J. Gen. Appl. Microbiol., 52, 55–62 (2006)

Short Communication

Identification of strains assigned to the genus 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 ; Asaia bogorensis; Asaia krungthepensis; Asaia siamensis; restriction analysis;

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 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 (AA01), BCC 15661 (AA35), BCC 15664 (AA38), BCC 15669 (AA44), BCC 15670 (AA49), BCC 15681 (AA98), BCC 15696 (AB30), BCC 15703 (AB40), BCC 15704 (AB41), BCC 15711 (AB58), BCC 15713 (AB73), BCC 15725 (AB82), and BCC 15806 (AC73). 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 NBRC 16594T, NRIC 0311T I Ab Ab A A. siamensis BCC 12268T NBRC 16457T, NRIC 0323T II As As B A. krungthepensis BCC 12978T NBRC 100057T, NRIC 0535T III Ak Ak C A. bogorensis BCC 15641 AA01, flower of Hibiscus sp., Bangkok VI ab F BCC 15661 AA35, flower of Plumeria acutifolia, Bangkok I Ab Ab D BCC 15664 AA38, flower of Citharexylum spinosum, Bangkok IV Ab As A BCC 15669 AA44, flower of Jasuminum sambac, Ratchaburi I Ab Ab D BCC 15696 AB30, flower of Ixora sp., Suratthani IV Ab As A BCC 15703 AB40, unknown flower, Samutsakorn VI ab D BCC 15711 AB58, flower of orchid, Bangkok I Ab Ab D BCC 15725 AB82, flower of Allamanda catharitica, Chiang Mai IV Ab As D BCC 15806 AC73, flower of Ipomoea digitata, Kanchanaburi I Ab Ab G A. siamensis BCC 15670 AA49, flower of Canna sp., Ratchaburi V As Ab H BCC 15681 AA98, flower of Canna sp., Ratchaburi V As Ab H A. krungthepensis BCC 15704 AB41, flower of Coccinia grandis, Nonthaburi III Ak Ak E BCC 15713 AB73, 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 (NBRC 16594TNRIC 0311T), Asaia siamensis BCC acetic acid bacteria from flowers collected in Bangkok, 12268T (NBRC 16457TNRIC 0323T), and Asaia Thailand. Most of the isolates were phenotypically krungthepensis BCC 12978T (NBRC 100057TNRIC 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 (MvaI), 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 (MvaI) 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;BstNI). 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 (AA01) and BCC 15703 (

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 (AA01) and BCC 15703 (AB40) 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 Bsr Taq Cvi

Pair-wise sequence similarities (%) in 16S-23S Scr rDNA ITS sequences were calculated. The calculated NI ( Restriction Bst pair-wise sequence similarities between Asaia bo- endonuclease 58 YUKPHAN et al. Vol. 52

Fig. 1. Digestion of 16S-23S rDNA ITS PCR products of 13 representative strains assigned to the genus Asaia with restriction endonucleases TaqI and MvaI. For estimation of digestion fragments, 50-bp DNA markers were used in agarose gel electrophoresis. Abbreviations: 1, Asaia bogorensis BCC 12264T; 2, Asaia siamensis BCC 12268T; 3, Asaia krungthepensis BCC 12978T; 4, BCC 15641 (AA01); 5, BCC 15661 (AA35); 6, BCC 15664 (AA38); 7, BCC 15669 (AA44); 8, BCC 15670 (AA49); 9, BCC 15681 (AA98); 10, BCC 15696 (AB30); 11, BCC 15703 (AB40); 12, BCC 15704 (AB41); 13, BCC 15711 (AB58); 14, BCC 15713 (AB73); 15, BCC 15725 (AB82); 16, BCC 15806 (AC73); M, 50-bp DNA marker. gorensis BCC 12264T for 790 bases and Asaia sia- BCC 12268T, and Asaia krungthepensis BCC 12978T, mensis BCC 12268T for 790 bases, between Asaia bo- constituted separate and independent clusters from gorensis BCC 12264T and Asaia krungthepensis BCC one another in a phylogenetic tree based on 16S-23S 12978T for 739 bases, and between Asaia siamensis rDNA ITS sequences (the 16S-23S rDNA ITS tree) BCC 12268T and Asaia krungthepensis BCC 12978T (Fig. 2). However, the 13 representative strains exam- were respectively 94.5, 91.2, and 89.6% in 16S-23S ined gave very complicated topology beyond the spe- rDNA ITS regions. Of the 13 representative strains cific level. Strains BCC 15664 (AA38) and BCC tested, strains BCC 15641 (AA01) for 737 bases and 15696 (AB30) were located in Cluster A, along with BCC 15703 (AB40) for 738 bases showed 91.2, the type strain of Asaia bogorensis. Strains BCC 89.8, and 97.8% and 91.1, 89.6, and 98.3% respec- 15704 (AB41) and BCC 15713 (AB73) were in- tively to the type strains of Asaia bogorensis, Asaia cluded in Cluster E, which appeared to be related to siamensis, and Asaia krungthepensis. Between strains Cluster C of the type strain of Asaia krungthepensis. BCC 15641 (AA01) and BCC 15703 (AB40), the The other nine strains constituted their own four clus- pair-wise sequence similarity was 98.7%. ters, designated as Cluster D, Cluster F, Cluster G, Multiple alignments of the 16S-23S rDNA ITS se- and Cluster H. In Cluster B, there was no strain except quences obtained were made, and distance matrices for the type strain of Asaia siamensis. for the aligned sequences were calculated, as de- Extraction and isolation of bacterial DNAs and DNA- scribed previously (Yukphan et al., 2004a, b). Compar- DNA hybridization were done as described previously ison of the aligned sequences was made for 683 (Yukphan et al., 2004c, d, 2005). Isolated, single- bases in constructing a phylogenetic tree by the neigh- stranded, and labeled DNAs were hybridized with bor-joining method of Saitou and Nei (1987). Robust- DNAs from test strains in 2 SSC and 50% formamide ness for individual branches was estimated by boot- at 48.0°C for 15 h. DNA-DNA similarity (%) was deter- strapping with 1,000 replications (Felsenstein, 1985). mined by the colorimetric method (Verlander, 1992). The type strains of the three known Asaia species, When DNAs from the 13 representative strains were i.e., Asaia bogorensis BCC 12264T, Asaia siamensis hybridized with labeled DNAs from the type strains of 2006 Identification of Asaia strains 59

Fig. 2. A phylogenetic tree based on 16S-23S rDNA ITS sequences for 13 representative strains assigned to the genus Asaia. The phylogenetic tree was constructed by the neighbor-joining method. Abbreviations: A., Asaia; G., Gluconobacter. the three known Asaia species, strains BCC 15641 the type strains of the three known Asaia species were (AA01), BCC 15661 (AA35), BCC 15664 (AA38), grouped into the following six groups (Table 1). BCC 15669 (AA44), BCC 15696 (AB30), BCC Group I was composed of the type strain of A. bo- 15703 (AB40), BCC 15711 (AB58), BCC 15725 gorensis, which showed the A. bogorensis types of (AB82), and BCC 15806 (AC73) showed high patterns with TaqI and MvaI. Four strains, BCC 15661 DNA-DNA similarities respectively of 91, 96, 96, 92, (AA35), BCC 15669 (AA44), BCC 15711 (AB58), 91, 74, 90, 94, and 93% to Asaia bogorensis BCC and BCC 15806 (AC73) were accommodated in 12264T and low DNA-DNA similarities of 25–56% to Group I. These four strains were distributed in Cluster Asaia siamensis BCC 12268T and Asaia krungthepen- D and Cluster G in the 16S-23S rDNA ITS tree (Fig. sis BCC 12978T. However, two strains, BCC 15670 2), and showed high DNA-DNA similarities of 96–90% (AA49) and BCC 15681 (AA98) gave DNA-DNA to the type strain of Asaia bogorensis. Therefore, similarities respectively of 95 and 92% to A. siamensis members of Group I were unequivocally identified as BCC 12268T and DNA-DNA similarities of 44–54% to Asaia bogorensis. the type strains of Asaia bogorensis and Asaia Group II was composed of the type strain of Asaia krungthepensis. In addition, the remaining two strains, siamensis, which showed the A. siamensis types of BCC 15704 (AB41) and BCC 15713 (AB73) had patterns with TaqI and MvaI, and constituted Cluster B DNA-DNA similarities of 97 and 97% to Asaia or the A. siamensis cluster. However, there was no krungthepensis BCC 12978T and DNA-DNA similari- strain that had only the A. siamensis types of patterns ties of 50–52% to the type strains of Asaia bogorensis in the 13 representative strains. Therefore, members and Asaia siamensis. Between the type strains of the of Group II were to be identified as Asaia siamensis. three known Asaia species, i.e., Asaia bogorensis, Group III