70319 (090) Biosci. Biotechnol. Biochem., 72, 70319-1–5, 2008 Tanticharoenia sakaeratensis gen. nov., sp. nov., a New Osmotolerant Acetic Acid Bacterium in the -Proteobacteria Pattaraporn YUKPHAN,1 Taweesak MALIMAS,1 Yuki MURAMATSU,2 Mai TAKAHASHI,2 Mika KANEYASU,2 Somboon TANASUPAWAT,3 Yasuyoshi NAKAGAWA,2 Ken-ichiro SUZUKI,2 y Wanchern POTACHAROEN,1 and Yuzo YAMADA1; ,* 1BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand 2Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation (NITE), Kisarazu 292-0818, Japan 3Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Received May 22, 2007; Accepted November 19, 2007; Online Publication, March 7, 2008 [doi:10.1271/bbb.70319] AdvanceTanticharoenia sakaeratensis gen. nov., sp. View nov. is 1980,1) 10 genera of acetic acid bacteria are presently proposed for three strains isolated from soil collected in recognized: Acetobacter Beijerinck 1898,2) Glucono- Thailand. The three strains, AC37T, AC38, and AC39, bacter Asai 1935,2) Acidomonas Urakami et al. 1989,3,4) were included within a lineage comprising the genera Gluconacetobacter Yamada et al. 1998,5,6) Asaia Asaia, Kozakia, Swaminathania, Neoasaia, Acetobacter, Yamada et al. 2000,7) Kozakia Lisdiyanti et al. 2002,8) Gluconobacter, and Saccharibacter in a phylogenetic tree Swaminathania Loganathan and Nair 2004,9) Sacchari- based on 16S rRNA gene sequences, but formed a quite bacter Jojima et al. 2004,10) Neoasaia Yukphan et al. different, independent cluster. Pair-wise sequence sim- 2006,11,12) and Granulibacter Greenberg et al. 2006.13) ilarities of strain AC37T were 96.5–92.1% to the type Of these 10, the genera Asaia, Kozakia, and Neoasaia strains of Acetobacter aceti, Gluconobacter oxydans, were described forProofs strains isolated from isolation sources Acidomonas methanolica, Gluconacetobacter liquefa- collected in Thailand and Indonesia, Southeast Asia. ciens, Asaia bogorensis, Kozakia baliensis, Swaminathania During the course of studies on microbial diversity salitolerans, Saccharibacter floricola, Neoasaia chiang- in the natural environment of Thailand, we isolated maiensis, and Granulibacter bethesdensis. The three phylogenetically and phenotypically unique acetic acid strains had DNA base compositions comprising respec- bacteria on August 17, 2002. tively 65.6, 64.5, and 65.6 mol % G+C with a range of This paper proposes Tanticharoenia sakaeratensis 1.1 mol %, and formed a single species. Phenotypically, gen. nov., sp. nov. for the isolates in the family the three strains did not oxidize acetate or lactate, but Acetobacteraceae. grew on 30% D-glucose (w/v). Chemotaxonomically, they had Q-10. The type strain is AC37T (= BCC Materials and Methods 15772T = NBRC 103193T). Three strains, AC37T, AC38, and AC39, were used in Key words: Tanticharoenia sakaeratensis gen. nov., sp. this study. They were isolated from soil collected at nov.; acetic acid bacteria; 16S rRNA gene Sakaerat, Nakhon Ratchasima, Thailand by an enrich- sequences; Acetobacteraceae; taxonomy ment culture approach with glucose/ethanol/acetic acid medium, which contained 1.5% D-glucose, 0.5% In the family Acetobacteraceae Gillis and De Ley ethanol (v/v), 0.8% peptone, 0.5% yeast extract, and y To whom correspondence should be addressed. Tel/Fax: +81-54-635-2316; E-mail: [email protected] * JICA Senior Overseas Volunteer, Japan International Cooperation Agency (JICA), Shibuya-ku, Tokyo 151-8558, Japan; Professor Emeritus, Shizuoka University, Shizuoka 422-8529, Japan Abbreviations: BCC, BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, Thailand; NBRC, NITE Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, Kisarazu, Chiba, Japan; NRIC, NODAI Research Institute Culture Collection Center, Tokyo University of Agriculture, Tokyo, Japan; IFO, Institute for Fermentation, Osaka (IFO), Osaka, Japan; LMG, Laboratorium voor Microbiologie, Universiteit Gent, Ghent, Belgium; ATCC, American Type Culture Collection, Rockville, Maryland, USA; NCIB, National Collection of Industrial Bacteria, Aberdeen, Scotland, UK; DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany 70319-2 P. YUKPHAN et al. 0.3% acetic acid (v/v), and was adjusted to pH 3.5, 100% among the three strains. The phylogenetic data among the four kinds of media used.7,8,11,14–16) Aceto- obtained above indicate that the three strains are bacter aceti NBRC 14818T, Gluconobacter oxydans distinguished at the generic level from the above- NBRC 14819T, Gluconacetobacter liquefaciens NBRC mentioned taxa. 12388T, Acidomonas methanolica NRIC 0498T, Asaia The three strains, AC37T, AC38, and AC39, had bogorensis