J. Gen. Appl. Microbiol., 51, 301–311 (2005)

Full Paper

Neoasaia chiangmaiensis gen. nov., sp. nov., a novel osmotolerant acetic acid bacterium in the a-

Pattaraporn Yukphan,1 Taweesak Malimas,1 Wanchern Potacharoen,1 Somboon Tanasupawat,2 Morakot Tanticharoen,1 and Yuzo Yamada1,*,†

1 BIOTEC Culture Collection (BCC), BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand 2 Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand

(Received April 5, 2005; Accepted July 14, 2005)

An acetic acid bacterium, designated as isolate AC28T, was isolated from a flower of red ginger (khing daeng in Thai; Alpinia purpurata) collected in Chiang Mai, Thailand, at pH 3.5 by use of a glucose/ethanol/acetic acid (0.3%, w/v) medium. A phylogenetic tree based on 16S rRNA gene sequences for 1,376 bases showed that isolate AC28T constituted a cluster along with the type strain of baliensis. However, the isolate formed an independent cluster in a phylogenetic tree based on 16S-23S rDNA internal transcribed spacer (ITS) region sequences for 586 bases. Pair-wise sequence similarities of the isolate in 16S rRNA gene sequences for 1,457 bases were 93.0–88.3% to the type strains of Asaia, Kozakia, Swaminathania, Acetobacter, Gluconobacter, Gluconacetobacter, Acidomonas, and Saccharibacter . Restriction analysis of 16S-23S rDNA ITS regions discriminated isolate AC28T from the type strains of Asaia and Kozakia species. Cells were non-motile. Colonies were pink, shiny, and smooth. The isolate produced acetic acid from ethanol. Oxidation of acetate and lactate was negative. The isolate grew on glu- tamate agar and mannitol agar. Growth was positive on 30% D-glucose (w/v) and in the presence

of 0.35% acetic acid (w/v), but not in the presence of 1.0% KNO3 (w/v). Ammoniac nitrogen was hardly assimilated on a glucose medium or a mannitol medium. Production of dihydroxyacetone from glycerol was weakly positive. The isolate did not produce a levan-like polysaccharide on a sucrose medium. Major isoprenoid quinone was Q-10. DNA base composition was 63.1 mol% GC. On the basis of the results obtained, Neoasaia gen. nov. was proposed with Neoasaia chi- angmaiensis sp. nov. The type strain was isolate AC28T (BCC 15763T NBRC 101099T).

Key Words—16S rRNA gene sequences; 16S-23S rDNA ITS region sequences; acetic acid ; BccI; Hin6I; MboII; Neoasaia chiangmaiensis gen. nov., sp. nov.; Tsp509I

* Address reprint requests to: Dr. Yuzo Yamada, BIOTEC Cul- Center for Genetic Engineering and Biotechnology, National ture Collection, National Center for Genetic Engineering and Science and Technology Development Agency, Pathumthani, Biotechnology, National Science and Technology Development Thailand; NBRC, NITE Biological Resource Center (NBRC), Agency, 113 Thailand Science Park, Phaholyothin Road, Klong Department of Biotechnology, National Institute of Technology 1, Klong Luang, Pathumthani 12120, Thailand. and Evaluation, Kisarazu, Chiba, Japan; IFO, Institute for Fer- E-mail: [email protected]; [email protected] mentation, Osaka (IFO), Osaka, Japan; NRIC, NODAI Re- † JICA Senior Overseas Volunteer, Japan International Coop- search Institute Culture Collection Center, Tokyo University of eration Agency (JICA), Shibuya-ku, Tokyo 151–8558, Japan; Agriculture, Tokyo, Japan; NCIB, National Collection of Indus- Professor Emeritus, Shizuoka University, Shizuoka 422–8529, trial Bacteria, Aberdeen, Scotland, UK; ATCC, American Type Japan; Visiting Professor, Faculty of Applied Bioscience, Tokyo Culture Collection, Rockville, Maryland, USA; LMG, Laborato- University of Agriculture, Setagaya-ku, Tokyo 156–8502, Japan. rium voor Microbiologie, Universiteit Gent, Gent, Belgium. Abbreviations: BCC, BIOTEC Culture Collection, National 302 YUKPHAN et al. Vol. 51

Introduction trasted with strains of the three Asaia species.

