Asaia Lannaensis Sp. Nov., a New Acetic Acid Bacterium in the Alphaproteobacteria

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 the View 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. 1A). The phylogenetic distances was 100%. between isolate AB92T and the type strains of the three A phylogenetic tree based on 23S rDNA sequences Asaia lannaensis sp. nov. 70233-3

T 100 A. lannaensis isolate AB92 (AB286052) T A. bogorensis isolate 71 (AB025928) A. lannaensis isolate AB93 (AB286053) T 57 53 A. siamensis isolate S60-1 (AB025932) 91 T A T B A. bogorensis BCC 12264 (AB208551) A. krungthepensis isolate AA08 (AB025928) 94 68 A. krungthepensis BCC 12978T (AB208553) A. lannaensis isolate AB92T (AB286050) 99 98 100 T A. lannaensis isolate AB93 (AB286051) A. siamensis BCC 12268 (AB208552) 52 T S. salitolerans strain PA51T (AF459454) S. salitolerans LMG 21291 (AB220163) T T 76 N. chiangmaiensis isolate AC28 (AB208549) N. chiangmaiensis isolate AC28 (AB208550) T K. baliensis isolate Yo-3 (AB056321) K. baliensis BCC 12275T (AB208554) G. oxydans ATCC 19357T (X73820) G. oxydans NBRC 14819T (AB111901) Knuc. 0.005 Knuc. 0.05

A. bogorensis BCC 12264T (AB255429) A. lannaensis isolate AB92T 66 100 T 81 A. krungthepensis BCC 12978 (AB255431) 57 A. lannaensis isolate AB93 T T C S. salitolerans LMG 21291 (AB255434) D 82 A. siamensis BCC 12268 T T 100 A. siamensis BCC 12268 (AB255429) A. bogorensis BCC 12264 100 A. lannaensis isolate AB92T (AB290027) 96 A. krungthepensis BCC 12978T 100 100 100 T A. lannaensis isolate AB93 (AB290026) S. salitolerlans LMG 21291 K. baliensis BCC 12275T (AB255432) K. baliensis BCC 12275T T N. chiangmaiensis BCC 15763 (AB255433) N. chiangmaiensis BCC 15763T T G. oxydans NBRC 14819 (AB257083) G. oxydans NBRC 14819T Knuc. Knuc. 0.005 0.01

T T 43 A. krungthepensis isolate AA08 (AB025928) 37 A. bogorensis isolate 71 (AB025928) T 56 A. bogorensis isolate 71T (AB025928) 42 A. krungthepensis isolate AA08 (AB025928) T T E 44 A. siamensis isolate S60-1 (AB025932) F 46 A. siamensis isolate S60-1 (AB025932) A. lannaensis isolate AB92T (AB286050) A. lannaensis isolate AB92T (AB286050) 84 64 92 83 A. lannaensis isolate AB93 (AB286051) A. lannaensis isolate AB93 (AB286051) T S. salitolerans strain PA51T (AF459454) S. salitolerans strain PA51 (AF459454) T T 42 N. chiangmaiensis isolate AC28 (AB208549) 70 K. baliensis isolate Yo-3 (AB056321) T AdvanceK. baliensis View isolate Yo-3T (AB056321) N. chiangmaiensis isolate AC28 (AB208549) G. oxydans ATCC 19357T (X73820) G. oxydans ATCC 19357T (X73820) Knuc. Knuc. 0.002 0.002

Fig. 1. Phylogenetic Relationships of Asaia lannaensis Isolates AB92T and AB93. Phylogenetic trees based on 16S rDNA (A), 16S-23S rDNA ITS (B), 23S rDNA (C), and 16S rDNA/16S-23S rDNA ITS/23S rDNA (D) sequences were constructed by the neighbor-joining method. Two additional phylogenetic trees based on 16S rDNA sequences were constructed by the maximum likelihood method (E) and the maximum parsimony method (F). The type strain of Gluconobacter oxydans was used as an outgroup. Numbers at nodes indicate bootstrap percentages derived from 1,000 replications. Abbreviations: A., Asaia; K., Kozakia; S., Swaminathania; N., Neoasaia: G., Gluconobacter Proofs Table 1. DNA-DNA Hybridization of Asaia lannaensis Isolates AB92T and AB93

DNA G+C DNA-DNA similarity (%) of Labeled DNA from (mol %) 123456 Asaia lannaensis isolate AB92T 60.8 100 100 13 14 29 3 Asaia lannaensis isolate AB93 60.9 100 100 18 9 23 2 Asaia bogorensis BCC 12264T 60.2 22 30 100 19 29 2 Asaia siamensis BCC 12268T 59.3 23 26 11 100 27 6 Asaia krungthepensis BCC 12978T 60.3 12 20 14 24 100 2

