Microbes Environ. Vol. 24, No. 2, 135–143, 2009 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME08564 Identification of Acetobacter, Gluconobacter, and Asaia Strains Isolated in Thailand Based on 16S-23S rRNA Gene Internal Transcribed Spacer Restriction and 16S rRNA Gene Sequence Analyses SOMBOON TANASUPAWAT1*, JINTANA KOMMANEE1, TAWEESAK MALIMAS2, PATTARAPORN YUKPHAN2, YASUYOSHI NAKAGAWA3, and YUZO YAMADA2, 4, 5 1Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Wangmai, Bangkok 10330, Thailand; 2BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand; 3Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation (NITE), 2-5-8, Kazusakamatari, Kisarazu Chiba 292-0818, Japan; 4Japan International Cooperation Agency (JICA Senior Overseas Volunteer), 2-1-1 Yoyogi, Shibuya-ku, Tokyo 151-8558, Japan; and 5Shizuoka University (Professor Emeritus), 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan (Received December 7, 2008—Accepted March 8, 2009—Published online April 10, 2009) Twenty-six strains of acetic acid bacteria were isolated from fruits, flowers and related materials collected in Thai- land. They were divided into three genera, Acetobacter, Gluconobacter and Asaia, by phenotypic characterization and 16S rRNA gene sequence analyses. On the basis of 16S-23S rRNA gene internal transcribed spacer (16S-23S rDNA ITS) restriction and 16S rRNA gene sequence analyses, fourteen isolates assigned to the genus Acetobacter were divided into five groups: 1) Group 1A or A. tropicalis (one isolate); 2) Group 2A or A. orientalis (four isolates); 3) Group 3A or A. pasteurianus (five isolates); 4) Group 4A or A. syzygii (one isolate); and 5) Group 5A or A. ghanensis (three isolates). The eleven isolates assigned to the genus Gluconobacter were divided into three groups: 6) Group 1B or G. frateurii (four isolates); 7) Group 2B or G. japonicus (six isolates); and 8) Group 3B or unidentified (one isolate). The remaining isolate was placed into: 9) Group 1C or unidentified, which was assigned to the genus Asaia and con- sidered to constitute a new species on the basis of the 16S rRNA gene sequence analysis and DNA-DNA hybridization. Key words: Acetic acid bacteria, Acetobacter, Asaia, Gluconobacter, 16S-23S rDNA ITS restriction analyses Acetic acid bacteria (AAB) are Gram-negative, aerobic, cerinus (9) isolated from fruits, flowers and other materials rod-shaped bacteria assigned to the Alphaproteobacteria. collected in Thailand have been found. In the course of iso- These bacteria have undergone considerable changes, con- lating, identifying and classifying acetic acid bacteria in sequently increasing the number of novel genera and species. Thailand, we have continued the taxonomic study of Thai Presently, there are a total of 11 genera (Euzéby, 2008, isolates based on 16S-23S rRNA gene internal transcribed URL: http://www.bacterio.net): Acetobacter, Gluconobacter, spacer (16S-23S rDNA ITS) restriction and 16S rRNA gene Acidomonas, Gluconacetobacter, Asaia, Kozakia, Swami- sequence analyses as well as phenotypic characterization and nathania, Saccharibacter, Neoasaia, Granulibacter, and DNA-DNA hybridization. This paper is concerned with the Tanticharoenia (7, 10, 12, 13, 18, 19, 25, 28, 33, 38, 39, identification of novel isolates from fruits, flowers, and other 42–45). These bacterial strains have been isolated from materials. fruits, flowers, vinegar, grapes, wine, sake, cider, beer, soft drink, nata, sugarcane, bee hives, tea fungus, plant materials Materials and Methods and so on (13). Many new acetic acid bacteria have been reported in Thai- Isolation of acetic acid bacteria and reference strains land, including the genera Neoasaia and Tanticharoenia, and Thirteen fruits, two other materials collected at markets and two the species Asaia krungthepensis, Gluconobacter albidus, kinds of flowers that fell from trees were cut into small pieces and enriched using a glucose/ethanol/yeast extract (GEY) medium. An Gluconobacter thailandicus, Neoasaia chiangmaiensis, isolation source was incubated at pH 4.5 and 30°C for 3–5 days in a Gluconobacter kondonii, Gluconobacter roseus, Asaia liquid medium (15 mL tube−1) composed of 2.0% D-glucose, 5.0% lannaensis, Tanticharoenia sakaeratensis, and Gluconobacter ethanol and 1.0% yeast extract (all by w/v). When microbial growth japonicus (11, 21–24, 31, 39, 40, 42–45). In addition, Aceto- was seen, the culture was streaked onto a GEY-agar plate contain- bacter pasteurianus, Gluconacetobacter liquefaciens, Asaia ing 0.3% CaCO3 (w/v) (36). Acetic acid bacteria were selected as acid-producing bacterial strains, which formed clear zones bogorensis, Asaia siamensis, Acetobacter orientalis, Aceto- T around colonies on the agar plate. A. indonesiensis NBRC 16471 , bacter lovaniensis, Gluconobacte frateurii (15, 20, 27, 41, 44), A. cibinongensis NBRC 16605T, A. lovaniensis NBRC 13753T, and all seven groups of Gluconobacter except Gluconobacter A. tropicalis NBRC 16470T, A. orientalis NBRC 16606T, A. pasteurianus TISTR 1056T, A. aceti NBRC 14818T, A. syzygii * Corresponding author. E-mail: [email protected]; Tel: NBRC 16604T, A. ghanensis LMG 23848, G. frateurii NBRC +662–2188376; Fax: +662–2188376. 