was composed of the type strain of Asaia Asaia siamensis, and Asaia krungthepensis, used for krungthepensis, which showed the A. krungthepensiss reference strains, the calculated DNA-DNA similarities types of patterns with TaqI and MvaI. Two strains, of 39–52% were low. BCC 15704 (AB41) and BCC 15713 (AB73) were In combination with the resulting restriction patterns included in Group III. These two strains were distrib- with the two restriction endonucleases, TaqI and MvaI uted in Cluster E (Fig. 2) and showed high DNA-DNA (BstNI), the 13 representative strains examined and similarities of 97 and 97% to the type strain of Asaia 60 YUKPHAN et al. Vol. 52 krungthepensis. Therefore, members of Group III were Asaia bogorensis and low DNA-DNA similarities re- unequivocally identified as Asaia krungthepensis. spectively of 50 and 30% to the type strain of Asaia Group IV was composed of strains, which showed krungthepensis, and were therefore identified as Asaia the A. bogorensis type and the A. siamensis type of bogorensis. These phenomena indicated the priority of patterns respectively with TaqI and MvaI. Three DNA-DNA similarity over pair-wise sequence similarity strains, BCC 15664 (AA38), BCC 15696 (AB30), in 16S-23S rDNA ITS sequences in the species-level and BCC 15725 (AB82) were included in Group IV. identification and classification. Of the three, two strains, BCC 15664 (AA38) and The 13 representative strains gave six groups in the BCC 15696 (AB30) were distributed in Cluster A or restriction analysis by use of the two restriction en- the A. bogorensis cluster, and one strain BCC 15725 donucleases, TaqI and MvaI as well as eight clusters (AB82) was in Cluster D (Fig. 2). These three strains in the 16S-23S rDNA ITS tree, along with the type showed high DNA-DNA similarities of 96–91% to the strains of the three known Asaia species, i.e., Asaia type strain of Asaia bogorensis. Therefore, members bogorensis, Asaia siamensis, and Asaia krungthepen- of Group IV were unequivocally identified as Asaia bo- sis. These phenomena contained the following two gorensis. problems: 1) The six groups and the eight clusters ob- Group V was composed of strains which showed the tained were too many for the only three known Asaia A. siamensis type and the A. bogorensis type of pat- species, especially when only two restriction endonu- terns respectively with TaqI and MvaI. Two strains, cleases were used; 2) The numbers of the groups and BCC 15670 (AA49) and BCC 15681 (AA98) were the clusters obtained above did not coincide with each included in Group V. These two strains were distrib- other. uted in Cluster H (Fig. 2), and showed high DNA-DNA The 16S-23S rDNA ITS sequences of the 13 repre- similarities of 95–92% to the type strain of Asaia sia- sentative strains in the genus Asaia appeared to be mensis. Therefore, members of Group V were un- abnormally variable, as contrasted with those in the equivocally identified as Asaia siamensis. genus Gluconobacter, which showed three clusters Group VI was composed of strains which showed that were correlated well to the three known Glu- the unidentified a type and the unidentified b type of conobacter species, i.e., Gluconobacter oxydans, Glu- patterns respectively with TaqI and MvaI. Two strains, conobacter cerinus, and Gluconobacter frateurii BCC 15641 (AA01) and BCC 15703 (AB40), were (Yukphan et al., 2004a, b). In addition, the phyloge- included in Group VI. These two strains were distrib- netic branches found in the 16S-23S rDNA ITS tree of uted respectively in Cluster F and Cluster D (Fig. 2), the strains in the three known Asaia species were ab- and showed high DNA-DNA similarities of 91–74% to normally short, when compared with those of Glu- the type strain of Asaia bogorensis. Therefore, mem- conobacter species (Yukphan et al., 2005). These ab- bers of Group VI were unequivocally identified as normalities are assumed to exhibit the abnormally Asaia bogorensis. The results obtained indicated