The genus Asaia Yamada et al. 2000 was intro- Materials and Methods duced as the fifth genus in the family Acetobacte- raceae Gillis and De Ley 1980 with a single species, Bacterial strains. An acetic acid bacterium, desig- Asaia bogorensis Yamada et al. 2000 (Yamada et al., nated as isolate AC28T, was used in this study, which 2000). Recently, the additional two species, Asaia sia- was deposited and maintained at BIOTEC Culture Col- mensis Katsura et al. 2001 and Asaia krungthepensis lection (BCC), Pathumthani, Thailand as strain BCC Yukphan et al. 2004 were described (Katsura et al., 15763T and at Culture Collection NBRC, Kisarazu, 2001; Yukphan et al., 2004c). The genus Kozakia Lis- Japan as strain NBRC 101099T. The strain was iso- diyanti et al. 2002 was subsequently proposed as the lated in September 2002 from a flower of red ginger sixth genus with a single species, (khing daeng in Thai; Alpinia purpurata) collected in Lisdiyanti et al. 2002 (Lisdiyanti et al., 2002). Very re- Chiang Mai, Thailand, at pH 3.5 by the enrichment cul- cently, the seventh and the eighth genera were de- ture approach using a glucose/ethanol/acetic acid scribed: Saccharibacter Jojima et al. 2004 and Swami- (0.3%, w/v) medium, but not either a sorbitol medium, nathania Loganathan and Nair 2004 with a single a dulcitol medium, or a sucrose medium (Katsura et species each, Swaccharibacter floricola Jojima et al., 2001; Lisdiyanti et al., 2002; Yamada et al., 1976, al. 2004 and Swaminathania salitolerans Loganathan 1999, 2000; Yukphan et al., 2004c). The type strains and Nair 2004, respectively (Jojima et al., 2004; of Asaia bogorensis (BCC 12264TNBRC 16594T Loganathan and Nair, 2004). Among the four genera NRIC 0311T), Asaia siamensis (BCC 12268TNBRC mentioned above, the three genera, Asaia, Kozakia, 16457TNRIC 0323T), Asaia krungthepensis (BCC and Swaminathania were related phylogenetically to 12978TNBRC 100057TNRIC 0535T), Kozakia each other. baliensis (BCC 12275TNBRC 16664TNRIC 0488T), The genus Asaia was characterized morphologically Gluconobacter oxydans (NBRC 14819T), Acetobacter by peritrichous flagellation and physiologically by no or aceti (NBRC 14818T), Gluconacetobacter liquefaciens weak oxidation of ethanol to acetic acid and no growth (NBRC 12388T), and Acidomonas methanolica (NRIC in the presence of 0.35% acetic acid (w/v). The genus 0498T) were used as reference strains. was quite differentiated in these respects from the Sequencing of 16S rRNA genes. Isolate AC28T genera Kozakia and Swaminathania, which showed no was sequenced for 16S rRNA genes, as described motility, the ability to oxidize ethanol to acetic acid, and previously (Yukphan et al., 2004a, b). A gene fragment growth in the presence of 0.35% acetic acid (w/v) (Ka- specific for 16S rRNA gene-coding regions was ampli- tsura et al., 2001; Lisdiyanti et al., 2002; Loganathan fied by means of a PCR amplification. Two primers, and Nair, 2004; Yamada et al., 2000; Yukphan et al., 20F (5-GAGTTTGATCCTGGCTCAG-3; positions 9– 2004c). 27) and 1500R (5-GTTACCTTGTTACGACTT-3; po- During the course of our studies on microbial diver- sitions 1509–1492) were used. The positions in the sity of acetic acid bacteria in Thailand, we isolated an rRNA gene fragment were based on the Escherichia osmotolerant acetic acid bacterium, which was related coli numbering system (accession number V00348; phylogenetically to the type strains of Asaia bogoren- Brosius et al., 1981). The amplified and purified 16S sis, Asaia siamensis, Asaia krungthepensis, and Koza- rRNA genes were sequenced directly with an ABI kia baliensis in 16S rRNA gene sequences, but differ- PRISM BigDye Terminator v3.1 Cycle Sequencing entiated at the generic level from the genera Asaia and Ready Reaction Kit on an ABI PRISM model 310 Ge- Kozakia. In addition, the isolate formed an indepen- netic Analyzer (Applied Biosystems, Foster City, Cali- dent cluster in a phylogenetic tree based on 16S-23S fornia, USA). The following primers were used for se- rDNA internal transcribed spacer (ITS) region se- quencing: 20F, 1500R, 520F (5-CAGCAGCCGCG- quences. GTAATAC-3; positions 519–536), 520R (5-GTATTAC- This paper describes Neoasaia chiangmaiensis gen. CGCGGCTGCTG-3; positions 536–519), 920F (5- nov., sp. nov., for the acetic acid bacterium that AAACTCAAATGAATTGACGG-3; positions 907–926), showed oxidation of ethanol to acetic acid and growth and 920R (5-CCGTCAATTCATTTGAGTTT-3; posi- in the presence of 0.35% acetic acid (w/v), as con- tions 926–907). 2005 Neoasaia chiangmaiensis gen. nov., sp. nov. 303

