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-

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 . 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 , , , and 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 , Gluconobacter 16911, Indonesia oxydans, Gluconacetobacter liquefaciens and . 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 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: gen. nov., sp. nov., acetic acid bacteria, , 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 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 , 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, (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, , 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., Gluconobacter; K., Kozakia.

Strain GjC content DNA–DNA similarity (%) with strain: (mol%) 1234567891011

1. K. baliensis NRIC 0488T 57n2 100 87 80 81 8 11 3 11 12 14 9 2. K. baliensis NRIC 0485 56n8 8810078989166961410 3. K. baliensis NRIC 0486 57n0 94 99 100 98 10 18 9 9 16 13 4. K. baliensis NRIC 0487 57n2 859992100 9176 51712 5. As. bogorensis NRIC 0311T 60n2 1287810035 2 14 5 6 6. As. siamensis NRIC 0323T 59n3 25 13 7 10 30 100 8658 7. Ga. liquefaciens IFO 12388T 63n6 11192215412100 22 9 16 8. Ga. xylinus IFO 15237T 62n5 16 21 15 100 9. Ga. hansenii LMG 1527T 58n7 12 7 10 7 4 10 11 100 11 6 10. G. oxydans IFO 14819T 61n7 25241618 109 1710017 11. A. aceti IFO 14818T 57n2 78872 0 5100

Gluconobacter oxydans IFO 14819T, Acetobacter aceti values in the phylogenetic tree based on 16S rRNA IFO 14818T and Frateuria aurantia IFO 3245T, re- gene sequences. spectively (Yamada et al., 1969). The major quinone of the four isolates was Q-10. The quinone system was The sequence similarities were determined to be composed of 91n5–100% Q-10, 0–0n8% Q-9 and 97n2–97n4% between the four isolates and the type 0–0n8% Q-8 in all the isolates; 98n4% Q-10 and 1n6% strain of Asaia bogorensis. These similarities were Q-9 were estimated for isolate Ri-1, 91n5% Q-10 and higher than the sequence similarity (96n5%) between 8n5% Q-9 for isolate Wa-5, 100% Q-10 for isolate the type strains of Asaia bogorensis and Gluconace- Wa-2 and 96n9% Q-10, 2n3% Q-9 and 0n8% Q-8 for tobacter liquefaciens. However, these four isolates isolate Yo-3T. differed from the type strain of Asaia bogorensis, the type species of the genus Asaia, in production of acetic Morphological, physiological and biochemical charac- acid from ethanol, growth in the presence of 0n35% teristics were examined as described previously (Asai acetic acid at pH 3n5 (both negative for Asaia strains), et al., 1964; Yamada et al., 1976, 1999, 2000; Lisdiyanti absence of growth on 30% -glucose and absence of et al., 2000). Vitamin requirements were determined by assimilation of (NH%)#SO% on glucose medium or a method reported previously (Lisdiyanti et al., 2000). mannitol medium without vitamins (both positive for Utilization of methanol was tested by the use of Asaia strains). These data indicate that the four iso- Hoyer–Frateur medium (De Ley & Frateur, 1974), lates are distinguished phenotypically from the Asaia Frateur-modified Hoyer medium (De Ley et al., 1984) strains. and medium C (Urakami et al., 1989). In addition to the characteristics listed in the genus description below, The four isolates oxidized acetate and lactate to carbon the following observations were made. Cellulose was dioxide and water, but the activity was weak, as with not produced. All isolates assimilated (NH%)#SO% on the type strains of Asaia bogorensis and Asaia sia- mannitol medium with vitamins but did not assimilate mensis. These characteristics contrast with those of the it on ethanol; two isolates (Ri-1 and Wa-2) assimilated type strains of Acetobacter aceti, Gluconobacter oxy- (NH%)#SO% on glucose medium with vitamins. γ- dans and Gluconacetobacter liquefaciens. Intense oxi- Pyrone production was weak for strain Wa-2. Acid dation of acetate and lactate is found in the type strains was produced from -arabinose by isolates Wa-5 and of Acetobacter aceti and Gluconacetobacter lique- Wa-2 and from sucrose by isolates Ri-1 and Yo-3T. faciens and no oxidation is found in the type strain of Gluconobacter oxydans. The four isolates differ from The acetic acid bacteria are currently classified into the genus Acidomonas, since they did not utilize five genera in the family Acetobacteraceae; Acetobacter methanol on any of the media tested. (the type genus), Gluconobacter, Acidomonas, Gluco- nacetobacter and Asaia (Yamada et al., 1997, 2000). The isolates differ from the genera Asaia, Gluconaceto- The four isolates are distinguished from these five bacter, Gluconobacter, Acidomonas and Acetobacter in genera of acetic acid bacteria at the generic level, since the production of acid from raffinose. Interestingly, the isolates were located on a sublineage separate from the four isolates produced a large amount of levan-like the genera Acetobacter, Gluconobacter, Acidomonas, mucous substance(s) from sucrose and -fructose. The Gluconacetobacter and Asaia with high bootstrap production of mucous substance(s) will be a probable http://ijs.sgmjournals.org 815 P. Lisdiyanti and others

