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Kozakia Baliensis Gen. Nov., Sp. Nov., a Novel Acetic Acid Bacterium in The International Journal of Systematic and Evolutionary Microbiology (2002), 52, 813–818 DOI: 10.1099/ijs.0.01982-0 Kozakia baliensis gen. nov., sp. nov., a novel NOTE acetic acid bacterium in the α-Proteobacteria 1 Laboratory of General and Puspita Lisdiyanti,1 Hiroko Kawasaki,2 Yantyati Widyastuti,3 Applied Microbiology, 3 2 1 1 Department of Applied Susono Saono, Tatsuji Seki, Yuzo Yamada, † Tai Uchimura Biology and Chemistry, and Kazuo Komagata1 Faculty of Applied Bioscience, Tokyo University of Agriculture, Author for correspondence: Yuzo Yamada. Tel\Fax: j81 54 635 2316. 1-1-1 Sakuragaoka, e-mail: yamada-yuzo!mub.biglobe.ne.jp Setagaya-ku, Tokyo 156- 8502, Japan 2 The International Center Four bacterial strains were isolated from palm brown sugar and ragi collected for Biotechnology, Osaka in Bali and Yogyakarta, Indonesia, by an enrichment culture approach for University, 2-1 Yamadaoka, Suita, Osaka 565-0871, acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences Japan showed that the four isolates constituted a cluster separate from the genera 3 Research and Development Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter and Asaia with a Centre for Biotechnology, high bootstrap value in a phylogenetic tree. The isolates had high values of Indonesian Institute of DNA–DNA similarity (78–100%) between one another and low values of the Sciences (LIPI), Jalan Raya Bogor Km 46, Cibinong similarity (7–25%) to the type strains of Acetobacter aceti, Gluconobacter 16911, Indonesia oxydans, Gluconacetobacter liquefaciens and Asaia bogorensis. The DNA base composition of the isolates ranged from 568to572 mol% GMC with a range of 04 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-3T (l NRIC 0488T l JCM 11301T l IFO 16664T l DSM 14400T) 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 1999), four strains seemed to be interesting because from Indonesian sources, a number of strains be- they oxidized acetate and lactate to carbon dioxide and longing to the genera Acetobacter, Gluconobacter and water and had Q-10 as the major quinone but their Gluconacetobacter were isolated (Yamada et al., 1999), taxonomic allocation was not within the genus Gluco- and the genus Asaia was introduced as the fifth genus nacetobacter. This paper deals with the detailed charac- in the family Acetobacteraceae, with a single species, terization of these four strains and proposal of Kozakia Asaia bogorensis (Yamada et al., 2000), followed by a baliensis gen. nov., sp. nov. second species, Asaia siamensis (Katsura et al., 2001). In addition, several novel species and new combina- An enrichment culture approach was employed for the tions were described in the genus Acetobacter for isolation of acetic acid bacteria (Yamada et al., 1976, isolates from Indonesian sources (Lisdiyanti et al., 1999, 2000; Lisdiyanti et al., 2000). The medium was 2000, 2001). composed of 1n0% -glucose, 0n5% ethanol, 0n3% acetic acid, 1n5% peptone, 0n8% yeast extract and Among the strains tentatively identified as Gluconace- 100 p.p.m. cycloheximide (w\v), and adjusted to pH tobacter species in a previous paper (Yamada et al., 3n5 with HCl. Isolates were maintained on agar slants ................................................................................................................................................. of AG medium composed of 0n1% -glucose, 1n5% Published online ahead of print on 28 January 2002 as DOI 10.1099/ glycerol, 0n5% peptone, 0n5% yeast extract, 0n2% malt ijs.0.01982-0. extract, 0n7% CaCO$ and 1n5% agar (w\v). † Present address: 2-3-21 Seinancho, Fujieda, Shizuoka 426-0063, Japan. The DDBJ accession numbers for the 16S rRNA gene sequences of isolates Yamada et al. (1999) tentatively identified eight Gluco- Ri-1, Wa-5, Wa-2 and Yo-3T are respectively AB056318–AB056321. nacetobacter strains isolated from Indonesian sources 01982 # 2002 IUMS Printed in Great Britain 813 P. Lisdiyanti and others Brosius et al., 1981). Amplified 16S rRNA genes were sequenced directly with an ABI PRISM Big Dye Ter- minator cycle sequencing ready reaction kit on an ABI PRISM model 310 Genetic Analyzer. The follow- ing six primers were used: 20F, 1500R, 520F (5h-CA- GCAGCCGCGGTAATAC-3h, positions 519–536), 520R(5h-GTATTACCGCGGCTGCTG-3h,536–519), 920F (5h-AAACTCAAATGAATTGACGG-3h, 907– 926) and 920R (5h-CCGTCAATTCATTTGAGTTT- 3h, 926–907). Multiple alignments of the sequences were carried out with the program (version 1.6) (Thompson et al., 1994). Distance matrices