Sequencing of 16S-23S rDNA ITS regions. Isolate HhaI). AC28T was sequenced for 16S-23S rDNA ITS regions, DNA base composition and DNA-DNA hybridization. along with the type strains of Asaia bogorensis (BCC Extraction and isolation of bacterial DNAs were made 12264T), Asaia siamensis (BCC 12268T), Asaia by the modified method of Marmur (Ezaki et al., 1983; krungthepensis (BCC 12978T), and Kozakia baliensis Marmur, 1961; Saito and Miura, 1963). DNA base (BCC 12275T) by the modified method of Trcˇek and composition was determined by the method of Teuber (2002), as described previously (Yukphan et Tamaoka and Komagata (1984). DNA-DNA hybridiza- al., 2004a, b). A PCR amplification was made by use tion was carried out by the photobiotin-labeling method of two primers, which were 5-TGCGG(C/T)TGGAT- with microdilution wells, as described by Ezaki et al. CACCTCCT-3 (positions 1522–1540 on 16S rDNA by (1989). Isolated, single-stranded, and labeled DNAs the Escherichia coli numbering system; Brosius et al., were hybridized with DNAs from test strains in 2 1981) and 5-GTGCC(A/T)AGGCATCCACCG-3 (po- SSC and 50% formamide at 48.0°C for 15 h. DNA-DNA sitions 38–22 on 23S rDNA) (Trcˇek and Teuber, 2002). similarities (%) were determined by the colorimetric The purified PCR products were directly sequenced by method (Verlander, 1992). use of the following four primers: The so-called 1522F Isoprenoid quinone.Isoprenoid quinone of isolate and 38R (respectively 5-TGCGG(C/T)TGGATCAC- AC28T was extracted and purified by the method of CTCCT-3; positions 1522–1540 on 16S rDNA and 5- Yamada et al. (1969). Ubiquinone isoprenologue was GTGCC(A/T)AGGCATCCACCG-3 (positions 38–22 qualified by paper chromatography with two different on 23S rDNA), TAlaf (5-AGAGCACCTGCTTTGCAA- solvent systems comprised of ethanol/ethyl 3; positions 285–302 on 16S-23S rDNA ITS by the acetate/water (5 : 3 : 1, by volume) and N,N-dimethyl Gluconacetobacter hansenii numbering system; Trcˇek formamide/water (97 : 3, by volume) (Yamada et al., and Teuber, 2002), and TAlar (5-ACCCCCTGCTTG- 1969). CAAA-3; positions 311–296 on 16S-23S rDNA ITS). Phenotypic characteristics. Morphological, physio- Sequence analyses. The multiple alignments of logical, and biochemical characteristics were tested by the sequences obtained were made with the program the methods of Asai et al. (1964), Yamada et al. (1976, CLUSTAL X (version 1.81) (Thompson et al., 1997). 1999, 2000), Katsura et al. (2001), and Yukphan et al. Gaps and ambiguous bases were eliminated. The (2004c). comparison of the aligned sequences was made for Base sequence deposition numbers.All the base 1,376 bases of 16S rRNA gene sequences and 586 sequences determined were deposited in the DDBJ bases of 16S-23S rDNA ITS region sequences in con- databases. The base sequence of 16S rRNA genes of structing phylogenetic trees by the neighbor-joining isolate AC28T was filed under the accession number method of Saitou and Nei (1987). Distance matrices AB208549. The base sequences of 16S-23S rDNA for the aligned sequences were calculated by the two- ITS regions were under the accession numbers parameter method of Kimura (1980). Robustness for AB208550 for isolate AC28T, AB208551 for Asaia bo- individual branches was estimated by bootstrapping gorensis BCC 12264T, AB208552 for Asaia siamensis with 1,000 replications (Felsenstein, 1985). Pair-wise BCC 12268T, AB208553 for Asaia krungthepensis sequence similarities (%) of isolate AC28T were calcu- BCC 12978T, and AB208554 for Kozakia baliensis lated in 18S rRNA gene sequences for 1,457 bases BCC 12275T. and in 16S-23S rDNA ITS region sequences for 769 bases. Results and Discussion Restriction analyses.A computerized restriction analysis of 16S-23S rDNA ITS regions was made by In a phylogenetic tree based on 16S rRNA gene se- use of the program NEBcutter (version 2.0; New Eng- quences for 1,376 bases, isolate AC28T constituted a land BioLabs, Beverly, Massachusetts, USA). The pu- cluster, along with the type strain of Kozakia baliensis rified 16S-23S rDNA ITS region PCR products ob- in a lineage composed of the genera Asaia, Kozakia, tained above were digested with the following four re- and Swaminathania (Fig. 1). However, the phyloge- striction endonucleases, Tsp509I (New England Bio- netic branch between the two type strains was much Labs), MboII (Fermentas, Hanover, Maryland, USA), longer than that between the type strains of Asaia bo- BccI (New England BioLabs), and Hin6I (Fermentas, gorensis, the type species of the genus Asaia, and 304 YUKPHAN et al. Vol. 51