3264T, G. thailandicus BCC 14116T, G. japonicus NBRC 3271T, G. 136 TANASUPAWAT et al. cerinus NBRC 3267T, G. oxydans NBRC 14819T, G. albidus NBRC and subjected to thin-layer chromatography on a silica gel plate T T T 3250 , G. kondonii NBRC 3266 ; Asaia lannaensis BCC 15733 , (20×20 cm, silica gel 60F254, Art 5715, E. Merck, Darmstadt, Ger- Asaia bogorensis BCC 12264T, Asaia siamensis BCC 12268T, and many) with a solvent system of pure benzene (35). The purified Asaia krungthepensis BCC 12978T were used as reference strains. ubiquinone preparation was subjected to reversed-phase paper chromatography (35, 37) and to high performance liquid chromato- Phenotypic characterization graphy (29). Phenotypic characterization was carried out by incubating isolates at 30°C and pH 6.8 for two days on glucose/yeast extract/ 16S rRNA gene sequence analyses peptone/glycerol (GYPG) agar, which was composed of 1.0% D- The 16S rRNA genes of strains were amplified by PCR with Taq glucose, 1.0% glycerol, 0.5% yeast extract, 1.0% peptone, and 1.5% DNA polymerase and primers; 20F (5'-GAGTTTGATCCTGGCT- agar (all by w/v), unless otherwise mentioned. For the Gram stain- CAG’-3, the Escherichia coli numbering system (2) and 1500R (5'- ing of bacterial cells, the Hucker-Conn modified method was used GTTACCTTGTTACGACTT’-3) and sequenced, as described pre- (8). Physiological and biochemical characterizations were made by viously (31, 40, 43). The sequencing was carried out with an ABI the methods of Asai et al. (1), Gosselé et al. (6), and Yamada et al. PRISM BigDye Terminator v3.1 cycle sequencing kit on an ABI (36). Isolates were grown in GEY medium on a rotary shaker (150– PRISM model 310 Genetic Analyzer (Applied Biosystems, Foster 200 rpm) at 30°C for 24 h. City, CA, USA). The following six primers were used for 16S rRNA gene sequencing; 20F, 1500R, 520F (5'-CAGCAG-CCGCG- Chemotaxonomic characterization GTAATAC-3', positions 519–536), 520R (5'-GTATTACCG- AAB were grown in GEY broth on a rotary shaker (150–200 CGGCTGCTG-3', positions 536–519), 920F (5'AAACTCAAAT- rpm) at 30°C for 24 h. Ubiquinone was extracted from freeze-dried GAATTGACGG-3', positions 907–926), and 920R (5'-CCGT- cells by shaking with a mixture of chloroform-methanol (2:1, v/v). CAATTCAT-TTGAGTTT-3', positions 926–907). Multiple align- Cells were removed by filtration, and the combined filtrates were ments of the 16S rRNA gene sequences determined were performed evaporated dry under reduced pressure on a rotary evaporator. The with the program CLUSTAL X (version 1.83) (32). Gaps and resulting residue was dissolved in a small volume of acetone, ambiguous bases were eliminated from the calculation. Distance Fig. 1. A phylogenetic tree based on 16S rRNA gene sequences for Thai isolates assigned to the genus Acetobacter. The phylogenetic tree was constructed by the neighbor-joining method. Numerals (%) at nodes indicate bootstrap values derived from 1,000 replications. Identification of Thai Acetic Acid Bacteria 137 Fig. 2. A phylogenetic tree based on 16S rRNA gene sequences for Thai isolates assigned to the genera Gluconobacter and Asaia. The phylo- genetic tree was constructed by the neighbor-joining method. Numerals (%) at nodes indicate bootstrap values derived from 1,000 replications. matrices for the aligned sequences were calculated by the two- intensity was measured at A450 on a model VersaMax microplate parameter method of Kimura (14). A phylogenetic tree based on reader (Molecular Devices, Sunnyvale, CA, USA). 16S rRNA gene sequences was constructed by the neighbor-joining method of Saitou and Nei (26) with the program MEGA (version Nucleotide sequence accession numbers 4.0) (30). The confidence values of individual branches in the The 16S rRNA gene sequences for the twenty-six strains in this phylogenetic tree were determined by using the bootstrap analysis study have been deposited in DDBJ under the accession numbers of Felsenstein (5) based on 1,000 replications. Pair-wise 16S rRNA AB470916 to AB470920, and AB489237 to AB489245 for Aceto- gene sequence similarities were calculated between phylogeneti- bacter