acid NOTE bacterium in the α-Proteobacteria Kazushige Katsura,1 Hiroko Kawasaki,2 Wanchern Potacharoen,3 Susono Saono,4 Tatsuji Seki,2 Yuzo Yamada,1† Tai Uchimura1 and Kazuo Komagata1 Author for correspondence: Yuzo Yamada. Tel\Fax: j81 54 635 2316. e-mail: yamada-yuzo!mub.biglobe.ne.jp 1 Laboratory of General and Five bacterial strains were isolated from tropical flowers collected in Thailand Applied Microbiology, and Indonesia by the enrichment culture approach for acetic acid bacteria. Department of Applied Biology and Chemistry, Phylogenetic analysis based on 16S rRNA gene sequences showed that the Faculty of Applied isolates were located within the cluster of the genus Asaia. The isolates Bioscience, Tokyo constituted a group separate from Asaia bogorensis on the basis of DNA University of Agriculture, 1-1-1 Sakuragaoka, relatedness values. Their DNA GMC contents were 586–597 mol%, with a range Setagaya-ku, Tokyo of 11 mol%, which were slightly lower than that of A. bogorensis (593–610 156-8502, Japan mol%), the type species of the genus Asaia. The isolates had morphological, 2 The International Center physiological and biochemical characteristics similar to A. bogorensis strains, for Biotechnology, but the isolates did not produce acid from dulcitol. On the basis of the results Osaka University, 2-1 Yamadaoka, Suita, obtained, the name Asaia siamensis sp. nov. is proposed for these isolates. Osaka 568-0871, Japan Strain S60-1T, isolated from a flower of crown flower (dok rak, Calotropis 3 National Center for gigantea) collected in Bangkok, Thailand, was designated the type strain Genetic Engineering and ( l NRIC 0323T l JCM 10715T l IFO 16457T). Biotechnology, National Science and Technology Development Agency, 73/1 Rama VI Road, Keywords: Asaia siamensis sp. nov., acetic acid bacteria, Acetobacteraceae, Bangkok 10400, Thailand Proteobacteria 4 Research and Development Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), Jalan Raya Bogor Km 46, Cibinong 16911, Indonesia The genus Asaia was introduced with a single species, These strains were maintained on agar slants of AG Asaia bogorensis, in the family Acetobacteraceae medium composed of 0n1% -glucose, 1n5% glycerol, (Yamada et al., 2000). In contrast with strains of the 0n5% peptone, 0n5% yeast extract, 0n2% malt extract, genera Acetobacter, Gluconobacter and Gluconaceto- 0n7% CaCO$ and 1n5% agar (w\v). Acetobacter aceti bacter, the strains assigned to this genus are charac- IFO 14818T, Gluconobacter oxydans IFO 14819T, terized by no or very weak capability for oxidizing Gluconacetobacter liquefaciens IFO 12388T and Asaia ethanol to acetic acid and no growth in the presence of bogorensis JCM 10569T, NRIC 0317 and NRIC 0318 acetic acid (0n35%, v\v). This paper describes the were used as reference strains. proposal of Asaia siamensis sp. nov., the second species Morphological, physiological and biochemical charac- of the genus Asaia, for strains isolated from tropical teristics were examined according to the methods flowers collected in Thailand and Indonesia. et al et al T reported by Asai . (1964) and Yamada . (1976, Five bacterial strains (S60-1 , D4-1, Y85, i36 and 1999, 2000). Cells of all the isolates were Gram- B28S-3) were isolated from tropical flowers collected negative, strictly aerobic and rod-shaped, measuring in Thailand and Indonesia by the enrichment culture 0n6–1n0i1n0–4n5 µm. The cells were motile by means of approach using a sorbitol medium and a dulcitol peritrichous flagella. Colonies were pink, shiny, medium at pH 3n5 (Table 1) (Yamada et al., 2000). smooth and raised with an entire margin on AG agar ................................................................................................................................................. plates. All the isolates grew well at pH 3n0 and 3n5 and † Present address: 2-3-21 Seinancho, Fujieda, Shizuoka 426-0063, Japan. at 30 mC on a CaCO$-free AG medium. The isolates The DDBJ accession numbers for the 16S rRNA gene sequences of isolates oxidized acetate and lactate to carbon dioxide and S60-1T and Y85 are AB035416 and AB025932, respectively. water but, as with strains of Asaia bogorensis, their 01495 # 2001 IUMS 559 K. Katsura and others Table 1 Strain designations and isolation sources ................................................................................................................................................................................................................................................................................................................. NRIC, NODAI Culture Collection Center, Tokyo University of Agriculture (NODAI), 