J. Gen. Appl. Microbiol., 52, 55–62 (2006) Short Communication Identification of strains assigned to the genus Asaia Yamada et al. 2000 based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions Pattaraporn Yukphan,1 Taweesak Malimas,1 Wanchern Potacharoen,1 Somboon Tanasupawat,2 Morakot Tanticharoen,1 and Yuzo Yamada1,*,† 1 BIOTEC Culture Collection, National Center for Genetic Engineering and Biotechnology, Pathumthani 12120, Thailand 2 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand (Received August 29, 2005; Accepted December 1, 2005) Key Words——16S-23S rDNA ITS sequences; acetic acid bacteria; Asaia bogorensis; Asaia krungthepensis; Asaia siamensis; restriction analysis; taxonomy The genus Asaia Yamada et al. 2000 was intro- acetic acid. duced with a single species, Asaia bogorensis Ya- For the species-level identification and classification mada et al. 2000 in the family Acetobacteraceae Gillis of acetic acid bacteria, including strains assigned to and De Ley 1980 (Yamada et al., 2000). Since the the genus Asaia, phenotypic characteristics such as second and the third species described were Asaia acid production from sugars and sugar alcohols and siamensis Katsura et al. 2001 and Asaia krungthepen- assimilation of carbon compounds are generally uti- sis Yukphan et al. 2004, three species are in total re- lized (Asai et al., 1964; Katsura et al., 2001; Lisdiyanti ported (Katsura et al., 2001; Yamada et al., 2000; et al., 2002; Yamada et al., 1976, 1999, 2000; Yukphan et al., 2004c). The species assigned to the Yukphan et al., 2004c, d). However, data obtained by genus Asaia were characterized phenotypically by no phenotypic characterization are not only difficult but oxidation or very weak oxidation of ethanol to acetic also very often inaccurate. The phenotypic characteris- acid and by no growth in the presence of 0.35% (w/v) tics obtained are sometimes unreliable. On the other * Address reprint requests to: Dr. Yuzo Yamada, BIOTEC Culture Collection, National Center for Genetic Engineering and Biotech- nology, National Science and Technology Development Agency, 113 Thailand Science Park, Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand. E-mail: [email protected] † JICA Senior Overseas Volunteer, Japan International Cooperation Agency (JICA), Shibuya-ku, Tokyo 151–8558, Japan; Profes- sor Emeritus, Shizuoka University, Shizuoka 422–8529, Japan; Visiting Professor, Faculty of Applied Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156–8502, Japan. Abbreviations: BCC, BIOTEC Culture Collection, National Center for Genetic Engineering and Biotechnology, 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. All the base sequences determined of 16S-23S rDNA ITS regions were deposited in the DDBJ databases under the accession numbers AB230999–AB231011 respectively for strains BCC 15641 (ϭAA01), BCC 15661 (ϭAA35), BCC 15664 (ϭAA38), BCC 15669 (ϭAA44), BCC 15670 (ϭAA49), BCC 15681 (ϭAA98), BCC 15696 (ϭAB30), BCC 15703 (ϭAB40), BCC 15704 (ϭAB41), BCC 15711 (ϭAB58), BCC 15713 (ϭAB73), BCC 15725 (ϭAB82), and BCC 15806 (ϭAC73). 56 YUKPHAN et al. Vol. 52 Table 1. Grouping of 13 representative strains assigned to the genus Asaia by restriction analysis of 16S-23S rDNA ITS regions. Restriction pattern with Cluster in Species and strain Isolate, source, and other designation Group 16S-23S rDNA TaqI MvaI ITS tree A. bogorensis BCC 12264T ϭNBRC 16594T, ϭNRIC 0311T I Ab Ab A A. siamensis BCC 12268T ϭNBRC 16457T, ϭNRIC 0323T II As As B A. krungthepensis BCC 12978T ϭNBRC 100057T, ϭNRIC 0535T III Ak Ak C A. bogorensis BCC 15641 ϭAA01, flower of Hibiscus sp., Bangkok VI ab F BCC 15661 ϭAA35, flower of Plumeria acutifolia, Bangkok I Ab Ab D BCC 15664 ϭAA38, flower of Citharexylum spinosum, Bangkok IV Ab As A BCC 15669 ϭAA44, flower of Jasuminum sambac, Ratchaburi I Ab Ab D BCC 15696 ϭAB30, flower of Ixora sp., Suratthani IV Ab As A BCC 15703 ϭAB40, unknown flower, Samutsakorn VI ab D BCC 15711 ϭAB58, flower of orchid, Bangkok I Ab Ab D BCC 15725 ϭAB82, flower of Allamanda catharitica, Chiang Mai IV Ab As D BCC 15806 ϭAC73, flower of Ipomoea digitata, Kanchanaburi I Ab Ab G A. siamensis BCC 15670 ϭAA49, flower of Canna sp., Ratchaburi V As Ab H BCC 15681 ϭAA98, flower of Canna sp., Ratchaburi V As Ab H A. krungthepensis BCC 15704 ϭAB41, flower of Coccinia grandis, Nonthaburi III Ak Ak E BCC 15713 ϭAB73, flower of Logenaria siceraria, Nonthaburi III Ak Ak E Abbreviations: a, the unidentified a type of patterns; b, the unidentified b type of patterns; Ab, the A. bogorensis type of patterns; As, the A. siamensis type of patterns; Ak, the A. krungthepensis type of patterns. hand, DNA-DNA hybridization was required for a deci- al., 2004a, b, d). This study aims to identify, as the sive criterion in the species-level classification and second model, representative strains isolated from identificationofAsaiastrains(Katsura