Abbreviations:1,Asaia lannaensis isolate AB92T;2,Asaia lannaensis isolate AB93; 3, Asaia bogorensis BCC 12264T;4,Asaia siamensis BCC 12268T;5,Asaia krungthepensis BCC 12978T;6,Acetobacter aceti NBRC 14818T The DNA G+C contents of the type strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis were cited respectively from Yamada et al.,1) Katsura et al.,2) and Yukphan et al.3) respectively. showed that the two isolates were located within the ilarity between the two isolates was 100% for 2,600 lineage of the genus Asaia and formed a small cluster bases. When a phylogenetic tree was constructed on the most distantly from the type strain of Asaia bogorensis, basis of the combined 16S rDNA/16S-23S rDNA ITS/ the type species of the genus Asaia, with a bootstrap 23S rDNA sequences, similar phylogenetic relationships value of 100% (Fig. 1C). The type strain of Swamina- were obtained (Fig. 1D), as found in the phylogenetic thania salitolerans Loganathan and Nair 200426) was trees based on 16S rDNA sequences and 16S-23S rDNA likewise within the Asaia cluster, as reported previous- ITS sequences. ly.10) The calculated pair-wise sequence similarities in The DNA G+C contents of the two isolates, AB92T 23S rDNA sequences of isolate AB92T were 99.3, 99.1, and AB93, were respectively 60.8 and 60.9 mol %. 99.2, and 98.9% respectively to the type strains of Asaia The calculated values were slightly higher than those of bogorensis, Asaia siamensis, Asaia krungthepensis, and the type strains of Asaia bogorensis, Asaia siamensis, Swaminathania salitolerans for 2,600 bases. The sim- and Asaia krungthepensis (Table 1). 70233-4 T. MALIMAS et al.

AB Asaia krungthepensis, and Acetobacter aceti. When isolate AB93 was labeled, the DNA-DNA similarities were 100, 100, 18, 9, 23, and 2% respectively. The type strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis gave DNA-DNA similarities of 12–30% to the two isolates (Table 1). These data indicate that the two isolates constitute a new species in the genus Asaia genetically. In addition to the phylogenetic tree shown in Fig. 1A, two phylogenetic trees were constructed on the basis CD of 16S rDNA sequences by the maximum likelihood method (Fig. 1E) and maximum parsimony method (Fig. 1F). In the three phylogenetic trees based on 16S rDNA sequences constructed, similar clustering was found for the two isolates. These data indicate that the presence of the new species is unequivocally confirmed molecular-phylogenetically. In the two isolates, ubiquinone-10 was determined as a major quinone, as found in the type strains of the Fig. 2. Restriction of 16S rDNA PCR Products of Asaia lannaensis three Asaia species. Isolates AB92T and AB93 by Digestion with Four Restriction The phenotypic characteristics of the two isolates are Endonucleases. described in the species description. AdvanceThe four restriction endonucleases used were StyI (A), ViewBsaJI (B), It is noted that the isolates produced acetic acid from SnaBI (C), and HpaII (D). Abbreviations:1,Asaia bogorensis BCC ethanol on ethanol/calcium carbonate agar, differing in 12264T (16S rDNA restriction group A); 2, Asaia siamensis BCC 12268T (16S rDNA restriction group B); 3, Asaia krungthepensis this respect from the type strains of Asaia bogorensis, BCC 12978T (16S rDNA restriction group C); 4, Asaia lannaensis Asaia siamensis, and Asaia krungthepensis (Table 2). isolate AB92T (16S rDNA restriction group F); 5, Asaia lannaensis However, the isolates did not grow in the presence of isolate AB93 (16S rDNA restriction group F); M, 50-bp DNA ladder 0.35% acetic acid (v/v), as found for the type strains of the three Asaia species. The isolates produced 2-keto-D- The labeled DNA from isolate AB92T showed gluconate and 5-keto-D-gluconate but not 2,5-diketo-D- DNA-DNA similarities of 100, 100, 13, 14, 29, and gluconate from D-glucose. In acid production from 3% respectively to isolate AB92T, isolate AB93, and ethanol and L-arabitol,Proofs the two isolates were weakly the type strains of Asaia bogorensis, Asaia siamensis, positive. These characteristics differentiated the two

Table 2. Characteristics Diﬀerentiating Asaia lannaensis Isolates AB92T and AB93 from the Type Strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis

A. bogorensis A. siamensis A. krungthepensis Isolate Characteristic isolate 71T isolate S60-1T isolate AA08T AB92T AB93 Oxidation of acetate w w w w w and lactate Production of acetic acid ++ from ethanol on ethanol/calcium carbonate agar Dihydroxyacetone w + + w w production from glycerol Acid production from Dulcitol + +ww L-Arabitol + + + w w Ethanol w w w Maltose w 16S rDNA restriction A B C F F group DNA G+C content 60.2 59.3 60.3 60.8 60.9 (mol %)