for the aligned sequences were determined by using the two- parameter method of Kimura (1980). The neighbour- joining method was used for construction of a phy- logenetic tree (Saitou & Nei, 1987). The sequence data obtained were compared on the basis of 1416 bases. The robustness of individual branches was estimated by bootstrapping with 1000 replicates (Felsenstein, 1985). The species, type strains and accession numbers of the sequences taken from databases are included in Fig. 1. Sequence similarities (%) among acetic acid bacteria including the novel isolates were determined for pairs of sequences of the 1416 bases. The four isolates constituted a cluster separate from the genera Acetobacter, Gluconobacter, Acidomonas, Glucona- cetobacter and Asaia with high bootstrap values in a phylogenetic tree (Fig. 1). The isolates showed 99n8–100% sequence similarity to one another. The T T sequence similarities of isolate Yo-3 (l NRIC 0488 ) ................................................................................................................................................. were respectively 95n9, 94n6, 95n3, 97n1 and 97n4% to Fig. 1. Phylogenetic relationships of acetic acid bacteria based the type strains of Acetobacter aceti, Gluconobacter on 16S rRNA gene sequences. Numerals at nodes indicate oxydans, Acidomonas methanolica, Gluconacetobacter bootstrap values derived from 1000 replications. Abbreviations liquefaciens and Asaia bogorensis. of genus names: A., Acetobacter; Ac., Acidomonas; As., Asaia; Ga., Gluconacetobacter; G., Gluconobacter; K., Kozakia. Extraction and isolation of bacterial DNA were carried out by the method of Saito & Miura (1963). DNA base composition was determined by the method of Tamaoka & Komagata (1984). DNA–DNA simi- by using the enrichment culture approach described larity was determined by fluorometric DNA–DNA above. Of these eight strains, four were used in this T T T hybridization in microdilution wells as described by study. Strain Yo-3 (l NRIC 0488 l IFO 16664 l T T Ezaki et al. (1989). Single-stranded and labelled DNAs JCM 11301 l DSM 14400 ) was isolated from palm were hybridized in 2iSSC and 50% formamide at brown sugar and Ri-1 (l NRIC 0485) from ragi 50 mC for 6 h. The DNA base composition of the four (starter) in Bali, Indonesia; Wa-5 (l NRIC 0486) and isolates ranged from 56n8to57n2mol%GjC, with a Wa-2 (l NRIC 0487) were isolated from ragi (starter) range of 0n4 mol% (Table 1). The GjC contents of in Yogyakarta, Indonesia. Acetobacter aceti IFO T T DNA of the isolates were lower than those of the type 14818 , Gluconobacter oxydans IFO 14819 , Gluco- strains of Asaia bogorensis (60n2 mol%) and Gluco- nacetobacter liquefaciens IFO 12388T, Gluconaceto- T nacetobacter liquefaciens (63n6 mol%). The isolates bacter xylinus IFO 15237 , Gluconacetobacter hansenii had high values of DNA–DNA similarity (78–100%) LMG 1527T, Asaia bogorensis NRIC 0311T and Asaia T to one another. Low values of similarity (7–25%) were siamensis NRIC 0323 were used as reference strains. found to the type strains of Acetobacter aceti, Gluco- nobacter oxydans, Gluconacetobacter liquefaciens and Gene fragments specific for the 16S rRNA-encoding Asaia bogorensis. These data indicate that the four regions of the four isolates were amplified by PCR as isolates constitute a single species. described previously (Kawasaki et al., 1993; Yamada et al., 2000; Lisdiyanti et al., 2000). Two primers, 20F Isoprenoid quinones were extracted and purified by (5h-GAGTTTGATCCTGGCTCAG-3h; positions 9– the method of Yamada et al. (1969). Ubiquinone 27) and 1500R (5h-GTTACCTTGTTACGACTT-3h; homologues were analysed quantitatively by HPLC 1509–1492), were used. The numbers of positions in with a Nova-Pak C18 3n9i150 mm column (Nihon the rRNA gene fragments were based on the Escheri- Waters) (Tamaoka et al., 1983). Standard preparations chia coli numbering system (accession number V00348; of Q-10, Q-9 and Q-8 were prepared from cells of 814 International Journal of Systematic and Evolutionary Microbiology 52 Kozakia baliensis gen. nov., sp. nov. Table 1. DNA base compositions and values of DNA–DNA similarity among acetic acid bacteria ................................................................................................................................................................................................................................................................................................................. Abbreviations: A., Acetobacter; As., Asaia; Ga., Gluconacetobacter; G.,
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