that vague 16S-23S rDNA ITS phylogenetic tree shown in the restriction analysis mentioned above is useful for this study, in which the 13 representative strains identi- the species-level identification and classification of fied as the three known Asaia species were randomly Asaia strains, since a single species was only recog- distributed beyond the specific level. nized in each group without any exception. By digestion with the two restriction endonucleases, It is of interest that strains BCC 15641 (AA01) of TaqI and MvaI, the 13 representative strains were Group VI and Cluster F and BCC 15703 (AB40) of grouped into six groups. These numbers in grouping Group VI and Cluster D, all of which were similar in the were not consistent with those in clustering, which number of bases (respectively 737 and 738 bases) to showed eight clusters in the 16S-23S rDNA ITS tree. the type strain of Asaia krungthepensis (739 bases), One example was previously reported, in which the showed relatively low pair-wise sequence similarities number of groups and the number of clusters coin- (91.2 and 91.1%) to the type strain of Asaia bogoren- cided with each other using six restriction endonucle- sis and high pair-wise sequence similarities (97.8 and ases, in strains once assigned to Gluconobacter 98.3%) to the type strain of Asaia krungthepensis. frateurii (Malimas et al., 2006). If additional restriction However, the two strains gave high DNA-DNA similari- endonucleases are utilized, the consistency in the ties respectively of 91 and 74% to the type strain of number of groups and the number of clusters will be 2006 Identification of Asaia strains 61 realized. However, these treatments will be practically Acknowledgments meaningless at the specific level for identification of This study was supported in part by the Biodiversity Re- isolates phenotypically assigned to the genus Asaia. search and Training Program, Bangkok, Thailand. Among the 13 representative strains examined, nine strains were identified as Asaia bogorensis, two strains References were identified as Asaia siamensis, and the remaining two strains were identified as Asaia krungthepensis. Asai, T., Iizuka, H., and Komagata, K. (1964) The flagellation From the results obtained above, the following conclu- and taxonomy of genera Gluconobacter and Acetobacter sions can be drawn: 1) Asaia bogorensis is one of the with reference to the existence of intermediate strains. J. Gen. Appl. Microbiol., 10, 95–126. most popular species in the genus Asaia. 2) Besides Felsenstein, J. (1985) Confidence limits on phylogenies: An ap- the three strains reported previously (Yukphan et al., proach using the bootstrap. Evolution, 39, 783–791. 2004c), two additional strains were newly isolated and Katsura, K., Kawasaki, H., Potacharoen, W., Saono, S., Seki, identified as Asaia krungthepensis. 3) Asaia siamensis T., Yamada, Y., Uchimura, T., and Komagata, K. (2001) is a rather rare species in the genus Asaia, since Asaia siamensis sp. nov., an acetic acid bacterium in the a- strains accommodated in this species were only two. . Int. J. Syst. Evol. Microbiol., 51, 559–563. When a certain isolate assigned phenotypically to Lisdiyanti, P., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T., the genus Asaia is, for example, identified at the Yamada, Y., Uchimura, T., and Komagata, K. (2002) Koza- species level, a 16S-23S rDNA ITS PCR product of kaia baliensis gen. nov., sp. nov., a novel acetic acid bac- terium in the a-Proteobacteria. Int. J. Syst. Evol. Microbiol., the isolate is digested first with restriction endonucle- 52, 813–818. ase TaqI. At this first digestion step, the isolate that Malimas, T., Yukphan, P., Takahashi, M., Potacharoen, W., gives either the A. bogorensis type (or the unidentified Tanasupawat, S., Nakagawa, Y., Tanticharoen, M., and Ya- a type), the A. siamensis type, or the A. krungthepen- mada, Y. (2006) Heterogeneity of strains assigned to Glu- sis type of patterns is recognized