J. Gen. Appl. Microbiol., 51, 301–311 (2005) Full Paper Neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the a-Proteobacteria Pattaraporn Yukphan,1 Taweesak Malimas,1 Wanchern Potacharoen,1 Somboon Tanasupawat,2 Morakot Tanticharoen,1 and Yuzo Yamada1,*,† 1 BIOTEC Culture Collection (BCC), BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand 2 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand (Received April 5, 2005; Accepted July 14, 2005) An acetic acid bacterium, designated as isolate AC28T, was isolated from a flower of red ginger (khing daeng in Thai; Alpinia purpurata) collected in Chiang Mai, Thailand, at pH 3.5 by use of a glucose/ethanol/acetic acid (0.3%, w/v) medium. A phylogenetic tree based on 16S rRNA gene sequences for 1,376 bases showed that isolate AC28T constituted a cluster along with the type strain of Kozakia baliensis. However, the isolate formed an independent cluster in a phylogenetic tree based on 16S-23S rDNA internal transcribed spacer (ITS) region sequences for 586 bases. Pair-wise sequence similarities of the isolate in 16S rRNA gene sequences for 1,457 bases were 93.0–88.3% to the type strains of Asaia, Kozakia, Swaminathania, Acetobacter, Gluconobacter, Gluconacetobacter, Acidomonas, and Saccharibacter species. Restriction analysis of 16S-23S rDNA ITS regions discriminated isolate AC28T from the type strains of Asaia and Kozakia species. Cells were non-motile. Colonies were pink, shiny, and smooth. The isolate produced acetic acid from ethanol. Oxidation of acetate and lactate was negative. The isolate grew on glu- tamate agar and mannitol agar. Growth was positive on 30% D-glucose (w/v) and in the presence of 0.35% acetic acid (w/v), but not in the presence of 1.0% KNO3 (w/v). Ammoniac nitrogen was hardly assimilated on a glucose medium or a mannitol medium. Production of dihydroxyacetone from glycerol was weakly positive. The isolate did not produce a levan-like polysaccharide on a sucrose medium. Major isoprenoid quinone was Q-10. DNA base composition was 63.1 mol% -G؉C. On the basis of the results obtained, Neoasaia gen. nov. was proposed with Neoasaia chi .(NBRC 101099T؍ BCC 15763T؍) angmaiensis sp. nov. The type strain was isolate AC28T Key Words—16S rRNA gene sequences; 16S-23S rDNA ITS region sequences; acetic acid bacteria; BccI; Hin6I; MboII; Neoasaia chiangmaiensis gen. nov., sp. nov.; Tsp509I * Address reprint requests to: Dr. Yuzo Yamada, BIOTEC Cul- Center for Genetic Engineering and Biotechnology, National ture Collection, National Center for Genetic Engineering and Science and Technology Development Agency, Pathumthani, Biotechnology, National Science and Technology Development Thailand; NBRC, NITE Biological Resource Center (NBRC), Agency, 113 Thailand Science Park, Phaholyothin Road, Klong Department of Biotechnology, National Institute of Technology 1, Klong Luang, Pathumthani 12120, Thailand. and Evaluation, Kisarazu, Chiba, Japan; IFO, Institute for Fer- E-mail: [email protected]; [email protected] mentation, Osaka (IFO), Osaka, Japan; NRIC, NODAI Re- † JICA Senior Overseas Volunteer, Japan International Coop- search Institute Culture Collection Center, Tokyo University of eration Agency (JICA), Shibuya-ku, Tokyo 151–8558, Japan; Agriculture, Tokyo, Japan; NCIB, National Collection of Indus- Professor Emeritus, Shizuoka University, Shizuoka 422–8529, trial Bacteria, Aberdeen, Scotland, UK; ATCC, American Type Japan; Visiting Professor, Faculty of Applied Bioscience, Tokyo Culture Collection, Rockville, Maryland, USA; LMG, Laborato- University of Agriculture, Setagaya-ku, Tokyo 156–8502, Japan. rium voor Microbiologie, Universiteit Gent, Gent, Belgium. Abbreviations: BCC, BIOTEC Culture Collection, National 302 YUKPHAN et al. Vol. 51 Introduction trasted with strains of the three Asaia species. The genus Asaia Yamada et al. 2000 was intro- Materials and Methods duced as the fifth genus in the family Acetobacte- raceae Gillis and De Ley 1980 with a single species, Bacterial strains. An acetic acid bacterium, desig- Asaia bogorensis Yamada et al. 2000 (Yamada et al., nated as isolate AC28T, was used in this study, which 2000). Recently, the additional two species, Asaia sia- was deposited and maintained at BIOTEC Culture Col- mensis Katsura et al. 2001 and Asaia krungthepensis lection (BCC), Pathumthani, Thailand as strain BCC Yukphan et al. 2004 were described (Katsura et al., 15763T and at Culture Collection NBRC, Kisarazu, 2001; Yukphan et al., 2004c). The genus Kozakia Lis- Japan as strain NBRC 101099T. The strain was iso- diyanti et al. 2002 was subsequently proposed as the lated in September 2002 from a flower of red ginger sixth genus