Fig. 1. Phylogenetic trees based on 16S rRNA gene sequences and 16S-23S rDNA ITS region sequences for Neoasaia chiangmaiensis. The non-rooted phylogenetic trees based on 16S rRNA gene sequences (a) and 16S-23S rDNA ITS region se- quences (b) were constructed by the neighbor-joining method. Abbreviations: N., Neoasaia; As., Asaia; K., Kozakia; Sw., Swaminathania; A., Acetobacter; G., Gluconobacter; Ac., Acidomonas; Ga., Gluconacetobacter; Sa., Sacchari- bacter.

Swaminathania salitolerans. In a phylogenetic tree in 16S rRNA gene sequences for 1,457 bases were based on 16S-23S rDNA ITS region sequences for calculated to be 92.9, 92.9, 93.0, 93.2, 92.3, 90.7, 586 bases, the isolate constituted an independent 91.2, 92.0, 91.6, and 88.3 respectively to the type cluster in a lineage composed of the genera Asaia and strains of Asaia bogorensis, Asaia siamensis, Asaia Kozakia (Fig. 1). The confidence values by bootstrap- krungthepensis, Kozakia baliensis, Swaminathania ping in rooted phylogenetic trees based on 16S rRNA salitolerans, Acetobacter aceti, Gluconobacter oxy- gene sequences and 16S-23S rDNA ITS region se- dans, Gluconacetobacter liquefaciens, Acidomonas quences were 97% and 97% respectively between iso- methanolica, and Saccharibacter floricola. On the late AC28T and the type strain of Kozakia baliensis other hand, pair-wise sequence similarities (%) in 16S- and between isolate AC28T and the type strains of 23S rDNA ITS region sequences for 769 bases were Kozakia baliensis and the three known Asaia species, 69.2, 68.2, 73.0, 71.1, 54.1, and 60.6 respectively to when Acidiphilium cryptum ATCC 33463T (D30773) the type strains of Asaia bogorensis, Asaia siamensis, and Roseovarius tolerans EL-172T (AJ012700) were Asaia krungthepensis, Kozakia baliensis, Acetobacter respectively used as outgroup (data not shown). aceti, and Gluconobacter oxydans. These data indi- These phylogenetic data suggested that the 16S-23S cated that an additional genus can be set up in the lin- rDNA ITS region sequences can be utilized for the eage composed of the genera Asaia, Kozakia, and genus-level classification or identification of microor- Swaminathania (Fig. 1). ganisms, as found in the 16S rRNA gene sequences, The 16S-23S rDNA ITS region of isolate AC28T for in addition to the species-level classification and identi- 769 bases gave two restriction fragments of 628 and fication. 141 bp with one restriction site at position 629 for Pair-wise sequence similarities (%) of isolate AC28T Tsp509I (5-AATT-3), four restriction fragments of 366, 2005 Neoasaia chiangmaiensis gen. nov., sp. nov. 305