NBRC 16594T, Kozakia baliensis NBRC DNA base compositions respectively of 65.6, 64.5, 16664T, Swaminathania salitolerans LMG 21291T, and and 65.6 mol % G+C with a range of 1.1 mol %. The Neoasaia chiangmaiensis BCC 15763T were used as calculated DNA G+C contents were higher in the acetic reference strains. acid bacteria tested (Table 1). PCR amplification for 16S rRNA genes was made, Labeled DNA of strain AC37T represented DNA- and amplified 16S rRNA genes were sequenced and DNA similarities of 100, 100, 100, and 2% respectively analyzed, as described previously.16–18) Multiple se- to DNAs from strains AC37T, AC38, and AC39, and quence alignment was made with the program CLUSTAL Gluconacetobacter liquefaciens NBRC 12388T, which X (version 1.8).19) Distance matrices for the aligned was used as a reference strain. When isolate AC38 was sequences were calculated using the two-parameter labeled, the calculated DNA-DNA similarities were 97 method of Kimura.20) A phylogenetic tree based on and 100% respectively to strains AC37T and AC38. 16S rRNA gene sequences for 1,374 bases was Similarly, the DNA-DNA similarity to strain AC37T constructed by the neighbor-joining method of Saitou was 100% when strain AC39 was labeled. The genetic and Nei.21) The robustness of individual branches was data obtained above indicate that the three isolates are to estimated by bootstrapping with 1,000 replications.22) be classified in an identical species. In addition, pair-wise sequence similarities were deter- The phenotypic characteristics of strains AC37T, mined for 1,390 bases. AC38, and AC39 are described in the genus and the AdvanceChromosomal DNA was prepared as described View pre- species descriptions. viously.11,16,17) DNA base composition was determined The three strains were especially unique physiolog- by the method of Tamaoka and Komagata.23) Levels ically in that they showed no oxidation of acetate or of DNA-DNA similarity (%) were determined by the lactate, vigorous growth on 30% D-glucose (w/v), and fluorometric method, as described by Ezaki et al.24) no assimilation of ammoniac nitrogen on D-glucose, Isolated, single-stranded, labeled DNAs were hybridized D-mannitol, or ethanol. They had Q-10 as a major with DNAs from test strains at 50.0 C for 3 h in 2 Â isoprenoid quinone. They were also unique ecologically, SSC containing 50% formamide. Fluorescence intensity in that they were isolated from soil, differing in this was measured with Fluoroskan Ascent (Thermo Lab- respect from most acetic acid bacteria. Systems, Helsinki, Finland) at wavelengths of 335 nm In the acetic acidProofs bacteria, 10 genera are presently for excitation and 460 nm for emission. The highest and described in the family Acetobacteraceae: Acetobacter, lowest values obtained in each test sample were Gluconobacter, Acidomonas, Gluconacetobacter, Asaia, excluded, and the mean of the remaining three values Kozakia, Swaminathania, Saccharibacter, Neoasaia, was taken as the DNA-DNA similarity value. and Granulibacter. The three strains form the 11th Phenotypic characteristics were determined by the genus. methods of Asai et al.,25) Yamada et al.,7,14) Katsura Morphologically, the three strains are discriminated by et al.,26) Lisdiyanti et al.,8) and Yukphan et al.11,16,17) an absence of motility and of flagellation from peri- Ubiquinone isoprenologue was determined by the trichously and polarly flagellated, motile strains of the method of Yamada et al.27) genera Acetobacter, Gluconobacter, Acidomonas, Glu- conacetobacter, Asaia, and Swaminathania (Table 1). Results and Discussion Physiologically, an absence of oxidation of acetate and lactate distinguished the three strains from strains of In a phylogenetic tree based on 16S rRNA gene Acetobacter, Acidomonas, Gluconacetobacter, Asaia, sequences, the three strains, AC37T, AC38, and AC39, Kozakia, Swaminathania, Saccharibacter, and Granuli- were included within a lineage comprising the genera bacter species. In addition, the three strains are distin- Asaia, Kozakia, Swaminathania, Neoasaia, Acetobacter, guished from strains of the remaining two genera, Gluconobacter, and Saccharibacter, but formed a quite Gluconobacter and Neoasaia. Growth on 30% D-glucose different, independent cluster (Fig. 1). Pair-wise se- (w/v) was found in the three isolates but not in quence similarities of strain AC37T were calculated to Gluconobacter strains, and a water-soluble brown pig- be 95.3, 95.8, 95.6, 96.3, 96.5, 95.8, 95.6, 92.5, 92.1, and ment was intensely produced in the three isolates but 94.6% respectively to the type strains of Acetobacter