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Ameyamaea Chiangmaiensis Gen. Nov., Sp. Nov., an Acetic Acid Bacterium in the -Proteobacteria

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Ameyamaea Chiangmaiensis Gen. Nov., Sp. Nov., an Acetic Acid Bacterium in the -Proteobacteria Biosci. Biotechnol. Biochem., 73 (10), 2156–2162, 2009 Ameyamaea chiangmaiensis gen. nov., sp. nov., an Acetic Acid Bacterium in the -Proteobacteria Pattaraporn YUKPHAN,1 Taweesak MALIMAS,1 Yuki MURAMATSU,2 Mai TAKAHASHI,2 Mika KANEYASU,2 Wanchern POTACHAROEN,1 Somboon TANASUPAWAT,3 Yasuyoshi NAKAGAWA,2 Koei HAMANA,4 Yasutaka TAHARA,5 Ken-ichiro SUZUKI,2 y Morakot TANTICHAROEN,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 4School of Health Sciences, Faculty of Medicine, Gunma University, Maebashi 371-8514, Japan 5Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan Received January 27, 2009; Accepted July 8, 2009; Online Publication, October 7, 2009 [doi:10.1271/bbb.90070] Two isolates, AC04T and AC05, were isolated from Key words: Ameyamaea chiagmaiensis gen. nov., sp. the flowers of red ginger collected in Chiang Mai, nov.; acetic acid bacteria; 16S rRNA gene Thailand. In phylogenetic trees based on 16S rRNA sequences; 16S rRNA gene restriction anal- gene sequences, the two isolates were included within a ysis; Acetobacteraceae lineage comprised of the genera Acidomonas, Glucona- cetobacter, Asaia, Kozakia, Swaminathania, Neoasaia, In acetic acid bacteria, several new genera have been Granulibacter, and Tanticharoenia, and they formed an reported for strains isolated from isolation sources independent cluster along with the type strain of obtained in Southeast Asia. The first was the genus Tanticharoenia sakaeratensis. The calculated pair-wise Asaia Yamada et al. 2000, with a single species, Asaia sequence similarities of isolate AC04T were 97.8–92.5% bogorensis Yamada et al. 2000.2) Asaia siamensis to the type strains of the type species of the 11 genera Katsura et al. 2001,3) Asaia krungthepensis Yukphan of acetic acid bacteria. The DNA base composition et al. 2004,4) and Asaia lannensis Malimas et al. 20085,6) was 66.0–66.1 mol % G+C with a range of 0.1 mol %. were additionally described. The second and the third A single-stranded, labeled DNA from isolate AC04T were the genus Kozakia Lisdiyanti et al. 20027) and the presented levels of DNA-DNA hybridization of 100, 85, genus Neoasaia Yukphan et al. 2006,8,9) with Kozakia 4, and 3% respectively to DNAs from isolates AC04T baliensis Lisdiyanti et al. 2002 and Neoasaia chiang- and AC05 and the type strains of Tanticharoenia maiensis Yukphan et al. 2006 respectively. The fourth sakaeratensis and Gluconacetobacter liquefaciens. The was recently described as the genus Tanticharoenia two isolates were unique morphologically in polar Yukphan et al. 200810,11) with Tanticharoenia sakaer- flagellation and physiologically in intense acetate oxida- atensis Yukphan et al. 2008. tion to carbon dioxide and water and weak lactate In the course of studies on microbial diversity in the oxidation. The intensity in acetate oxidation almost natural environment of Thailand, a chance to isolate two equaled that of the type strain of Acetobacter aceti. The types of unique acetic acid bacteria was given on the two isolates had Q-10. Isolate AC04T was discriminated same day, September 20, 2002, from the same kind of from the type strains of the type species of the 11 genera isolation sources obtained at the same place, the campus by 16S rRNA gene restriction analysis using restriction of Chiang Mai University, Chiang Mai, in the northern endonucleases TaqI and Hin6I. The unique phylogenet- district of Thailand. One was the type strain of Neoasaia ic, genetic, morphological, physiological, and biochem- chiagmaiensis,8) and the others were two isolates ical characteristics obtained indicate that the two discussed below. isolates can be classified into a separate genus, and This paper proposes Ameyamaea chiangmaiensis Ameyamaea chiangmaiensis gen. nov., sp. nov. is pro- gen. nov., sp. nov. for the two isolates accommodated posed. The type strain is isolate AC04T (¼ BCC 15744T, to the family Acetobacteraceae Gillis and De Ley ¼ NBRC 103196T), which has a DNA G+C content of 1980.12) 66.0 mol %. 