Fig. 2. Restriction of the 16S-23S rDNA ITS region PCR product of Neoasaia chiangmaiensis by digestion with re- striction endonucleases Tsp509I, MboII, BccI, and Hin6I. For estimation of digestion fragments, 50-bp DNA markers were used in agarose gel electrophoresis. (a) Restriction patterns by digestion with restriction endonuclease Tsp509I. (b) Restriction patterns by digestion with restriction en- donuclease MboII. (c) Restriction patterns by digestion with restriction endonuclease BccI. (d) Restriction patterns by digestion with restriction endonuclease Hin6I. Abbreviations: 1, Asaia bogorensis BCC 12264T; 2, Asaia siamensis BCC 12268T; 3, Asaia krungthepensis BCC 12978T; 4, Kozakia baliensis BCC 12275T; 5, Neoasaia chiangmaiensis isolate AC28T; M, 50-bp DNA marker.

311, 70, and 22 bp with three restriction sites at posi- restriction endonucleases mentioned above, the result- tions 354, 376, and 446 for MboII (5-GAAGA-3), ing restriction fragments coincided in their numbers seven restriction fragments of 404, 112, 95, 69, 48, 32, and their molecular sizes with those calculated theo- and 9 bp with six restriction sites at positions 396, 442, retically (Fig. 2). By digestion with Tsp509I and MboII, 537, 546, 594, and 706 for BccI (5-CCATC-3/5- the type strains of the three Asaia species gave identi- GATGG-3), and three restriction fragments of 366, cal restriction patterns, designated as the Asaia bo- 284, and 119 bp with two restriction sites at positions gorensis type of patterns (Table 1). Other restriction 117 and 483 for Hin6I (5-GCGC-3), when analyzed patterns were designated as the Neoasaia chiang- with the program NEBcutter (version 2.0). The posi- maiensis type of patterns and the Kozakia baliensis tions were based on the specified Neoasaia chiang- type of patterns. By digestion with BccI and Hin6I, re- maiensis numbering system, in which position 1 corre- spective restriction patterns were given, designated as sponds to position 1522 on 16S rDNA by the Esch- the Asaia bogorensis type of patterns, the Asaia sia- erichia coli numbering system (Brosius et al., 1981). mensis type of patterns, the Asaia krungthepensis type When 16S-23S rDNA ITS region PCR products of of patterns, the Kozakia baliensis type of patterns, and isolate AC28T for 769 bp were digested with the four the Neoasaia chiangmaiensis type of patterns. The re- 306 YUKPHAN et al. Vol. 51 nd b v w nd nd w/ Peritrichous None c ww w w w w nd ww w w w ww . 16 17 18 19 20 vw a v v v nd nd nd nd nd w nd nd in the family wnd wndw wnd nd Neoasaia (d) (d) (d) ww (d) w (d) w (d) (d) 2223456789 wwwww wwwww (d) (d) 1 10 11 12 13 14 15 able 1. the genus Characteristics differentiating T a (w/v) 3 Characteristic -glucose (w/v) -Erythritol D -Glucose -Mannitol-Xylose -Arabinose-Fructose -Mannitol-Sorbitol w w w -Rhamnose-Sorbose w from glycerol Melibiose Glycerol Maltose Ethanol Dulcitolmeso w 0.35% acetic acid (w/v) 1.0% KNO Glutamate agar Mannitol agar Methanol D D D D L D L D D Sucrose Acetate Lactate 30% ater-soluble brown pigment production Assimilation of ammoniac nitrogen on Production of levan-like polysaccharide Production of dihydroxyacetone w w W Production of acetic acid from ethanol Acid production from Flagellation None Peritrichous Peritrichous Peritrichous None None Peritrichous Polar Polar Oxidation of Growth on 2005 Neoasaia chiangmaiensis gen. nov., sp. nov. 307 b BCC NRIC Neoa- 52.3 type; 23, ; 10, j 64.5 Kozakia baliensis Saccharibacter Asaia bogorensis c ; 14, dndnd T ndnd nd nd nd nd ; 9, 62 Acidomonas methanolica ; 18, T j d e type; 22, the BCC 12978 60.6 Gluconacetobacter j type. NBRC 14819 ; 8, 16 17 18 19 20 Asaia krungthepensis a dndndn nd Acidomonas Neoasaia chiangmaiensis ; 13, . T 57.6–59.9 58.6 ; 7, T i Gluconobacter oxydans ; 21, the T Gluconobacter oxydans 57.2 BCC 12268 ; 17, isolate AC28 T Gluconobacter h type; 26, the strain S-877 ; 6, 60.3 NBRC 14818 able 1. continued. T Asaia siamensis g Acetobacter 32425n (d) w ; 12, 59.3 T ; 5, Kozakia baliensis Neoasaia chiangmaiensis Saccharibacter floricola f Acetobacter aceti (d) 2223456789 BCC 12264 ; 20, T type; 25, the Swaminathania ; 16, T ; 4, 1 10 11 12 13 14 15 NBRC 12388 Kozakia strain PA51 Asaia bogorensis ; 3, ; 11, ; 11, T Asaia krungthepensis Asaia ; 2, isolate AC28 type; 24, the Neoasaia , negative, w, weakly positive; vw, very weakly positive; d, delayed; v, variable; nd, not determined. very weakly positive; d, delayed; v, weakly positive; vw, , negative, w, Characteristic Swaminathania salitolerans C mol% 63.1 60.2 Gluconacetobacter liquefaciens II 21 22 22 22 25 nd nd 26 I21222 509I 21 22 22 22 25 nd nd 26 6I 21 22 23 24 25 nd nd nd nd nd nd ; 15, T ; 19, T Raffinose Mbo Bcc Hin Ethanol Tsp , positive; Asaia siamensis Cited from Loganathan and Nair (2004), except for the data of Cited from Jojima et