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 F149-1T ( BCC 14116T NBRC 100600T JCM 12310T TISTR 1533T PCU 225T), which had
* 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
55.8 mol% G C, isolated from a flower of the Indian cork tree (Millingtonia hortensis) collected in 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 (5 -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-3 , the Escherichia coli numbering system, 296 on 16S–23S rDNA). Brosius et al., 1981) and 1541R (5 -AAGGAGGT- Phylogenetic analyses based on 16S–23S rDNA ITS GATCCAGCC-3 ). 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). (5 -CTCCTACGGGAGGCAGCAG-3 ), 357R (5 -CT- The neighbor-joining method was used for construct- GCTGCCTCCCGTAG-3 ), 785F (5 -GGATTAGATAC- ing a phylogenetic tree (Saitou and Nei, 1987). The CCTGGTAGTC-3 ), 802R (5 -TACCAGGGTATCTAA- comparison of the sequences was made on 676 TCC-3 ), 1099F (5 -GCAACGAGCGCAACCC-3 ), and bases. Confidence values of individual branches in the 1115R (5 -AGGGTTGCGCTCGTTG-3 ). 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: 5 -TGCGG(C/T)TGGATCAC- Kimura (1980). A phylogenetic tree was constructed by CTCCT-3 (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 5 -GTGCC(A/T)AGGCATCCACCG-3 (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. The bases) were digested separately with restriction en- 16S–23S rDNA ITS regions of the isolates were se- donucleases Bsp1286I and MboII, as described previ- quenced, as described previously (Yukphan et al., ously (Yukphan et al., 2004). In addition, the restriction 2004). PCR products for sequencing of 16S–23S analysis was made by digestion with restriction en- rDNA ITS regions were obtained by using two primers, donuclease AvaII (Fermentas, Hanover, Maryland, which were: 5 -CGTGTCGTGAGATGTTGG-3 (posi- USA). The digestion reactions of the three restriction tions 1071–1087 on 16S rDNA by the Escherichia coli endonucleases followed the manufacturer’s instruc- numbering system; Brosius et al., 1981) and 5 - tions. The resulting reaction products were analyzed CGGGGTGCTTTTCACCTTTCC-3 (positions 488– by 2.5% (w/v) agarose gel electrophoresis developed 468 on 23S rDNA) (Trcˇek and Teuber, 2002). The di- at 100 V for 40 min in 1 Tris-acetate running buffer. rect sequencing of the purified PCR products was car- DNA base composition and DNA-DNA hybridization. ried out with the following two primers: TA af (5 - The DNAs of the isolates were extracted and purified AGAGCACCTGCTTTGCAA-3 , positions 285–302 on from their whole cells by the phenol method of Saito 16S–23S rDNA by the Gluconacetobacter hansenii and Miura (1963). DNA base composition was deter- numbering system; Trcˇek and Teuber, 2002) and mined by the method of Tamaoka and Komagata TA ar (5 -ACCCCCTGCTTGCAAA-3 , positions 311– (1984). DNA-DNA hybridization was carried out by the 162 TANASUPAWAT et al. Vol. 50 photobiotin-labeling method with microdilution wells in Results 2 SSC and 50% formamide at 47°C for 16 h (Ezaki et al., 1989). DNA-DNA similarity was determined col- Phylogenetic analyses based on 16S rRNA gene se- orimetrically, as described previously (Tanasupawat et quences al., 2000). In a phylogenetic tree based on 16S rRNA gene se- Ubiquinone analysis. The ubiquinone system of quences, G. thailandicus F149-1T and F142-1, all of the isolates was determined, as described previously which were isolated from flowers, were located in the (Seearunruangchai et al., 2004). Ubiquinone was ex- lineage of the genus Gluconobacter and constituted a tracted from freeze-dried cells by shaking with a mix- cluster separate from the type strains of the three ture of chloroform-methanol (2 : 1, v/v) and purified by known species, G. oxydans, G. cerinus, and G. thin-layer chromatography on a silica gel plate frateurii (Fig. 1). A sequence similarity was 99.8% be- T (20 20 cm, silica gel 60F254, Art 5715, E. Merck, tween the two isolates, F149-1 and F142-1. Glu- Darmstdt, Germany) with a solvent system of pure conobacter thailandicus F149-1T