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-Proteobacteria
1 BIOTEC Culture Collection, BIOTEC Central Research Unit, National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand
2 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Wangmai, Pathumwan, Bangkok 10330, Thailand
3 JICA (Japan International Cooperation Agency, Tokyo, Japan) Senior Overseas Volunteer; Laboratory of General and Applied Microbiology, Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
Correspondence
Yuzo Yamada
yamada-yuzo{at}mub.biglobe.ne.jp
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Asaia krungthepensis AA08T, AA09 and AA06 are respectively AB102953, AB102954 and AB102955.
Present address: 2-3-21 Seinancho, Fujieda, Shizuoka-ken 426-0063, Japan. 
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). Asaia siamensis, the second species, was described by Katsura et al. (2001). Strains assigned to the genus Asaia are characterized by poor or non-existent production of acetic acid from ethanol and by the absence of growth in the presence of 0·35 % acetic acid (v/v). On the other hand, strains of the genera Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter and Kozakia produce large amounts of acetic acid from ethanol and grow in the presence of 0·35 % acetic acid. According to Yamada et al. (2000) and Katsura et al. (2001), strains of the genus Asaia have been isolated mostly from tropical flowers. During the course of our studies on acetic acid bacteria isolated from flowers collected in Thailand, we found that three isolates constitute a novel species; this paper describes Asaia krungthepensis sp. nov. Three bacterial strains were isolated from heliconia flowers (‘paksaasawan’ in Thai; Heliconia sp.) collected in Bangkok, Thailand, by an enrichment-culture approach using a sucrose medium, which was composed of 2·0 % sucrose, 0·3 % peptone, 0·3 % yeast extract and 0·3 % acetic acid (v/v) and adjusted to pH 3·5. When bacterial growth was found in the sucrose medium, micro-organisms were streaked on agar plates containing 2·0 % D-glucose, 0·5 % ethanol (v/v), 0·3 % peptone, 0·3 % yeast extract, 0·7 % CaCO3 and 1·2 % agar (w/v). These acetic acid bacteria were selected as acid-producing bacterial strains that formed clear zones around colonies on agar plates. The three isolates, AA08T (=BCC 12978T), AA09 (=BCC 12979) and AA06 (=BCC 12977), were cultured at 30 °C on agar slants comprising glucose/ethanol/CaCO3/agar medium (GECA medium) that contained 2·5 % D-glucose, 0·5 % ethanol, 0·5 % peptone, 0·3 % yeast extract, 0·7 % CaCO3 and 1·2 % agar and were maintained at 4 °C. Asaia bogorensis IFO 16594T and Asaia siamensis IFO 16457T were used as reference strains, together with Acetobacter aceti IFO 14818T, Gluconobacter oxydans IFO 14819T and Gluconacetobacter liquefaciens IFO 12388T.
Morphological, physiological and biochemical characteristics were examined by using the methods reported by Asai et al. (1964), Yamada et al. (1999, 2000) and Katsura et al. (2001). The phenotypic characteristics of the three isolates are described in the species description. The isolates were different from the type strains of Asaia bogorensis and Asaia siamensis in acid production from maltose and growth on maltose: the isolates produced acid weakly from maltose, with one exception, and grew on maltose, but their growth was not intense (Table 1).
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. Ubiquinone isoprenologues were quantified by reversed-phase HPLC (Tamaoka et al., 1983). The three isolates (AA08T, AA09 and AA06) had Q10 as the major quinone. The ubiquinone system of isolate AA08T was composed of 1 % Q7, 2 % Q8, 16 % Q9 and 81 % Q10.
Gene fragments specific for 16S rRNA-encoding regions were amplified by means of a PCR as described previously (Yamada et al., 2000; Katsura et al., 2001; Kawasaki et al., 1993). Two primers, 20F (5'-GAGTTTGATCCTGGCTCAG-3', positions 9–27) and 1500R (5'-GTTACCTTGTTACGACTT-3', positions 1509–1492), were used. Positions in the rRNA gene fragments were based on the Escherichia coli numbering system (accession number V00348; Brosius et al., 1981).
Amplified 16S rRNA genes were sequenced directly with an ABI PRISM BigDye Terminator cycle sequencing ready reaction kit on an ABI PRISM model 310 Genetic Analyzer (both from Applied Biosystems). The following primers were used for sequencing: 20F, 1500R, 520F (5'-CAGCAGCCGCGGTAATAC-3'; positions 519–536), 520R (5'-GTATTACCGCGGCTGCTG-3'; positions 536–519), 920F (5'-AAACTCAAATGAATTGACGG-3'; positions 907–926) and 920R (5'-CCGTCAATTCATTTGAGTTT-3'; positions 926–907). Multiple alignments of the sequences were carried out with the program CLUSTAL X (version 1.81) (Thompson et al., 1997). Distance matrices for the aligned sequences were calculated by using the two-parameter method of Kimura (1980). The neighbour-joining method was used to construct a phylogenetic tree (Saitou & Nei, 1987). Comparison of the sequence data obtained was made on 1376 bases (Yamada et al., 2000) and the robustness for individual branches was estimated by bootstrapping with 1000 replications (Felsenstein, 1985). Asaia krungthepensis sp. nov. isolates AA08T, AA09 and AA06 were located in the lineage of the genus Asaia, but constituted a cluster separate from the type strains of the known species of the genus Asaia, Asaia bogorensis and Asaia siamensis (Fig. 1).
