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Gluconobacter Thailandicus Sp. Nov., an Acetic Acid Bacterium in the Α-Proteobacteria

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Gluconobacter Thailandicus Sp. Nov., an Acetic Acid Bacterium in the Α-Proteobacteria J. Gen. Appl. Microbiol., 50, 159–167 (2004) Full Paper Gluconobacter thailandicus sp. nov., an acetic acid bacterium in the a-Proteobacteria Somboon Tanasupawat,1,* Chitti Thawai,1 Pattaraporn Yukphan,5 Duangtip Moonmangmee,2 Takashi Itoh,3 Osao Adachi,4 and Yuzo Yamada5,** 1 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 2 Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology, Thonburi, Bangkok 10140, Thailand 3 Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351–0198, Japan 4 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753–8515, Japan 5 BIOTEC Culture Collection, BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand (Received March 1, 2004; Accepted June 27, 2004) Four strains of acetic acid bacteria were isolated from flowers collected in Thailand. In phyloge- netic trees based on 16S rRNA gene sequences and 16S–23S rDNA internal transcribed spacer (ITS) region sequences, the four isolates were located in the lineage of the genus Gluconobacter and constituted a separate cluster from the known Gluconobacter species, Gluconobacter oxy- dans, Gluconobacter cerinus, and Gluconobacter frateurii. In addition, the isolates were distin- guished from the known species by restriction analysis of 16S–23S rDNA ITS region PCR prod- ucts using three restriction endonucleases Bsp1286I, MboII, and AvaII. The DNA base composi- -tion of the isolates ranged from 55.3–56.3 mol% G؉C. The four isolates constituted a taxon sepa rate from G. oxydans, G. cerinus, and G. frateurii on the basis of DNA-DNA similarities. Morpho- logically, physiologically, and biochemically, the four isolates were very similar to the type strains of G. oxydans, G. cerinus, and G. frateurii; however, the isolates were discriminated in their growth at 37°C from the type strains of G. cerinus and G. frateurii, and in their growth on L- arabitol and meso-ribitol from the type strain of G. oxydans. The isolates showed no acid pro- duction from myo-inositol or melibiose, which differed from the type strains of the three known species. The major ubiquinone homologue was Q-10. On the basis of the results obtained, Glu- conobacter thailandicus sp. nov. was proposed for the four isolates. The type strain is isolate PCU 225T), which had؍TISTR 1533T؍JCM 12310T؍NBRC 100600T؍BCC 14116T؍) F149-1T * Address reprint requests to: Dr. Somboon Tanasupawat, Department of Microbiology, Faculty of Pharmaceutical Sciences, Chu- lalongkorn University, 254 Phayathai Road, Wangmai, Pathumwan, Bangkok 10330, Thailand. E-mail: [email protected] ** JICA Senior Overseas Volunteer; Visiting Professor, Laboratory of General and Applied Microbiology, Department of Applied Bi- ology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156–8502, Japan. Abbreviations: BCC, BIOTEC Culture Collection, BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand; NBRC, Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation, Kisarazu, Chiba, Japan; JCM, Japan Collec- tion of Microorganisms, RIKEN BioResource Center, Wako, Saitama, Japan; TISTR, Thailand Institute of Scientific and Technologi- cal Research, Bangkok, Thailand; PCU, Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. 160 TANASUPAWAT et al. Vol. 50 mol% G؉C, isolated from a flower of the Indian cork tree (Millingtonia hortensis) collected in 55.8 Bangkok, Thailand. Key Words——acetic acid bacteria; AvaII; Bsp1286I; Gluconobacter; Gluconobacter oxydans; Glu- conobacter thailandicus sp. nov.; MboII; 16S–23S rDNA ITS region sequence analyses; 16S rRNA gene sequences Introduction Materials and Methods In the genus Gluconobacter Asai 1935, 689, emend. Isolation of strains assigned to the genus Glu- mut. char. Asai et al. 1964, 100AL, three species are conobacter. Strains of acetic acid bacteria were iso- currently recognized: Gluconobacter oxydans (Hen- lated from natural sources by an enrichment culture neberg 1897) De Ley 1961, 304AL, Gluconobacter ceri- approach, as described previously (Seearunruangchai nus Yamada and Akita 1984, 503VP, and Gluconobac- et al., 2004). The collected flowers were incubated at ter frateurii Mason and Claus 1989, 182VP (Katsura et pH 4.5 and 30°C for 3–5 days in a liquid medium (15 al., 2002; Mason and Claus, 1989; Tanaka et al., 1999; ml/tube), which was composed of 2.0% D-glucose, Yamada and Akita, 1984; Yamada et al., 1999). Glu- 5.0% ethanol, and 1.0% yeast extract (GEY) (all by conobacter asaii Mason and Claus 1989, 183VP is a w/w). When microbial growth was found, the cultures subjective junior synonym of G. cerinus, since the type were streaked onto GEY-CaCO3 (0.3%, w/v) agar strain and the representative strains of G. asaii plates (Yamada et al., 1976, 1999). The acetic acid showed very high DNA-DNA similarities