represented 98, 98.9, benzene (Yamada et al., 1968). The purified 99.6, 94.9, 94.9, 95.9, and 95% similarities respec- ubiquinone preparations were analyzed for their homo- tively to the type strains of G. oxydans, G. cerinus, G. logues by reversed-phase paper chromatography frateurii, Acetobacter aceti, Acidomonas methanolica, (Yamada et al., 1968) and by HPLC on a Shimadzu Asaia bogorensis, and Kozakia baliensis. model LC-3A high performance liquid chromatograph (Tamaoka et al., 1983). Phylogenetic analyses based on 16S–23S rDNA ITS Phenotypic characteristics. The isolates were ex- region sequences amined for their phenotypic characteristics, as de- As shown in Fig. 2, the phylogenetic tree based on scribed previously (Seearunruangchai et al., 2004). the 16S–23S rDNA ITS region sequences showed that Oxidation of acetate and lactate to carbon dioxide and G. thailandicus F149-1T and F142-1 were located in water, oxidation of ethanol to acetic acid, growth on the lineage of the genus Gluconobacter and consti- glutamate agar and mannitol agar, and acid production tuted a cluster separate from the type strains of the from different carbon sources were tested by the meth- known species, G. oxydans, G. cerinus, and G. ods of Asai et al. (1964). Growth on D-arabitol, L-ara- frateurii. A sequence similarity was 99.9% between the bitol, meso-ribitol, dulcitol, and meso-erythritol was de- two isolates, F149-1T and F142-1. Gluconobacter thai- termined in a basal medium, as described by Katsura landicus F149-1T represented 87.2, 96.8, 99.1, and et al. (2002). Production of D-gluconate, 2-keto-D-glu- 69.7% sequence similarities respectively to the type conate, 5-keto-D-gluconate, and 2,5-diketo-D-glu- strains of G. oxydans, G. cerinus, G. frateurii, and A. conate from D-glucose and D-gluconate was detected aceti. by the methods of Gosselé et al. (1980). The effect of temperature on growth at 30, 37, and 40°C was tested Digestion of 16S–23S rDNA ITS region PCR products by using potato agar plates (Moonmangmee et al., with restriction endonucleases Bsp1286I, MboII, and 2000). The effect of different pH on growth at pH AvaII 3.0–8.0 was tested by using a GMYP broth, which was The 16S–23S rDNA ITS region PCR products (ca. composed of 2.0% glycerol, 2.0% D-mannitol, 0.5% 715) of the four isolates of G. thailandicus were di- yeast extract, and 0.5% peptone (all by w/v). gested separately with restriction endonucleases Base sequence deposition numbers. All the base Bsp1286I and MboII. The isolates showed different re- sequences determined were deposited in the DDBJ striction patterns from the type strains of G. oxydans database. The 16S rRNA gene sequences were under and G. cerinus, viz., respectively G. oxydans and G. the accession numbers AB128050 and AB128051 re- cerinus types of patterns, but gave restriction patterns spectively for isolates F149-1T and F142-1. The identical with one another, which were the same as 16S–23S rDNA ITS region sequences were under the those of the type strain of G. frateurii, viz., G. frateurii accession numbers AB127941 and AB127942 for iso- types of patterns (data not shown) (Yukphan et al., lates F149-1T and F142-1. 2004). However, the four isolates were completely dif- ferentiated from the type strain of G. frateurii by diges- tion with restriction endonuclease AvaII [5 - 2004 Gluconobacter thailandicus sp. nov. 163
Fig. 1. Phylogenetic relationships of strains of Gluconobacter thailandicus based on 16S rRNA gene sequences. The phylogenetic tree was constructed by the neighbor-joining method. The type strain of Acidiphilium cryptum was used as an outgroup. Numerals at nodes indicate bootstrap values derived from 1,000 replications.
Fig. 2. Phylogenetic relationships of strains of Gluconobacter thailandicus based on 16S–23S rDNA ITS region se- quences. The phylogenetic tree was constructed by the neighbor-joining method. The type strain of Acetobacter aceti was used as an outgroup. Numerals at nodes indicate bootstrap values derived from 1,000 replications.