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan; JCM, Japan Collection of Microorganisms, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; IFO, Institute for Fermentation, Osaka, 2-17-85 Juso Honmachi, Yodogawa-ku, Osaka 532-8686, Japan. Isolate Source NRIC JCM IFO S60-1T Flower of crown flower (dok rak, Calotropis gigantea), Bangkok, Thailand 0323T 10715T 16457T D4-1 Flower of spider lily (plub-plueng, Crinum asiaticum), Bangkok, Thailand 0324 Y85 Flower of spider lily (krinum bakung, Crinum asiaticum), Bogor, Indonesia 0325 10716 i36 Flower of spider lily (krinum bakung, Crinum asiaticum), Bogor, Indonesia 0326 B28S-3 Flower of ixora (soka, Ixora chinensis), Bogor, Indonesia 0327 Table 2 Physiological and biochemical characteristics, and ubiquinone composition of isolates ................................................................................................................................................................................................................................................................................................................. j, Positive; , weakly positive; k, negative. All strains were positive for growth at pH 3n5 and production of acid from - glucose; all strains were negative for growth on methanol. Strains: 1, Asaia siamensis S60-1T;2,Asaia siamensis Y85; 3, Asaia siamensis D4-1; 4, Asaia siamensis i36; 5, Asaia siamensis B28S-3; 6, Asaia bogorensis NRIC 0317; 7, Asaia bogorensis NRIC 0318; 8, Asaia bogorensis JCM 10569T;9,Acetobacter aceti IFO 14818T; 10, Gluconobacter oxydans IFO 14819T; 11, Gluconacetobacter liquefaciens IFO 12388T. Character 1 2 3 4 567891011 Oxidation of acetate and lactate jkj Production of water-soluble brown pigment on glucose\CaCO$ medium kkk kkkkkkkj Dihydroxyacetone formation from glycerol jj jjj Growth on dulcitol jjj jjjjjkkk Assimilation of ammonium sulfate on vitamin-free glucose medium jjj jjjjj k Growth at pH 3n5 in the presence of acetic acid (0n35%, v\v) kkk kkkkkjjj Acid production from: Ethanol kk kk kkjjj Dulcitol kkk kkjjjkkk -Mannitol and -sorbitol jjj jjjjjkjk Glycerol jjj jjjjjkj Ubiquinone composition (%):* Q-10 98 98 99 100 96 97 97 93 2 98 93 Q-9 211 033377927 Q-8 010 010001900 * The ubiquinone compositions of the type strains of Acetobacter aceti, Gluconobacter oxydans and Gluconacetobacter liquefaciens were cited from Urakami et al. (1989). The ubiquinone isoprenologues below 1% were omitted. oxidation capability was not intense (Table 2). The at pH 3n5 on CaCO$-free AG medium. Acid was production of dihydroxyacetone from glycerol was produced from -glucose, -mannose, -fructose, - variable depending upon the isolates; positive or sorbose, -xylose, -arabinose, -ribose, myo-inositol, weakly positive strains were present. All the isolates ribitol, -arabitol, xylitol, meso-erythritol, glycerol, grew on glutamate agar and mannitol agar, but did melibiose and sucrose, but not from lactose. The not grow on methanol. They showed vigorous growth isolates did not produce acid from dulcitol, but on a vitamin-free glucose\(NH%)#SO% medium, but assimilated dulcitol for growth. no growth was found on a vitamin-free ethanol\ (NH%)#SO% medium. The isolates produced 2-keto-- Isoprenoid quinones were extracted from bacterial gluconate and 5-keto--gluconate, but not 2,5-diketo- cells and purified by the method of Yamada et al. -gluconate, and a water-soluble brown pigment was (1969). Ubiquinone isoprenologues were quantitat- observed on a glucose\yeast extract\CaCO$ medium. ively determined by reversed-phase HPLC (Tamaoka Acetic acid was not produced on an ethanol\yeast et al., 1983). Standard preparations of Q-10, Q-9 and extract\CaCO$ agar. The presence of 0n35% (v\v) Q-8 were obtained from the cells of Gluconobacter acetic acid completely inhibited growth of the isolates cerinus NRIC 0229T, Acetobacter aceti IFO 14818T 560 International Journal of Systematic and Evolutionary Microbiology 51 Asaia siamensis sp. nov. Table 3 DNA base compositions and values of DNA relatedness ................................................................................................................................................................................................................................................................................................................. , Not tested. Species Strain GjC content DNA relatedness (%) with: (mol%) S60-1T Y85 JCM 10569T Asaia siamensis S60-1T 59n3 100 84 20 D4-1 58n7949224 Y85 59n7 86 100 20 i36 58n6799719 B28S-3 59n6 100 93 23 Asaia bogorensis JCM 10569T 60n2 33 27 100 NRIC 0317 59n7 27 68 NRIC 0318 59n3 36 87 Acetobacter aceti IFO 14818T 58n31274 Gluconobacter oxydans IFO 14819T