Downloaded from www.microbiologyresearch.org by IP: 54.162.133.179 On: Sat, 06 Feb 2016 16:57:45 International Journal of Systematic and Evolutionary Microbiology (2002), 52, 813–818 DOI : 10.1099/ijs.0.01982-0 NOTE Kozakia baliensis gen. nov., sp. nov., a novel acetic acid bacterium in the α-Proteobacteria 1 Laboratory of General and Applied Microbiology, Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156- 8502, Japan 2 The International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan 3 Research and Development Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), Jalan Raya Bogor Km 46, Cibinong 16911, Indonesia Puspita Lisdiyanti, 1 Hiroko Kawasaki, 2 Yantyati Widyastuti, 3 Susono Saono, 3 Tatsuji Seki, 2 Yuzo Yamada, 1 † Tai Uchimura 1 and Kazuo Komagata 1 Author for correspondence : Yuzo Yamada. TelFax : 81 54 635 2316. e-mail : yamada-yuzomub.biglobe.ne.jp Four bacterial strains were isolated from palm brown sugar and ragi collected in Bali and Yogyakarta, Indonesia, by an enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the four isolates constituted a cluster separate from the genera Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter and Asaia with a high bootstrap value in a phylogenetic tree. The isolates had high values of DNA–DNA similarity (78–100 %) between one another and low values of the similarity (7–25 %) to the type strains of Acetobacter aceti, Gluconobacter oxydans, Gluconacetobacter liquefaciens and Asaia bogorensis. The DNA base composition of the isolates ranged from 568 to 572 mol % GMC with a range of 04 mol %. The major quinone was Q-10. The isolates oxidized acetate and lactate to carbon dioxide and water, but the activity was weak, as with strains of Asaia bogorensis. The isolates differed from Asaia bogorensis strains in phenotypic characteristics. The name Kozakia baliensis gen. nov., sp. nov., is proposed for the four isolates. Strain Yo-3 T ( NRIC 0488 T  JCM 11301 T  IFO 16664 T  DSM 14400 T ) was isolated from palm brown sugar collected in Bali, Indonesia, and was designated as the type strain. Keywords : Kozakia baliensis gen. nov., sp. nov., acetic acid bacteria, Acetobacteraceae, Proteobacteria During the systematic study of acetic acid bacteria from Indonesian sources, a number of strains be- longing to the genera Acetobacter, Gluconobacter and Gluconacetobacter were isolated (Yamada et al., 1999), and the genus Asaia was introduced as the fifth genus in the family Acetobacteraceae, with a single species, Asaia bogorensis (Yamada et al., 2000), followed by a second species, Asaia siamensis (Katsura et al., 2001). In addition, several novel species and new combina- tions were described in the genus Acetobacter for isolates from Indonesian sources (Lisdiyanti et al., 2000, 2001). Among the strains tentatively identified as Gluconace- tobacter species in a previous paper (Yamada et al., ................................................................................................................................................. Published online ahead of print on 28 January 2002 as DOI 10.1099/ ijs.0.01982-0. † Present address : 2-3-21 Seinancho, Fujieda, Shizuoka 426-0063, Japan. The DDBJ accession numbers for the 16S rRNA gene sequences of isolates Ri-1, Wa-5, Wa-2 and Yo-3 T are respectively AB056318–AB056321. 1999), four strains seemed to be interesting because they oxidized acetate and lactate to carbon dioxide and water and had Q-10 as the major quinone but their taxonomic allocation was not within the genus Gluco- nacetobacter. This paper deals with the detailed charac- terization of these four strains and proposal of Kozakia baliensis gen. nov., sp. nov. An enrichment culture approach was employed for the isolation of acetic acid bacteria (Yamada et al., 1976, 1999, 2000 ; Lisdiyanti et al., 2000). The medium was composed of 10% -glucose, 05% ethanol, 03% acetic acid, 15% peptone, 08% yeast extract and 100 p.p.m. cycloheximide (wv), and adjusted to pH 35 with HCl. Isolates were maintained on agar slants of AG medium composed of 01% -glucose, 15% glycerol, 05 % peptone, 05 % yeast extract, 02 % malt extract, 07% CaCO  and 15% agar (wv). Yamada et al. (1999) tentatively identified eight Gluco- nacetobacter strains isolated from Indonesian sources 01982  2002 IUMS Printed in Great Britain 813