70233 (089) Biosci. Biotechnol. Biochem., 72, 70233-1–6, 2008 Asaia lannaensis sp. nov., a New Acetic Acid Bacterium in the Alphaproteobacteria Taweesak MALIMAS,1 Pattaraporn YUKPHAN,1 Mai TAKAHASHI,2 Mika KANEYASU,2 Wanchern POTACHAROEN,1 Somboon TANASUPAWAT,3 Yasuyoshi NAKAGAWA,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, Kisarazu 292-0818, Japan 3Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand Received April 19, 2007; Accepted November 21, 2007; Online Publication, March 7, 2008 [doi:10.1271/bbb.70233] AdvanceAsaia lannaensis sp. nov. was described View for two During the course of studies on acetic acid bacteria strains isolated from flowers of the spider lily collected distributed in the natural environment of Thailand, we in Chiang Mai, Thailand. The isolates produced acetic isolated two strains similar to the type strains of the acid from ethanol on ethanol/calcium carbonate agar, three Asaia species mentioned above. differing from the type strains of Asaia bogorensis, Asaia This paper describes Asaia lannaensis sp. nov. for siamensis, and Asaia krungthepensis, but did not grow in the two strains, which were isolated in Chiang Mai, the presence of 0.35% acetic acid (v/v). The new species Thailand on June 25, 2002. is the fourth of the genus Asaia, the family Acetobacter- aceae. Materials and Methods ProofsT Key words: 16S rDNA restriction analysis; 23S rDNA The two isolates, AB92 and AB93, deposited sequences; acetic acid bacteria; Acetobac- respectively as BCC 15733T (= NBRC 102526T) and teraceae; Asaia lannaensis sp. nov. BCC 15734 (= NBRC 102527), were isolated from flowers of the spider lily (Crynum asiaticum; plub- The genus Asaia Yamada et al. 2000 was set up with a plueng in Thai) by an enrichment culture approach single species, Asaia bogorensis Yamada et al. 2000 using a glucose/ethanol/acetic acid medium, which was as the fifth genus of acetic acid bacteria.1) The second composed of 1.5% D-glucose (w/v), 0.5% ethanol and the third species of the genus Asaia were Asaia (v/v), 0.3% acetic acid (v/v), 0.8% peptone (w/v), siamensis Katsura et al. 20012) and Asaia krungth- and 0.5% yeast extract (w/v), adjusted to pH 3.5, as epensis Yukphan et al. 2004.3) The phenotypic features described previously.1–5) Asaia bogorensis BCC 12264T of strains classified in the three species were charac- (= NBRC 16594T), Asaia siamensis BCC 12268T terized by either no or weak oxidation of ethanol to (= NBRC 16457T), Asaia krungthepensis BCC 12978T acetic acid or by no or very weak growth in the presence (= NBRC 100057T), and Acetobacter aceti NBRC of 0.35% acetic acid (v/v), differing widely in these 14818T were used as reference strains. respects from those of other acetic acid bacteria, such as The 16S rDNA, 16S-23S rDNA ITS, and 23S rDNA strains assigned to the genera Acetobacter, Glucono- sequences of the two isolates were determined, as de- bacter, and Gluconacetobacter. scribed respectively by Yukphan et al.,3,6) by Yukphan 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: ITS, internal transcribed spacer; 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; LMG, Laboratorium voor Microbiologie, Universiteit Gent, Ghent, Belgium; ATCC, American Type Culture Collection, Rockville, MD, USA The 16S rDNA, 16S-23S rDNA ITS, and 23S rDNA sequences of Asaia lannaensis isolates AB92T and AB93 are filed under DDBJ accession numbers AB286050 and AB286051, AB286052 and AB286053, and AB290027 and AB290026 respectively. 70233-2 T. MALIMAS et al. et al.,7,8) and Malimas et al.,9) and by Yukphan et al.10) Asaia species were almost identical with or longer than Multiple sequence alignments were made with the those between the type strains of the three Asaia species. program CLUSTAL X (version 1.81).11) Alignment The calculated pair-wise sequence similarities in 16S gaps and ambiguous bases were excluded from calcu- rDNA sequences of isolate AB92T were 99.6, 99.5, 99.4, lations of similarity. Sequence comparisons were made and 98.9% respectively to the type strains of Asaia for 1,373 bases (positions 28-1472 by the Escherichia bogorensis, Asaia siamensis, Asaia krungthepensis, and coli numbering system, accession no. V00348)12) of 16S Swaminathania salitolerans for 1,369–1,402 bases. The rDNA sequences, for 652 bases (from position 1 by the similarity between the two isolates was 100% for 1,411 specified Asaia bogorensis numbering system, accession bases. no. AB208551)8) of 16S-23S rDNA ITS sequences, for On computerized restriction analysis using the pro- 2,592 bases (position 22-2763 by the E. coli