GG(A/T)CC-3 ], which showed a unique restriction pat- DNA base composition and DNA-DNA similarity tern, viz., G. thailandicus type of patterns (Fig. 3). The The DNA base composition of the isolates ranged four isolates did not produce any restriction fragments, 55.3–56.3 mol% G C with a range of 1.0 mol% (Table although the type strain of G. frateurii produced two 2). These values were almost identical with those of fragments showing 610 and 105 bp. the type strains of G. cerinus and G. frateurii, but lower than that of the type strain of G. oxydans. 164 TANASUPAWAT et al. Vol. 50
As shown in Table 2, the four isolates had low DNA- Phenotypic characteristics DNA similarities (4–43%), when the type strains of G. The four isolates were Gram-negative and rod oxydans, G. cerinus, and G. frateurii were labeled. On shaped, measuring 0.6–1.0 1.0–2.5 mm on GEY- the other hand, high and low DNA-DNA similarities CaCO3 agar. All the isolates were non-motile. Colonies (79–102% and 4–38%) were found respectively were white, shiny, smooth, and raised with an entire among the isolates and to the type strains of G. oxy- margin on GEY-CaCO3 agar. They were strictly aero- dans, G. cerinus, and G. frateurii, when G. thailandicus bic. All isolates grew at pH 3.0, 7.5, and 37°C. How- F149-1T and F142-1 were labeled. ever, their growth at 37°C was not so intense but weak. No growth was found at pH 8.0 or 40°C. Ubiquinone system The four isolates did not oxidize acetate and lactate. The major ubiquinone homologue of the isolates They grew on mannitol agar but not on glutamate agar. was Q-10, as found in the type strains of G. oxydans, They produced D-gluconate, 2-keto-D-gluconate, and G. cerinus, and G. frateurii. The four isolates had 5-keto-D-gluconate from D-glucose, but not 2,5-diketo- 92.6–97.3% Q-10, 2.7–6.9% Q-9, and 0.5–0.6% Q-8 D-gluconate or a water-soluble brown pigment. All pro- (Table 1). duced 2-keto-D-gluconate, and 5-keto-D-gluconate from D-gluconate, but not 2,5-diketo-D-gluconate. The isolates produced dihydroxyacetone from glycerol. They grew on D-arabitol, L-arabitol, meso-ribitol, and meso-erythritol, but not on dulcitol; however, their growth was not so intense but weak in L-arabitol, and meso-ribitol. Acid was produced from L-arabinose, D-fructose, D- galactose, D-glucose, glycerol, D-mannitol (variable and weak), D-ribose, L-sorbose (sometimes weak), D- xylose, and ethanol. In contrast, no acid production was found from amygdalin, cellobiose, myo-inositol, Fig. 3. Restriction patterns of 16S–23S rDNA ITS region maltose, D-mannose, melibiose, melezitose, raffinose, PCR products of strains of Gluconobacter thailandicus by di- L-rhamnose, salicin, D-sorbitol, sucrose, or trehalose. gestion with restriction endonuclease AvaII. For estimation of digestion fragments produced from 16S–23S rDNA ITS region PCR products, 50-bp DNA markers Discussion were used in the agarose gel electrophoresis. Abbreviations: 1, G. frateurii NBRC 3264T; 2, G. thailandicus 149-1T; 3, G. thai- The acetic acid bacteria are currently classified in landicus 142-1; 4, G. thailandicus 145-1; 5, G. thailandicus 148- the six genera in the family Acetobacteraceae: Aceto- 1; M, 50-bp DNA marker. bacter Beijerinck 1898, 215AL (the type genus), Glu-
Table 2. DNA base composition and DNA-DNA similarity of Gluconobacter thailandicus.
DNA G C Percent similarity with labeled DNA of Species and strain content (mol%) F149-1T F142-1 NBRC 3264T NBRC 3267T NBRC 14819T
G. thailandicus F142-1 55.3 85 100 10 36 3 G. thailandicus F145-1 55.4 79 102 14 43 24 G. thailandicus F148-1 56.3 99 99 13 16 9 G. thailandicus F149-1T 55.8 100 100 16 18 4 G. frateurii NBRC 3264T 55.1 38 23 100 22 10 G. cerinus NBRC 3267T 55.9 9 15 22 100 12 G. oxydans NBRC 14819T 60.3 8 8 16 16 100 Ga. liquefaciens TISTR 1057T ND 0 2 10 6 6
ND, not determined. 2004 Gluconobacter thailandicus sp. nov. 165
Table 3. Differential characteristics of Gluconobacter thailandicus.