Table 2. Differential characteristics of the genera Kozakia, Asaia, Gluconacetobacter, Gluconobacter, Acidomonas and Acetobacter ...... Data for Acidomonas were taken from Urakami et al. (1989). Abbreviations: j, positive; k, negative; , weak; , not determined.

Characteristic Kozakia Asaia Gluconacetobacter Gluconobacter Acidomonas Acetobacter

Flagellation Non-motile Peritrichous or Peritrichous or Polar or Non-motile Peritrichous or non-motile non-motile non-motile non-motile Pigmentation kj\kk jkk Production of water soluble brown-pigment(s) kk j\kk\jk k Production of cellulose kk j\kkkk Production of mucous substance(s) from sucrose jk k kkk\j Oxidation of: Acetate  jkjj Lactate  jkkj Production of acetic acid from ethanol jk j jjj Growth in the presence of 0n35% (v\v) acetic acid jk j jjj in CaCO$-free AG broth at pH 3n5 Growth in the presence of 30% -glucose kj j\kk k Growth on YPM agar: Without -mannitol       With -mannitol jj j j  Growth on glutamate agar kj j k j Assimilation of ammoniacal nitrogen on Hoyer–Frateur medium with: -Glucose kj j\kk k -Mannitol kj j\kk k Ethanol kk k k j\k Assimilation of ammoniacal nitrogen on Frateur-modified Hoyer medium with: -Glucose j\kj j\kj k -Mannitol jj j\kj k Ethanol kk k k j\k Utilization of methanol kk k kjk* Ketogenesis from glycerol jj\kj\kjkj\k Production of γ-pyrones from: -Glucose kk j\kk\j  k -Fructose j\ k\jk j k Production of keto--gluconates from -glucose: 2-Keto--gluconate jj j j j\k 5-Keto--gluconate jj j\kj j\k 2,5-Diketo--gluconate kk j\kk\j  k Acid production from: -Arabinose jj j\kjkj\k -Arabinose j\kj k j  k -Xylose jj j\kjkj\k -Rhamnose kj\kk k k -Glucose jj j jjj\k -Galactose jj j jkj\k -Mannose jj j\kjkj\k -Fructose kj j jkk -Sorbose kj j\kj k Melibiose jj k j k Sucrose j\kj k j k k Raffinose jk k k k -Mannitol kj\kj\kjkk -Sorbitol kj\kk jkk Dulcitol kj k k k Glycerol jj j jkk Ethanol jk j jjj Major ubiquinone Q-10 Q-10 Q-10 Q-10 Q-10 Q-9 GjC content (mol%) 56–57 59–61 55–66 54–63 63–66 52–60

* was reported to utilize methanol weakly (Sokollek et al., 1998).

marker for differentiating the isolates from other Description of Kozakia gen. nov. genera. The differential characteristics of the four isolates from the other five genera are listed in Table 2. Kozakia (Ko.zahki.a. N. L. fem. n. Kozakia of Kozaki, to honour the Japanese microbiologist Michio Kozaki, As has been described above, the four isolates merit Professor Emeritus of Tokyo University of Agricul- assignment to a new genus, for which the name ture, in recognition of his contributions to the study of Kozakia gen. nov. is proposed. The type species is micro-organisms in tropical regions, especially South- Kozakia baliensis sp. nov. east Asia).