et al., 2001; Yama- Thai sources and assigned phenotypically to the da et al., 2000; Yukphan et al., 2004c). However, this genus Asaia at the species level on the basis of re- technique is laborious and impossible to construct striction analysis of 16S-23S rDNA ITS regions. databases. Thirteen representative strains were examined in During the course of studies on diversity of acetic this study (Table 1). Asaia bogorensis BCC 12264T acid bacteria in Thailand, we isolated a number of (ϭNBRC 16594TϭNRIC 0311T), Asaia siamensis BCC acetic acid bacteria from flowers collected in Bangkok, 12268T (ϭNBRC 16457TϭNRIC 0323T), and Asaia Thailand. Most of the isolates were phenotypically krungthepensis BCC 12978T (ϭNBRC 100057TϭNRIC identified as Asaia species at the genus level. How- 0535T) were used for reference strains. ever, the species-level identification of the isolates has The 13 representative strains were sequenced for not yet been completed, because of the difficulty and 16S-23S rDNA ITS regions by the modified method of unreliability of phenotypic characterization. Trcˇek and Teuber (2002), as described previously In previous papers, we reported, as the first model, (Yukphan et al., 2004a, b). The 16S-23S rDNA ITS re- that restriction analysis of 16S-23S rDNA internal tran- gions sequenced were of 790 bases in Asaia bogoren- scribed spacer (ITS) regions was applicable to the sis BCC 12264T and Asaia siamensis BCC 12268T, species-level identification and classification of Glu- and 739 bases in Asaia krungthepensis BCC 12978T conobacter strains (Malimas et al., 2006; Yukphan et from position 1, which was based on the A. bogorensis 2006 Identification of Asaia strains 57 numbering system, as indicated in the genus Glu- conobacter (Yukphan et al., 2004b). When the 16S-23S rDNA ITS sequences obtained from the DDBJ databases in accession numbers T AB208551 for Asaia bogorensis BCC 12264T, AB208552 for Asaia siamensis BCC 12268T, and AB208553 for Asaia krungthepensis BCC 12978T were BCC 12978 analyzed with the program NEBcutter (version 2.0; New England BioLabs, Beverly, Massachusetts, USA), the following five restriction endonucleases, TaqI, , BstNI (ϭMvaI), ScrFI, BsrI, and CviJI discriminated the type strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis from one another (Table 2). For T example, restriction endonuclease TaqI cut the 16S- 23S rDNA ITS regions of the type strains of the three Asaia siamensis , known Asaia species mentioned above respectively at BCC 12268 3, 2, and 1 sites. On the other hand, restriction en- donuclease BstNI (ϭMvaI) cut respectively at 2, 1, and 3 sites. The purified 16S-23S rDNA ITS PCR products obtained above for the sequencing were digested with Asaia bogorensis the following two restriction endonucleases, TaqI (Fer- T mentas, Hanover, Maryland, USA) and MvaI (Fermen- . tas;ϭBstNI). As shown in Fig. 1, the restriction fragments result- BCC 12264 ing from digestion with restriction endonucleases TaqI 449, 226, 80, 35501, 289 449, 261, 80 501, 275, 14 450, 289 — 60, 43, 26, 9, 7, 6, 5, 5 44, 43, 27, 7, 5, 5 60, 43, 40, 7, 5, 5 and MvaI coincided in their sizes and their numbers with those calculated theoretically (Table 2). The type strains of the three known Asaia species, i.e., Asaia Asaia krungthepensis T bogorensis, Asaia siamensis, and Asaia krungthepen- and sis, gave their own restriction patterns: the A. bogoren- sis type, the A. siamensis type, and the A. krungthe- pensis type of patterns respectively. The A. bogorensis BCC 12978 type, the A. siamensis type, or the A. krungthepensis type of patterns was shown in the 13 representative T strains except for two strains. The exceptional two strains, BCC 15641 (ϭAA01) and BCC 15703 (ϭ AB40) represented a different kind of restriction pat- BCC 12268 terns, which was designated as the unidentified a type of patterns (Table 1). On digestion with restriction en- T Number of restriction sites in Molecular size of restriction 16S-23S rDNA ITS regions of fragments (bp) in able 2. Restriction endonucleases discriminating the three species, donuclease MvaI, all the 13 representative strains T 120 321 showed the A. bogorensis type, the A. siamensis type, or the A. krungthepensis type of patterns with two ex- BCC 12264 A. bogorensis A. siamensis A. krungthepensis A. bogorensis A. siamensis A. krungthepensis ceptions (Fig. 1). The exceptional two strains, BCC 15641 (ϭAA01) and BCC 15703 (ϭAB40) represented a different kind of restriction patterns, which was des- I) 2 1 3 201 378, 211, 590, 200 294, 212, 201, 32 Mva I I ignated as the unidentified b type of patterns (Table 1). JI 12 11 9 192, 188, 94, 89, 66, 202, 193, 94, 66, 60, 44, 203, 193, 94, 89, FI 5 2 5 340, 201, 198, 38, 12, 1 341, 249, 200 256, 201, 199, 38, 32, 13 ϭ Bsr Taq Cvi Pair-wise sequence similarities (%) in 16S-23S Scr rDNA ITS sequences were calculated.