al. (2004). et al. (2004d) Cited from Yukphan et al. (2004b) Cited from Yukphan Cited from Katsura et al. (2001). et al. (2004c). Cited from Yukphan Cited from Yamashita et al. (2004). Cited from Yamashita Cited from Yamada et al. (2000). Cited from Yamada Cited from Lisdiyanti et al. (2002). et al. (1984). Cited from Yamada DNA G a b c d e f g h i j Abbreviations: 1, Restriction pattern type of 16S-23S rDNA ITS region PCR products with Major isoprenoid quinone Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-9 Q-10 Q-10 Q-10 Q-10 the 12275 0498 saia chiangmaiensis 308 YUKPHAN et al. Vol. 51 striction data obtained above differentiated isolate duced in a glucose/peptone/yeast extract/CaCO3 AC28T molecular-biologically from the type strains of medium. The isolate did not produce a levan-like poly- Asaia bogorensis, Asaia siamensis, Asaia krung- saccharide on a sucrose medium, which differed from thepensis, and Kozakia baliensis. Kozakia strains. DNA G+C content of isolate AC28T was estimated to Acid was produced from D-glucose, D-mannose, D- be 63.1 mol%. This value was higher than those of the galactose, D-xylose, D-arabinose (weakly positive), L- type strains of Asaia bogorensis (60.2 mol%), Asaia arabinose, L-rhamnose (weakly positive), D-fructose siamensis (59.3 mol%), Asaia krungthepensis (60.3 (delayed), D-mannitol (weakly positive), D-sorbitol (de- mol%), Kozakia baliensis (57.2 mol%), and Swami- layed), dulcitol (weakly positive), meso-erythritol, glyc- nathania salitolerans (57.6–59.9 mol%) (Katsura et al., erol, melibiose, sucrose, raffinose, or ethanol, but not 2001; Lisdiyanti et al., 2002; Loganathan and Nair, from L-sorbose, maltose, or lactose. The isolate grew 2004; Yamada et al., 2000; Yukphan et al., 2004c). in a liquid medium containing D-glucose, D-mannose Isolate AC28T gave low DNA-DNA similarities of 8, (weakly positive), D-galactose, D-xylose, D-arabinose 12, 9, 19, and 5% respectively to Asaia bogorensis (weakly positive), L-arabinose, D-fructose, L-sorbose, BCC 12264T, Asaia siamensis BCC 12268T, Asaia D-mannitol, D-sorbitol, dulcitol, meso-erythritol, glyc- krungthepensis BCC 12978T, Kozakia baliensis BCC erol, sucrose, or raffinose, but not from L-rhamnose, 12275T, and Gluconobacter oxydans NBRC 14819T, ethanol, maltose, lactose, or melibiose. These data when DNA from the isolate was labeled. On the other were, on the whole, similar to those of Asaia and hand, labeled DNAs from Asaia bogorensis BCC Kozakia strains. 12264T, Asaia siamensis BCC 12268T, Asaia krung- As described above, isolate AC28T formed a lin- thepensis BCC 12978T, and Kozakia baliensis BCC eage, together with members of the three genera, 12275T gave 3–1% DNA-DNA similarities to DNA from Asaia, Kozakia, and Swaminathania, in the phyloge- the isolate. These data indicated that isolate AC28T netic tree based on 16S rRNA gene sequences. How- can be accommodated in a separate taxon. ever, the phylogenetic branch between the isolate and Isolate AC28T had Q-10 as major isoprenoid the type strain of Kozakia baliensis was much longer quinone, as found in the type strains of Asaia bogoren- than that between the type strains of Asaia bogorensis sis, Asaia siamensis, Asaia krungthepensis, Kozakia and Swaminathania salitolerans (Fig. 1). The isolate baliensis, and Swaminathania salitolerans (Katsura et constituted an independent cluster from members of al., 2001; Lisdiyanti et al., 2002; Loganathan and Nair, the three genera in the phylogenetic tree based on 2004; Yamada et al., 2000; Yukphan et al., 2004c). 16S-23S rDNA ITS region sequences (Fig. 1). Addi- Cells of isolate AC28T were Gram-negative rods and tionally, the isolate had low sequence similarities of non-motile, measuring 0.8–1.01.0–2.0 mm. Colonies 92.3–93.2% in 16S rRNA gene sequences to mem- were pink, shiny, smooth, raised, and with an entire bers of the three genera. The calculated sequence margin, as found in Asaia strains. The isolate pro- similarities were much lower than those of the type duced acetic acid from ethanol, which was different strain of Swaminathania salitolerans to the type strains from Asaia strains but not from Kozakia strains. Oxida- of Kozakia baliensis (96.5%) and Asaia siamensis tion of acetate and lactate was negative. The isolate (98.6%) reported by Loganathan and Nair (2004). grew on glutamate agar and mannitol agar. Growth on These phylogenetic data indicated that the name of 30% D-glucose (w/v) was positive, as found in Asaia Neoasaia is taxonomically recognized, in comparison strains but not in Kozakia strains. The isolate grew in with the name of Swaminathania. the presence of 0.35% acetic acid (w/v), which differed It is noteworthy that such a high sequence similarity from Asaia strains, but did not grow in the presence of of 98.6% was calculated for the genus Swaminathania.