G. thailandicus G. frateurii G. cerinus G. oxydans Characteristic F142-1 F145-1 F148-1 F149-1T NBRC 3264T NBRC 3267T NBRC 14819T
Growth at 37°C wwww Growth at pH 3.0 Growth on D-Arabitol L-Arabitol wwwww meso-Ribitol wwwww Acid production from D-Mannose w D-Sorbitol myo-Inositol w Maltose Melibiose w Sucrose Restriction-pattern type with Bsp1286I G. frateurii G. frateurii G. frateurii G. frateurii G. frateurii G. cerinus G. oxydans MboII G. frateurii G. frateurii G. frateurii G. frateurii G. frateurii G. cerinus G. oxydans AvaII G. thailandicus G. thailandicus G. thailandicus G. thailandicus G. frateurii ND ND DNA G C content 55.3 55.4 56.3 55.8 55.1 55.9 60.3 (mol%)
ND, not determined. All the isolates and the type strains of the three known Gluconobacter species produced dihydroxyacetone from glycerol and 2- keto-D-gluconate and 5-keto-D-gluconate from D-glucose, and acid from D-glucose, D-galactose, D-xylose, L-arabinose, D-ribose, D- fructose, L-sorbose, D-mannitol, glycerol, and ethanol, and grew on mannitol agar, meso-erythritol, and at pH 3.5 and 7.5, but did not produce 2,5-diketo-D-gluconate or a water-soluble brown pigment from D-glucose or acid from amygdalin, cellobiose, melezitose, raf- finose, L-rhamnose, salicin, and trehalose, and did not grow on glutamate agar or dulcitol, at pH 8.0, or at 40°C. conobacter, Acidomonas Urakami et al. 1989, 54VP, frateurii. However, the two species were completely Gluconacetobacter Yamada et al. 1998, 327VP, Asaia differentiated from each other by digestion with restric- Yamada et al. 2000, 828VP, and Kozakia Lisdiyanti et tion endonuclease AvaII (Table 3). al. 2002. 817VP (Lisdiyanti et al., 2002; Urakami et al., The DNA base composition of the four isolates 1989; Yamada et al., 1997, 2000). Gluconobacter thai- (55.3–56.3 mol% G C) was similar to that of the type landicus was proposed as the fourth species of the strains of G. cerinus (55.9 mol% G C) and G frateurii genus Gluconobacter. (55.1 mol% G C) and lower than that of the type In phylogenetic trees based on 16S rRNA gene se- strain of G. oxydans (60.3 mol% G C). The low DNA- quences and 16S–23S rDNA ITS region sequences, DNA similarities (4–43%) of the isolates showed a the isolates (F149-1T and F142-1) of G. thailandicus taxon separate from G. oxydans, G. cerinus, or G. were located in the lineage of the genus Gluconobac- frateurii. Yamada and Akita (1984) recognized that ter, especially in the sublineage that was comprised of strains assigned to the genus Gluconobacter are di- the type strains of G. cerinus and G. frateurii, but con- vided into two groups, viz., the higher DNA G+C con- stituted a cluster separate from the type strains of G. tent-having group including G. oxydans and the lower oxydans, G. cerinus, and G. frateurii. DNA G C content group including G. cerinus (and G. On digestion of 16S–23S rDNA ITS region PCR frateurii, Mason and Claus, 1989) on the basis of DNA products with restriction endonucleases Bsp1286I and base composition. The four isolates were actually an MboII, the four isolates of G. thailandicus gave the additional member of the lower DNA G C content same restriction pattern as the type strain of G. group. 166 TANASUPAWAT et al. Vol. 50
Phenotypically, the four isolates were distinguished References in growth at 37°C from the type strains of G. cerinus Asai, T., Iizuka, H., and Komagata, K. (1964) The flagellation and G. frateurii and in growth on L-arabitol and meso- and taxonomy of genera Gluconobacter and Acetobacter ribitol from the type strains of G. cerinus and G. oxy- with reference to the existence of intermediate strains. J. dans, but their growth was not so intense. In acid pro- Gen. Appl. Microbiol., 10, 95–126. duction from myo-inositol, D-mannose, and melibiose, Brosius, J., Dult, T. J., Sleeter, D. D., and Noller, H. F. (1981) the four isolates were distinguished in no acid produc- Gene organization and primary structure of a ribosomal tion from the type strains of G. cerinus and G. frateurii. RNA operon from Escherichia coli. J. Mol. Biol., 148, 107– On the basis of the results obtained above, the four 127. Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric isolates can be discriminated genetically, molecular-bi- deoxyribonucleic acid-deoxyribonucleic acid hybridization ologically, and phenotypically from the known species in microdilution wells as an alternation to membrane filter of the genus Gluconobacter, and should be classified hybridization in which radioisotopes are used to determine as a new species (Table 3). The name of Gluconobac- genetic relatedness among bacterial strains. Int. J. Syst. ter thailandicus is proposed for the four isolates. Bacteriol., 39, 224–229. Felsenstein, J. (1985) Confidence limits on phylogenies: An ap- Description of Gluconobacter thailandicus sp. nov. proach using the bootstrap. Evolution, 39, 783–791. Gluconobacter thailandicus (tha.i.lan’di.cus. N. L. Gosselé, F., Swings, J., and De Ley, J. (1980) A rapid, simple masc. adj. thailandicus of Thailand, where the type and simultaneous detection of 2-keto-, 5-keto- and 2,5- diketogluconic acids by thin layer chromatography in cul- strain was isolated). ture media of acetic acid bacteria. Zbl. Bakt. Hyg., I. Abt. Cells are Gram-negative and rod-shaped, measur- Orig. C, 1, 178–181. ing 0.6–1.0 1.0–2.5 mm and are nonmotile. Colonies Katsura, K., Yamada, Y., Uchimura, T., and Komagata, K. are white, shiny, smooth, and raised. Strictly aerobic. (2002) Gluconobacter asaii Mason and Claus 1989 is a ju- Grows at pH 3.0 and 7.5. Growth at 37°C is positive nior subjective synonym of Gluconobacter cerinus Yamada but weak. Does not oxidize acetate or lactate. Grows and Akita 1994. Int. J. Syst. Evol. Microbiol., 52, 1635– on mannitol agar but not on glutamate agar. Produces 1640. Kimura, M. (1980) A simple method for estimating evolutionary 2-keto-D-gluconate and 5-keto-D-gluconate from D-glu- rates of base substitutions through comparative studies of cose and D-gluconate but not 2,5-diketo-D-gluconate or nucleotide sequences. J. Mol. Evol., 16, 111–120. a water-soluble brown pigment. Production of dihy- Kumar, S., Tamura, K., Jakobsen, I. B., and Nei, M. (2001) droxyacetone from glycerol is positive. Acid is pro- MEGA2: Molecular evolutionary genetics analysis software, duced from L-arabinose, D-fructose, D-galactose, D-glu- Bioinformatics, 17, 1244–1245. cose, glycerol, D-mannitol (variable and weak), D-ri- Lisdiyanti, P., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T., bose, L-sorbose (sometimes weak), D-xylose, and Yamada, Y., Uchimura, T., and Komagata, K. (2002) Koza- ethanol. Acid is not produced from amygdalin, cel- kia baliensis gen. nov., sp. nov., a novel acetic acid bac- lobiose, myo-inositol, maltose, D-mannose, melibiose, terium in the a-Proteobacteria. Int. J. Syst. Evol. Microbiol., 52, 813–818. melezitose, raffinose, L-rhamnose, salicin, D-sorbitol, Mason, L. M. and Claus, G. W. (1989) Phenotypic characteris- sucrose, or trehalose. The major ubiquinone is Q-10. tics correlated with deoxyribonucleic acid sequence similar- DNA base composition is 55.3–56.3 mol% G C with a ities for three species of Gluconobacter: G. oxydans (Hen- T range of 1.0 mol%. The type strain is F149-1 ( BCC neberg 1897) De Ley 1961, G. frateurii sp. nov., and G. T T T T 14116 NBRC 100600 JCM 12310 TISTR 1533 asaii sp. nov. Int. J. Syst. Bacteriol., 39, 174–184. PCU 225T), which had 55.8 mol% G C, isolated from Moonmangmee, D., Adachi, O., Ano, Y., Shinagawa, E., a flower of the Indian cork tree (Millingtonia hortensis) Toyama, H., Theeragool, G., Lotong, N., and Matsushita, K. collected in Bangkok, Thailand. (2000) Isolation and characterization of thermotolerant Glu- conobacter strains catalyzing oxidative fermentation at higher temperatures. Biosci. 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