numbering gram NEBcutter (version 2.0), the two isolates, AB92T system)12) of 23S rDNA sequences, and for 4,581 and AB93, showed calculated restriction fragments bases of 16S rDNA/16S-23S rDNA ITS/23S rDNA comprised of (1) 790, 327, 123, 91, and 83 bp with four sequences. Distance matrices were calculated by the restriction sites in StyI digestion; (2) 236, 205, 172, 148, two-parameter method of Kimura (Knuc).13) Phyloge- 123, 102, 91, 87, 87, 55, 29, 28, 24, 16, and 11 bp with netic trees were constructed by the neighbor-joining 14 restriction sites in BsaJI digestion; (3) no restriction method,14) maximum likelihood method,15) and maxi- fragment in SnaBI digestion; and (4) 445, 311, 216, 210, mum parsimony method.16) Robustness of individual 110, 58, 53, and 11 bp with seven restriction sites in branches was estimated by bootstrapping with 1,000 HpaII digestion. The type strains of Asaia bogoresnsis, replications.17) In addition, pair-wise sequence similar- Asaia siamensis, and Asaia krungthepensis showed ities were calculated. restriction fragments comprised respectively of 445, Computerized 16S rDNA restriction analysis of the 421, 216, 210, 55, 53, and 11 bp, 445, 421, 216, 210, 55, twoAdvance isolates was done theoretically with theView program 53, and 11 bp, and 445, 311, 216, 210, 110, 55, 53, and NEBcutter (version 2.0, New England BioLabs, Bever- 11 bp in HpaII digestion. ley, MA, USA).6) The 16S rDNA PCR products of The 16S rDNA PCR products of the two isolates, the two isolates were analyzed by digestion with four AB92T and AB93, were analyzed by digestion with restriction endonucleases of StyI (Fermentas, Hanover, four restriction endonucleases, StyI, BsaJI, SnaBI, and MD, USA), BsaJI (Fermentas), SnaBI (Fermentas), and HpaII. As shown in Fig. 2, the two isolates were HpaII (Fermentas), as described by Yukphan et al.6) practically discriminated from (1) the type strains of Bacterial DNAs were prepared by a modification of Asaia siamensis and Asaia krungthepensis by the the method of Marmur.18–20) DNA base composition was absence of a 214-bp fragment in StyI digestion; (2) determined by the method of Tamaoka and Komagata.21) the type strain ofProofsAsaia siamensis by the absence of a DNA-DNA hybridization was performed by the photo- 327-bp fragment in BsaJI digestion; (3) the type strain of biotin-labeling method with microplate wells, as de- Asaia krungthepensis by the absence of both 850-bp and scribed by Ezaki et al.22) Isolated, single-stranded, 561-bp fragments in SnaBI digestion; and (4) the type labeled DNAs were hybridized with DNAs from test strains of Asaia bogorensis and Asaia siamensis by the strains in 2X SSC and 50% formamide at 48.0 C presence of a 311-bp fragment in HpaII digestion. To for 15 h. Levels of DNA-DNA similarity (%) were summarize, the two isolates showed 16S rDNA restric- determined colorimetrically.23) The color intensity was tion group F type of patterns and were completely T measured at A450 on a model VersaMax microplate distinguished from Asaia bogorensis BCC 12264 of reader (Molecular Devices, Sunnyvale, CA, USA). 16S rDNA restriction group A, Asaia siamensis BCC The isolates were examined for isoprenoid quinone 12268T of 16S rDNA restriction group B, or Asaia as described by Yamada et al.,24) and were examined krungthepensis BCC 12978T of 16S rDNA restriction for phenotypic characteristics, including morphological, group C by the combinations of restriction endonu- physiological, and biochemical characteristics, by the cleases detailed above. methods reported by Asai et al.,25) Yamada et al.,1,4) A phylogenetic tree based on 16S-23S rDNA ITS Katsura et al.,2) and Yukphan et al.3) sequences showed that the two isolates were located within the lineage of the genus Asaia and formed an Results and Discussion independent cluster from the type strains of the three Asaia species, with a bootstrap value of 57% (Fig. 1B). A phylogenetic tree based on 16S rDNA sequences The calculated pair-wise sequence similarities in 16S- showed that the two isolates, AB92T and AB93, were 23S rDNA ITS sequences of isolate AB92T were 96.0, located within the lineage of the genus Asaia, and 95.7, 96.7, and 94.7% respectively to the type strains of formed an independent cluster from the type strains of Asaia bogorensis, Asaia siamensis, Asaia krungthepen- the three Asaia species, viz., Asaia bogorensis, Asaia sis, and Swaminathania salitolerans for 643–700 bases. siamensis, and Asaia krungthepensis, with a bootstrap The similarity between the two isolates for 700 bases value of 68% (Fig.