1.0% KNO3 (w/v), which differed from Swaminathania This level of sequence similarities in 16S rRNA genes strains. Ammoniac nitrogen was hardly assimilated in is to be discussed generally for definition of bacterial the presence of D-glucose or D-mannitol, as contrasted strains in the species-level classification or identifica- with Asaia strains. Production of dihydroxyacetone tion (Stackebrandt and Goebel, 1994). It is of great in- from glycerol was weakly positive. 2-Keto-D-gluconate terest that members of the lineage comprised of Asaia, and 5-keto-D-gluconate were produced from D-glu- Kozakia, Swaminathania, and Neoasaia have very cose. A water-soluble brown pigment was not pro- short phylogenetic branches, as contrasted with mem- 2005 Neoasaia chiangmaiensis gen. nov., sp. nov. 309 bers of the lineage of Acetobacter, Gluconobacter, and of acetic acid bacteria; N. L. fem. n. Neoasaia new Saccharibacter (Fig. 1). In addition, the phylogenetic Asaia). branches among the three known species of the genus Gram-negative rods and non-motile, measuring Asaia, i.e., Asaia bogorensis, Asaia siamensis, and 0.8–1.01.0–2.0 mm. Colonies are pink, shiny, Asaia krungthepensis, were extremely short, when smooth, raised, and with an entire margin. Produces compared with, for example, those within the genus acetic acid from ethanol. Oxidation of acetate and lac- Gluconobacter. These phenomena may be attributed tate is negative. Grows on glutamate agar and manni- to whether either substitution rates of bases are ‘ex- tol agar. Growth on 30% D-glucose (w/v) is positive. ceptionally’ slow in the microorganisms in the lineage Grows in the presence of 0.35% acetic acid (w/v). of Asaia, Kozakia, Swaminathania, and Neoasaia or Does not grow in the presence of 1.0% KNO3 (w/v). the appearance of the microorganisms is not evolu- Ammoniac nitrogen is barely assimilated in the pres- tionally old but rather new. From only the phylogenetic ence of D-glucose or D-mannitol as carbon source. point of view, the possibility that members of the lin- Production of dihydroxyacetone from glycerol is eage including Asaia, Kozakia, and Swaminathania, weakly positive. Produces 2-keto-D-gluconate and 5- along with Neoasaia, are grouped into only one genus keto-D-gluconate from D-glucose. Does not produce ei- Asaia, because of the priority of the generic name ther a water-soluble brown pigment on a glucose/pep-