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Extraction and isolation of bacterial DNA were performed using the modified method of Marmur (1961) (Saito & Miura, 1963; Ezaki et al., 1983). DNA base composition was determined by the method of Tamaoka & Komagata (1984). The DNA G+C contents of the three isolates (AA08T, AA09 and AA06) were respectively 60·3, 60·2 and 60·5 mol%, with a range of 0·3 mol%.
DNA–DNA hybridization was carried out using the photobiotin-labelling method with microdilution wells as described by Ezaki et al. (1989). DNA–DNA relatedness (%) was determined using the colorimetric method (Verlander, 1992). Isolated single-stranded and labelled DNAs were hybridized with DNAs from test strains in 2x SSC and 50 % formamide at 48·0 °C for 12 h. The colour intensity was measured as A450 on a Bio-Rad model 3550 microplate reader. Labelled DNA from Asaia krungthepensis AA08T (=BCC 12978T) showed respectively 100, 100, 100, 38, 40 and 9 % DNA–DNA relatedness to isolates AA08T, AA09 and AA06, Asaia bogorensis IFO 16594T, Asaia siamensis IFO 16457T and Gluconobacter oxydans IFO 14819T. When isolate AA06 DNA was labelled, the DNA–DNA similarities were respectively 100, 100, 100, 31, 36 and 5 %. Asaia bogorensis IFO 16594T and Asaia siamensis IFO 16457T had respectively 27 and 17 %, 36 and 22 %, 29 and 18 %, 100 and 22 %, 32 and 100 % and 6 and 4 % DNA–DNA relatedness to isolates AA08T, AA09 and AA06, Asaia bogorensis 16594T, Asaia siamensis IFO 16457T and Gluconobacter oxydans IFO 14819T.
The acetic acid bacteria are currently classified into six genera: Acetobacter (the type genus), Gluconobacter, Acidomonas, Gluconacetobacter, Asaia and Kozakia (Yamada et al., 2000; Lisdiyanti et al., 2002). In the phylogenetic tree based on 16S rRNA-gene sequences, the three isolates (AA08T, AA09 and AA06) were located in the lineage of the genus Asaia, but constituted a cluster separate from the cluster comprising Asaia bogorensis IFO 16594T and Asaia siamensis IFO 16457T. The DNA base compositions of the isolates (60·2–60·5 mol% G+C) were almost identical to that of Asaia bogorensis IFO 16594T (60·2 mol% G+C). The DNA–DNA relatedness (40–17 %) of the isolates revealed a taxon separate from Asaia bogorensis and Asaia siamensis. Phenotypically, the three isolates can be distinguished from the type strains of the known species (Table 1
). The isolates were different from Asaia siamensis IFO 16457T in terms of acid production from dulcitol. The three isolates showed varying growth responses on maltose, which also differed from those of the type strains of the known species, and the growth was not intense for any strain. Consequently, the three isolates can be distinguished genetically and phenotypically from the known species of the genus Asaia, and should be classified as a novel species. The name Asaia krungthepensis sp. nov. is proposed for the three isolates.
Description of Asaia krungthepensis sp. nov.
Asaia krungthepensis (krung.thep.en'sis. N.L. fem. adj. krungthepensis of or pertaining to Krungthep, the Thai name of Bangkok, Thailand, where the type strain was isolated).
Cells are Gram-negative rods, measuring 0·6–1·0x1·0–2·5 µm on GECA medium. Motile with peritrichous flagella. Colonies are pink, shiny, smooth and raised with an entire margin on GECA medium. Strictly aerobic. Grows at pH 3·0 and 3·5 at 30 °C on CaCO3- and agar-free GECA medium. Oxidizes acetate and lactate to carbon dioxide and water, but the activity is not intense. No production of acetic acid from ethanol. No growth on methanol. Produces 2-keto-D-gluconate and 5-keto-D-gluconate from D-glucose, but not 2,5-diketo-D-gluconate. Grows on glutamate agar and mannitol agar. Vigorous growth on a vitamin-free glucose/(NH4)2SO4 medium, but not on a vitamin-free ethanol/(NH4)2SO4 medium. Produces dihydroxyacetone. Acid is produced from D-glucose, D-mannose, D-galactose, D-fructose, L-sorbose, D-xylose, L-arabinose, L-rhamnose, D-mannitol, D-sorbitol, dulcitol, meso-erythritol, glycerol, melibiose and sucrose. Acid production is weak from D-arabinose, maltose and raffinose and acid is not produced from lactose or ethanol. Grows on D-glucose, D-galactose, D-fructose, L-sorbose, D-xylose, L-arabinose, D-mannitol, D-sorbitol, dulcitol, meso-erythritol, glycerol and sucrose, but grows hardly at all on D-mannose, D-arabinose, L-rhamnose, melibiose and raffinose. No growth is shown on lactose and ethanol. Grows on maltose, but the growth is not intense. The major ubiquinone is Q10. The G+C content of the DNA is 60·2–60·5 mol%, with a range of 0·3 mol%.
The type strain, isolate AA08T, has a DNA G+C content of 60·3 mol% and was isolated from a heliconia flower collected in Bangkok, Thailand. The type strain has been deposited in the BIOTEC Culture Collection, BIOTEC Central Research Unit, National Centre for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand, as BCC 12978T, in the culture collection of the National Biological Resource Centre, Department of Biotechnology, National Institute of Technology and Evaluation, Kisarazu, Chiba-ken, Japan, as NBRC 100057T, in the NODAI Culture Collection, Tokyo University of Agriculture, Setagaya-ku, Tokyo, Japan, as NRIC 0535T and in the culture collection of the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand, as TISTR 1524T.
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ACKNOWLEDGEMENTS |
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