as a candidate to be conobacter frateurii Mason and Claus 1989 based on re- identified respectively as Asaia bogorensis, Asaia sia- striction analysis of 16S-23S rDNA internal transcribed mensis, or Asaia krungthepensis. At the second diges- spacer regions. Biosci. Biotehnol. Biochem., 70, 684–690. tion step, the restriction analysis with MvaI confirms Saitou, N. and Nei, M. (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. the result obtained above, although exceptional restric- Biol. Evol., 4, 406–425. tion patterns may be sometimes found, as in the A. Trcˇek, J. and Teuber, M. (2002) Genetic and restriction analysis siamensis pattern of strains BCC 15664 ( AA38) and of the 16S-23S rDNA internal transcribed spacer regions of BCC 15696 (AB30) and as in the A. bogorensis pat- the acetic acid bacteria. FEMS Microbiol. Lett., 208, 69–75. tern of strains BCC 15670 (AA49) and BCC 15681 Verlander, C. P. (1992) Detection of horseradish peroxidase by (AA98) (Table 1). However, these exceptional, con- colorimetry. In Nonisotopic DNA Probe Techniques, ed. by tradictory experimental data will act as additional data- Kricka, L. J., Academic Press, New York, pp.185–201. bases. If a restriction pattern different from the above- Yamada, Y., Hosono, R., Lisdiyanti, P., Widyastuti, Y., Saono, mentioned patterns is additionally found, the isolate S., Uchimura, T., and Komagata, K. (1999) Identification of acetic acid bacteria isolated from Indonesian sources, es- will be examined for a new taxon. pecially of isolates classified in the genus Gluconobacter. We isolated a number of Asaia strains from flowers J. Gen. Appl. Microbiol., 45, 23–28. collected in Bangkok as well as in other districts, Thai- Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, land. The restriction analysis of 16S-23S rDNA ITS re- S., Seki, T., Uchimura, T., and Komagata, K. (2000) Asaia gions is, as one of the fastest and the most accurate bogorensis gen. nov., sp. nov., an unusual acetic acid bac- methods, able to identify the isolates at the species terium in the a-Proteobacteria. Int. J. Syst. Evol. Microbiol., level. For example, the isolate that is assigned to one 50, 823–829. of the most popular species, Asaia bogorensis, has to Yamada, Y., Okada, Y., and Kondo, K. (1976) Isolation and characterization of “polarly flagellated intermediate strains” be rejected for selection of candidates representing a in acetic acid bacteria. J. Gen. Appl. Microbiol., 22, 237– new species or a new genus. For this purpose, the re- 245. striction analysis mentioned above will be utilized as Yukphan, P., Malimas, T., Potacharoen, W., Tanasupawat, S., one of the most effective tools. Tanticharoen, M., and Yamada, Y. (2005) Neoasaia chiang- maiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the a-Proteobacteria. J. Gen. Appl. Mi- 62 YUKPHAN et al. Vol. 52

crobiol., 51, 301–311. rDNA internal transcribed spacer regions. J. Gen. Appl. Mi- Yukphan, P., Malimas, T., Takahashi, M., Potacharoen, W., Bu- crobiol., 50, 9–15. sabun, T., Tanasupawat, S., Nakagawa, Y., Tanticharoen, Yukphan, P., Potacharoen, W., Tanasupawat, S., Tanticharoen, M., and Yamada, Y. (2004a) Re-identification of Glu- M., and Yamada, Y. (2004c) Asaia krungthepensis sp. nov., conobacter strains based on restriction analysis of 16S- an acetic acid bacterium in the a-Proteobacteria. Int. J. 23S rDNA internal transcribed spacer regions. J. Gen. Syst. Evol. Microbiol., 54, 313–316. Appl. Microbiol., 50, 189–195. Yukphan, P., Takahashi, M., Potacharoen, W., Tanasupawat, S., Yukphan, P., Potacharoen, W., Nakagawa, Y., Tanticharoen, M., Nakagawa, Y., Tanticharoen, M., and Yamada, Y. (2004d) and Yamada, Y. (2004b) Identification of strains assigned Gluconobacter albidus (ex Kondo and Ameyama 1958) sp. to the genus Gluconobacter Asai 1935 based on the nov., nom. rev., an acetic acid bacterium in the a-Pro- sequence and the restriction analyses of the 16S-23S teobacteria. J. Gen. Appl. Microbiol., 50, 235–242.