816 International Journal of Systematic and Evolutionary Microbiology 52 Kozakia baliensis gen. nov., sp. nov.

Cells are Gram-negative, non-motile and rod-shaped, organization and primary structure of a ribosomal RNA operon from measuring 0n6–0n8by2n0–3n0 µm. Strictly aerobic. Escherichia coli. J Mol Biol 148, 107–127. Catalase-positive. Oxidase-negative. Non-pigmented. De Ley, J. & Frateur, J. (1974). Genus Acetobacter Beijerinck 1898, 215. In Bergey’s Manual of Determinative Bacteriology, 8th edn, pp. Water-soluble brown pigment(s) is not produced from 276–278. Edited by R. E. Buchanan & N. E. Gibbons. Baltimore: -glucose or on CaCO$-containing agar slants. Grows Williams & Wilkins. ! at pH 3n0 and 30 mC. Produces levan-like mucous De Ley, J., Swings, J. & Gossele, F. (1984). Genus I. Acetobacter substance(s) from sucrose or -fructose. Does not Beijerinck 1898, 215AL.InBergey’s Manual of Systematic Bacteriology, produce gelatinase, H#S, indole or ammonia from - vol. 1, pp. 268–274. Edited by N. R. Krieg & J. G. Holt. Baltimore: arginine and does not reduce nitrate. Oxidizes acetate Williams & Wilkins. and lactate to carbon dioxide and water, but the Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric activity is weak. Produces acetic acid from ethanol. deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro- dilution wells as an alternative to membrane filter hybridization in Growth is not inhibited by 0n35% acetic acid at pH 3n5. which radioisotopes are used to determine genetic relatedness among Grows on mannitol agar but not on glutamate bacterial strains. Int J Syst Bacteriol 39, 224–229. agar. Does not grow on 30% -glucose. Ammoniacal Felsenstein, J. (1985). Confidence limits on phylogenies: an approach nitrogen is not assimilated on glucose medium, man- using the bootstrap. Evolution 39, 783–791. nitol medium or ethanol medium without vitamins. Katsura, K., Kawasaki, H., Potacharoen, W., Saono, S., Seki, T., Does not utilize methanol. Produces dihydroxyacetone Yamada, Y., Uchimura, T. & Komagata, K. (2001). Asaia siamensis from glycerol. Produces -gluconate, 2-keto--glu- sp. nov., an acetic acid bacterium in the α-Proteobacteria. Int J Syst Evol conate and 5-keto--gluconate from -glucose, but Microbiol 51, 559–563. not 2,5-diketo--gluconate. Produces γ-pyrones from Kawasaki, H., Hoshino, Y., Hirata, A. & Yamasato, K. (1993). Is -fructose but not from -glucose. Acid is produced intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among photosynthetic from -arabinose, -xylose, -glucose, -galactose, purple nonsulfur bacteria. Arch Microbiol 160, 358–362. -mannose, melibiose, raffinose, meso-erythritol, Kimura, M. (1980). A simple method for estimating evolutionary rates glycerol and ethanol, but not from -rhamnose, - of base substitutions through comparative studies of nucleotide fructose, -sorbose, lactose, -mannitol, -sorbitol or sequences. J Mol Evol 16, 111–120. dulcitol. Acid production from -arabinose and su- Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T. & crose is variable depending on the strain. Major Komagata, K. (2000). Systematic study of the genus Acetobacter with ubiquinone is Q-10. DNA base composition ranges descriptions of Acetobacter indonesiensis sp. nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb. nov., from 56n8to57n2mol% GjC with a range of Acetobacter lovaniensis (Frateur 1950) comb. nov. and Acetobacter 0n4 mol%. The type species is Kozakia baliensis. estunensis (Carr 1958) comb. nov. J Gen Appl Microbiol 46, 147–165. Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T. & Description of Kozakia baliensis sp. nov. Komagata, K. (2001). Identification of Acetobacter strains isolated from Indonesian sources, and proposals of Acetobacter syzygii sp. nov., Kozakia baliensis (ba.li.enhsis. N.L. fem. adj. baliensis Acetobacter cibinongensis sp. nov. and Acetobacter orientalis sp. nov. J pertaining to Bali, Indonesia, where the type strain was Gen Appl Microbiol 47, 119–131. isolated). Saito, H. & Miura, K. (1963). Preparation of transforming deoxyri- bonucleic acid by phenol treatment. Biochim Biophys Acta 72, 619–629. Characteristics are the same as those described for the Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new genus. The DNA base composition of the type strain is method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. 57n2mol%GjC. Sokollek, S. J., Hertel, C. & Hammes, W. P. (1998). Description of T T The type strain is strain Yo-3 (l IFO 16664 l JCM Acetobacter oboediens sp. nov. and Acetobacter pomorum sp. nov., two T T T new species isolated from industrial vinegar fermentations. Int J Syst 11301 l DSM 14400 l NRIC 0488 ), isolated from Bacteriol 48, 935–940. palm brown sugar collected in Bali, Indonesia, in 1996. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high performance liquid chromato- Acknowledgements graphy. FEMS Microbiol Lett 25, 125–128. Tamaoka, J., Katayama-Fujimura, Y. & Kuraishi, H. (1983). Analysis The authors wish to thank the Japan Bioindustry As- of bacterial menaquinone mixtures by high-performance liquid chroma- sociation, Tokyo, Japan, for support of this study and the tography. J Appl Bacteriol 54, 31–36. Institute for Fermentation, Osaka (IFO), Osaka, Japan, and Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).  : the NODAI Research Institute Culture Collection (NRIC), improving the sensitivity of progressive multiple sequence alignment Tokyo University of Agriculture, Tokyo, Japan, for sup- through sequence weighting, position-specific gap penalties and weight plying reference cultures. Thanks are also due to Sachiko matrix choice. Nucleic Acids Res 22, 4673–4680. Ohtsu, Laboratory of General and Applied Microbiology, Urakami, T., Tamaoka, J., Suzuki, K. & Komagata, K. (1989). Department of Applied Biology and Chemistry, Faculty Acidomonas gen. nov., incorporating Acetobacter methanolicus as of Applied Bioscience, Tokyo University of Agriculture, Acidomonas methanolica comb. nov. Int J Syst Bacteriol 39, 50–55. Tokyo, Japan, for her technical assistance. Yamada, Y., Aida, K. & Uemura, T. (1969). Enzymatic studies on the oxidation of sugar and sugar alcohol. V. Ubiquinone of acetic acid References bacteria and its relation to classification of Gluconobacter and Aceto- bacter, especially of the so-called intermediate strains. J Gen Appl Asai, T., Iizuka, H. & Komagata, K. (1964). The flagellation and Microbiol 15, 186–196. of genera Gluconobacter and Acetobacter with reference to Yamada, Y., Okada, Y. & Kondo, K. (1976). Isolation and characteri- the existence of intermediate strains. J Gen Appl Microbiol 10, 95–126. zation of ‘polarly flagellated intermediate strains’ in acetic acid bacteria. Brosius, J., Dull, T. J., Sleeter, D. D. & Noller, H. F. (1981). Gene J Gen Appl Microbiol 22, 237–245. http://ijs.sgmjournals.org 817 P. Lisdiyanti and others

Yamada, Y., Hoshino, K. & Ishikawa, T. (1997). The phylogeny of bacteria isolated from Indonesian sources, especially of isolates classi- acetic acid bacteria based on the partial sequences of 16S ribosomal fied in the genus Gluconobacter. J Gen Appl Microbiol 45, 23–28. RNA: the elevation of the subgenus Gluconoacetobacter to the generic Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, S., level. Biosci Biotechnol Biochem 61, 1244–1251. Seki, T., Uchimura, T. & Komagata, K. (2000). Asaia bogorensis gen. Yamada, Y., Hosono, R., Lisdiyanti, P., Widyastuti, Y., Saono, S., nov., sp. nov., an unusual acetic acid bacterium in the α-Proteobacteria. Uchimura, T. & Komagata, K. (1999). Identification of acetic acid Int J Syst Evol Microbiol 50, 823–829.

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