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, Suruga-ku, Shizuoka 422-8529, Japan The DDBJ accession numbers of the 16S rRNA gene sequences are AB303366 and AB303367 for isolates AC04T and AC05 respectively.1) Ameyamaea chiangmaiensis gen. nov., sp. nov. 2157 Materials and Methods ever, the phylogenetic distance between the isolates and the type strain of the Tanticharoenia species was much Isolates AC04T and AC05 were examined. They were isolated from longer than that between the type strains of the Asaia the flowers of red ginger (khing daeng in Thai, Alpinia purpurea) and Swaminathania species. In addition, the type strains collected in Chiang Mai, Thailand by an enrichment culture approach of Kozakia baliensis and Neoasaia chiangmaiensis gave using a sorbitol medium, which contained 2.0% D-sorbitol, 0.3% peptone, and 0.3% yeast extract (all by w/v) and was adjusted to a similar cluster, with a bootstrap value of 85%, to that pH 3.5, among the four media used.2–5,7,8,10,13,14) Acetobacter aceti of the isolates and the type strain of the Tanticharoenia NBRC 14818T (NBRC, NITE Biological Resource Center, Department species. In both clusters, the phylogenetic distances were of Biotechnology, National Institute of Technology and Evaluation, almost identical to each other. The cluster comprised of Kisarazu, Chiba, Japan), Gluconobacter oxydans NBRC 14819T, the two isolates and the type strain of the Tanticharoenia T Acidomonas methanolica NRIC 0498 (NRIC, NODAI Research species was independently connected to a large cluster Institute Culture Collection Center, Tokyo University of Agriculture, of the genera Asaia, Swaminathania, Kozakia, Neoasaia, Tokyo, Japan), Gluconacetobacter liquefaciens NBRC 12388T, Asaia bogorensis NBRC 16594T, Kozakia baliensis NBRC 16664T, Swami- Acidomonas, Granulibacter, and Gluconacetobacter, nathania salitolerans LMG 21291T (LMG, Laboratorium voor Micro- with a bootstrap value of 85%. biologie, Universiteit Gent, Ghent, Belgium), Saccharibacter floricola In a phylogenetic tree constructed by the maximum BCC 16445T (BCC, BIOTEC Culture Collection, National Center parsimony method, the cluster of the two isolates and the for Genetic Engineering and Biotechnology, National Science and type strain of the Tanticharoenia species had a bootstrap Technology Development Agency, Pathumthani, Thailand), Neoasaia value of 75%, connected itself with the cluster com- chiangmaiensis T Granulibacter bethesdensis BCC 15763 , ATCC BAA prised of the genera Kozakia, Neoasaia, Acidomonas, 1260T (ATCC, American Type Culture Collection, Rockville, MD, USA), and Tanticharoenia sakaeratensis BCC 15772T were used as Granulibacter, and Gluconacetobacter, with a bootstrap reference strains. value below 50%, and formed a large cluster together PCR amplification of the 16S rRNA genes was done, and the with the cluster of the genera Asaia and Swaminathania, amplified 16S rRNA genes were sequenced and analyzed as described with a bootstrap value of 53% (Fig. 1B). The phyloge- previously.4,8,15) Multiple sequence alignment was done with the netic distance between the two isolates and the type 16) program CLUSTAL X (version 1.8). Alignment gaps and unidenti- strain of the Tanticharoenia species was almost identical fied bases were eliminated. Distance matrices for the aligned sequences were calculated by the two-parameter method of Kimura.17) Phyloge- to that between the type strains of the Kozakia and netic trees were constructed for 1,374 bases by the neighbor-joining,18) Neosasaia species and much longer than that between maximum parsimony,19) and maximum likelihood20) methods. The the type strains of the Asaia and Swaminathania species, robustness of individual branches was estimated by bootstrapping21) as found in the phylogenetic tree constructed by the with the program MEGA (version 4.0).22) Bootstrap values below 50% neighbor-joining method. were not shown. In constructing the phylogenetic tree by the maximum In the phylogenetic tree constructed by the maximum likelihood method, the program PHYLIP (version 3.6; J. Felsenstein, likelihood method, the cluster of the two isolates and the University of Washington) was used. Pair-wise sequence similarities were calculated in 16S rRNA gene sequences of 1,390 bases. type strain of the Tanticharoenia species was connected A computerized 16S rRNA gene restriction analysis was done using to a cluster comprised of the genera Gluconacetobacter, NEBcutter (version 2.0; New England BioLabs, Ipswich, MA, USA) Acidomonas, and Granulibacter with a bootstrap value for isolate AC04T and the type strains of the type species of the 11 below 50%, and the resulting cluster formed a large genera of acetic acid bacteria. The 16S rRNA gene PCR products of cluster with the cluster of the genera Asaia, Swamina- isolate AC04T and the type strains were prepared and digested with 23) thania, Kozakia, and Neoasaia, with a bootstrap value restriction endonucleases
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