+, positive; , negative; w, weakly positive; w, weakly positive, but the color change occurred within three days. The characteristics of the type strains of the three Asaia species are cited from Yamada et al.,1) Katsura et al.,2) and Yukphan et al.,3) except for acid production from L-arabitol and the 16S rDNA restriction groups. The 16S rDNA restriction groups, D and E, have been represented by Yukphan et al.6) and Huong et al.27) respectively. Asaia lannaensis sp. nov. 70233-5 isolates from the type strains of the three Asaia species nology (BIOTEC), Pathumthani, Thailand, as BCC (Table 2). 15733T, and in the culture collection of The National As described above, the two isolates were genetically, Biological Resource Center (NBRC), Department of molecular-phylogenetically, chemotaxonomically, and Biotechnology, National Institute of Technology and phenotypically distinguished from the type strains of the Evaluation, Kisarazu, Chiba, Japan, as NBRC 102526T. three Asaia species and can be classified as a new species in the genus Asaia. The name Asaia lannaensis Acknowledgments is proposed for the two isolates. We express our sincere thanks to Dr. Watcharee Description of Asaia lannaensis sp. nov. Hanmoungjai and Dr. Saisamorn Lumyong, Professor, Asaia lannaensis (lan.na.en’sis. Lanna: L. fem. suffix Chiang Mai University, Chiang Mai, Thailand, for their -ensis indicating geographical origin; N. L. fem. adj. help in collecting isolation sources. This study was lannaensis of or pertaining to Lanna, the old name of supported in part by the Bioresources Research Network the region including Chiang Mai, Northern Thailand, (BRN), National Center for Genetic Engineering and Thailand, where the type strain was isolated). Biotechnology (BIOTEC), Pathumthani, Thailand. Cells are Gram-negative and rod-shaped, measuring 0:6{1:0 1:0{2:0 mm on glucose/ethanol/calcium car- References bonate agar. Peritrichous flagella when motile. Colonies are pink, shiny, smooth, and raised with an entire margin 1) Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T., Uchimura, T., and Komagata, K., on glucose/ethanol/calcium carbonate agar. Grows at Asaia bogorensis gen. nov., sp. nov., an unusual acetic pH 3.0 and 3.5. However, maximum growth occurs at acid bacterium in the -Proteobacteria. Int. J. Syst. Evol. pH 5.5. Grows on 30% glucose (w/v). No growth in Microbiol., 50, 823–829 (2000). theAdvance presence of either 0.35% acetic acid (v/v) View or 1.0% 2) Katsura, K., Kawasaki, H., Potacharoen, W., Saono, S., potassium nitrate (w/v). Methanol is not assimilated as Seki, T., Yamada, Y., Uchimura, T., and Komagata, sole carbon source. Oxidizes acetate and lactate to K., Asaia siamensis sp. nov., an acetic acid bacterium in carbon dioxide and water, but the activity is not intense. the -Proteobacteria. Int. J. Syst. Evol. Microbiol., 51, Acetic acid is produced on ethanol/calcium carbonate 559–563 (2001). agar. Produces 2-keto-D-gluconate and 5-keto-D-gluco- 3) Yukphan, P., Potacharoen, W., Tanasupawat, S., Tanticharoen, M., and Yamada, Y., Asaia krungth- nate, but not 2,5-diketo-D-gluconate from D-glucose. Grows on glutamate agar and mannitol agar. Grows on a epensis sp. nov., an acetic acid bacterium in the - Proteobacteria. Int. J. Syst. Evol. 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(weakly positive), L-arabinose, L-rhamnose, D-mannitol, 5) Yamada, Y., Hosono, R., Lisdiyanti, P., Widyastuti, Y., D-sorbitol (weakly positive), dulcitol (weakly positive), Saono, S., Uchimura, T., and Komagata, K., Identifica- D-arabitol, L-arabitol (weakly positive), meso-ribitol, tion of acetic acid bacteria isolated from Indonesian meso-erythritol, glycerol, melibiose, sucrose (weakly sources, especially of isolates classified in the genus positive in isolate AB92T), raffinose (weakly positive Gluconobacter. J. Gen. Appl. Microbiol., 45, 23–28 in isolate AB92T but negative in AB93), or ethanol (1999). 6) Yukphan, P., Malimas, T., Takahashi, M., Kaneyasu, (weakly positive). Grows on D-glucose, D-mannose, D- M., Potacharoen, W., Tanasupawat, S., Nakagawa, Y., galactose, D-fructose, L-sorbose, D-xylose, D-arabinose, Tanticharoen, M., and Yamada, Y., Identification of L-arabinose, L-rhamnose, D-mannitol, D-sorbitol, dulci- strains assigned to the genus Asaia Yamada et al. 2000 tol, D-arabitol, L-arabitol (weakly positive in isolate based on 16S rDNA restriction analysis. J. Gen. Appl. AB93), meso-ribitol, meso-erythritol, glycerol, meli- Microbiol., 52, 241–247 (2006). biose, sucrose (weakly positive in isolate AB92T), 7) Yukphan, P., Malimas, T., Takahashi, M., Potacharoen, raffinose (weakly positive in isolate AB92T but negative W., Busabun, T., Tanasupawat, S., Nakagawa, Y., in AB93), or ethanol (weakly positive), but not on Tanticharoen, M., and Yamada, Y., Re-identification of maltose or lactose. Shows 16S rDNA restriction group Gluconobacter strains based on restriction analysis of F-type of patterns. The major ubiquinone is Q-10. 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