with a single species, Kozakia baliensis (khing daeng in Thai; Alpinia purpurata) collected in Lisdiyanti et al. 2002 (Lisdiyanti et al., 2002). Very re- Chiang Mai, Thailand, at pH 3.5 by the enrichment cul- cently, the seventh and the eighth genera were de- ture approach using a glucose/ethanol/acetic acid scribed: Saccharibacter Jojima et al. 2004 and Swami- (0.3%, w/v) medium, but not either a sorbitol medium, nathania Loganathan and Nair 2004 with a single a dulcitol medium, or a sucrose medium (Katsura et species each, Swaccharibacter floricola Jojima et al., 2001; Lisdiyanti et al., 2002; Yamada et al., 1976, al. 2004 and Swaminathania salitolerans Loganathan 1999, 2000; Yukphan et al., 2004c). The type strains and Nair 2004, respectively (Jojima et al., 2004; of Asaia bogorensis (BCC 12264TϭNBRC 16594Tϭ Loganathan and Nair, 2004). Among the four genera NRIC 0311T), Asaia siamensis (BCC 12268TϭNBRC mentioned above, the three genera, Asaia, Kozakia, 16457TϭNRIC 0323T), Asaia krungthepensis (BCC and Swaminathania were related phylogenetically to 12978TϭNBRC 100057TϭNRIC 0535T), Kozakia each other. baliensis (BCC 12275TϭNBRC 16664TϭNRIC 0488T), The genus Asaia was characterized morphologically Gluconobacter oxydans (NBRC 14819T), Acetobacter by peritrichous flagellation and physiologically by no or aceti (NBRC 14818T), Gluconacetobacter liquefaciens weak oxidation of ethanol to acetic acid and no growth (NBRC 12388T), and Acidomonas methanolica (NRIC in the presence of 0.35% acetic acid (w/v). The genus 0498T) were used as reference strains. was quite differentiated in these respects from the Sequencing of 16S rRNA genes. Isolate AC28T genera Kozakia and Swaminathania, which showed no was sequenced for 16S rRNA genes, as described motility, the ability to oxidize ethanol to acetic acid, and previously (Yukphan et al., 2004a, b). A gene fragment growth in the presence of 0.35% acetic acid (w/v) (Ka- specific for 16S rRNA gene-coding regions was ampli- tsura et al., 2001; Lisdiyanti et al., 2002; Loganathan fied by means of a PCR amplification. Two primers, and Nair, 2004; Yamada et al., 2000; Yukphan et al., 20F (5Ј-GAGTTTGATCCTGGCTCAG-3Ј; positions 9– 2004c). 27) and 1500R (5Ј-GTTACCTTGTTACGACTT-3Ј; po- During the course of our studies on microbial diver- sitions 1509–1492) were used. The positions in the sity of acetic acid bacteria in Thailand, we isolated an rRNA gene fragment were based on the Escherichia osmotolerant acetic acid bacterium, which was related coli numbering system (accession number V00348; phylogenetically to the type strains of Asaia bogoren- Brosius et al., 1981). The amplified and purified 16S sis, Asaia siamensis, Asaia krungthepensis, and Koza- rRNA genes were sequenced directly with an ABI kia baliensis in 16S rRNA gene sequences, but differ- PRISM BigDye Terminator v3.1 Cycle Sequencing entiated at the generic level from the genera Asaia and Ready Reaction Kit on an ABI PRISM model 310 Ge- Kozakia. In addition, the isolate formed an indepen- netic Analyzer (Applied Biosystems, Foster City, Cali- dent cluster in a phylogenetic tree based on 16S-23S fornia, USA). The following primers were used for se- rDNA internal transcribed spacer (ITS) region se- quencing: 20F, 1500R, 520F (5Ј-CAGCAGCCGCG- quences. GTAATAC-3Ј; positions 519–536), 520R (5Ј-GTATTAC- This paper describes Neoasaia chiangmaiensis gen. CGCGGCTGCTG-3Ј; positions 536–519), 920F (5Ј- nov., sp. nov., for the acetic acid bacterium that AAACTCAAATGAATTGACGG-3Ј; positions 907–926), showed oxidation of ethanol to acetic acid and growth and 920R (5Ј-CCGTCAATTCATTTGAGTTT-3Ј; posi- in the presence of 0.35% acetic acid (w/v), as con- tions 926–907). 2005 Neoasaia chiangmaiensis gen. nov., sp. nov. 303 Sequencing of 16S-23S rDNA ITS regions. Isolate ϭHhaI). AC28T was sequenced for 16S-23S rDNA ITS regions, DNA base composition and DNA-DNA hybridization. along with the type strains of Asaia bogorensis (BCC Extraction and isolation of bacterial DNAs were made 12264T), Asaia siamensis (BCC 12268T), Asaia by the modified method of Marmur (Ezaki et al., 1983; krungthepensis (BCC 12978T), and Kozakia baliensis Marmur, 1961; Saito and Miura, 1963). DNA base (BCC 12275T) by the modified method of Trcˇek and composition was determined by the method of Teuber (2002), as described previously (Yukphan et Tamaoka and Komagata (1984). DNA-DNA hybridiza- al., 2004a, b). A PCR amplification was made by use tion was carried out by the photobiotin-labeling method of two primers, which were 5Ј-TGCGG(C/T)TGGAT- with microdilution wells, as described by Ezaki et al. CACCTCCT-3Ј (positions 1522–1540 on 16S rDNA by (1989). Isolated, single-stranded, and