to the type bacteria were selected as acid-producing bacterial strain of G. cerinus (Katsura et al., 2002; Tanaka et al., strains, which formed clear zones around colonies on 1999; Yamada et al., 1999). agar plates. The four isolated strains are listed in Table During the course of studies on diversity of acetic 1. acid bacteria in Thailand, we found that four strains Reference strains of acetic acid bacteria. Glu- isolated from flowers and assigned to the genus Glu- conobacter oxydans NBRC 14819T, G. cerinus NBRC conobacter constitute a new species on the basis of 3267T, G. frateurii NBRC 3264T, and Gluconacetobac- DNA-DNA similarities, 16S rRNA gene sequences, ter liquefaciens TISTR 1057T were used as reference and 16S–23S rDNA internal transcribed spacer (ITS) strains. region sequences. 16S rRNA gene sequences. The 16S rRNA genes This paper describes Gluconobacter thailandicus sp. of the isolates were sequenced, as described previ- nov., the fourth species of the genus Gluconobacter in ously (Seearunruangchai et al., 2004). The 16S rRNA the family Acetobacteraceae Gillis and De Ley 1980, genes were amplified by PCR with Taq DNA poly- 23VP. merase and primers 9F (5Ј-GAGTTTGATCCTG- Table 1. Isolates, designation, sources, and ubiquinone system of Gluconobacter thailandicus. Designation Ubiquinone system (%) Isolate Source BCC JCM NBRC TISTR PCU Q-8 Q-9 Q-10 F142-1 14113 12311 100601 1534 222 Flower of glossy ixora (Ixora lobbii ) 0.5 6.8 93.2 F145-1 14114 223 Flower of Barbados pride 0.6 6.2 93.2 (Caesalpinia pulcherrima) F148-1 14115 224 Flower of China box tree (Murraya paniculata) 0.5 6.9 92.6 F149-1T 14116T 12310T 100600T 1533T 225T Flower of Indian cork tree ND 2.7 97.3 (Millingtonia hortensis) ND, not detected. 2004 Gluconobacter thailandicus sp. nov. 161 GCTCAG-3Ј, the Escherichia coli numbering system, 296 on 16S–23S rDNA). Brosius et al., 1981) and 1541R (5Ј-AAGGAGGT- Phylogenetic analyses based on 16S–23S rDNA ITS GATCCAGCC-3Ј). A PCR amplification was carried region sequences. The 16S–23S rDNA ITS regions out on a GeneAmp PCR System 2400 (PEBiosystems, were sequenced for ca. 715 bases (from position 1 on Foster, California, USA). Double-stranded DNA was the 16S–23S rDNA ITS region by the specified Glu- cloned and sequenced with an ABI PRISM BigDye conobacter oxydans numbering system; Yukphan et Terminator v3.1 Cycle Sequencing Kit (Applied Biosys- al., 2004). The determined sequences were aligned tems, Foster, California, USA) according to the manu- along with the sequences obtained from the database facturer’s instruction, by the use of the following eight of Yukphan et al. (2004) by using the program primers; universal vector primers, T7 and SP6, 339F CLUSTAL X (version 1.81) (Thompson et al., 1997). (5Ј-CTCCTACGGGAGGCAGCAG-3Ј), 357R (5Ј-CT- The neighbor-joining method was used for construct- GCTGCCTCCCGTAG-3Ј), 785F (5Ј-GGATTAGATAC- ing a phylogenetic tree (Saitou and Nei, 1987). The CCTGGTAGTC-3Ј), 802R (5Ј-TACCAGGGTATCTAA- comparison of the sequences was made on 676 TCC-3Ј), 1099F (5Ј-GCAACGAGCGCAACCC-3Ј), and bases. Confidence values of individual branches in the 1115R (5Ј-AGGGTTGCGCTCGTTG-3Ј). The PCR phylogenetic tree were determined by using the boot- products were sequenced with an ABI PRISM 377 Ge- strap analysis of Felsenstein (1985) based on 1,000 netic Analyzer (Applied Biosystems). samplings. Sequence similarities among the type Phylogenetic analyses based on 16S rRNA gene se- strains of the new species and the known species of quences. The 16S rRNA gene sequences deter- the genus Gluconobacter were calculated on ca. 715 mined (ca. 1,440 bases) were aligned along with the bases. selected sequences obtained from the GenBank/ PCR amplification of 16S–23S rDNA ITS regions for EMBL/DDBJ databases by using the program digestion with restriction endonucleases. Amplifica- CLUSTAL X (version 1.81) (Thompson et al., 1997). tion of 16S–23S rDNA ITS regions for digestion with Gaps and ambiguous bases were eliminated from the restriction endonucleases were performed, as de- calculations. Distance matrices for aligned sequences scribed previously (Yukphan et al., 2004). The two were calculated by the two-parameter method of primers used were: 5Ј-TGCGG(C/T)TGGATCAC- Kimura (1980). A phylogenetic tree was constructed by CTCCT-3Ј (positions 1522–1540 on 16S rDNA by the the neighbor-joining method (Saitou and Nei, 1987) Escherichia coli numbering system; Brosius et al., with the program MEGA (version 2.1) (Kumar et al., 1981) and 5Ј-GTGCC(A/T)AGGCATCCACCG-3Ј (po- 2001). Confidence values of individual branches in the sitions 38–22 on 23S rDNA) (Trcˇek and Teuber, 2002). phylogenetic tree were determined by using the boot- Digestion of 16S–23S rDNA ITS region PCR prod- strap analysis of Felsenstein (1985) based on 1,000 ucts with restriction endonucleases. The purified samplings. 16S–23S rDNA ITS region PCR products (ca. 715 16S–23S rDNA ITS region sequences.
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