Asaia, is necessarily undeniable. However, these gen- tone/yeast extract/CaCO3 medium or a levan-like poly- era will be retained, because strains of the respective saccharide on a sucrose medium. Acid is produced genera have their own characteristic phenotypic fea- from D-glucose, D-mannose, D-galactose, D-xylose, D- tures that are clearly differentiated from one another. arabinose (weakly positive), L-arabinose, L-rhamnose Upon the proposal of the genus Swaminathania, Lo- (weakly positive), D-fructose (delayed), D-mannitol ganathan and Nair (2004) stressed the differences of (weakly positive), D-sorbitol (delayed), dulcitol (weakly the phenotypic features found in members of the positive), meso-erythritol, glycerol, melibiose, sucrose, genus, rather than the phylogenetic data obtained. As raffinose, or ethanol, but not from L-sorbose, maltose, shown in Fig. 1, the type strain of Swaminathania sali- or lactose. Grows in a liquid medium containing D-glu- tolerans was obviously located outside but never in- cose, D-mannose (weakly positive), D-galactose, D-xy- side the Asaia cluster, although the only three species lose, D-arabinose (weakly positive), L-arabinose, D- are at the present time described in the genus Asaia. fructose, L-sorbose, D-mannitol, D-sorbitol, dulcitol, The phylogenetic data mentioned above indicated that meso-erythritol, glycerol, sucrose, or raffinose, but not the name of Neoasaia is recognized, together with the from L-rhamnose, ethanol, maltose, lactose, or meli- name of Swaminathania. biose. Major isoprenoid quinone is Q-10. DNA base Phenotypically, isolate AC28T was discriminated es- composition is 63.1 mol% GC. The type species is pecially from Asaia and Kozakia strains (Table 1); it Neoasaia chiangmaiensis sp. nov. showed growth on 30% D-glucose (w/v), differing from Kozakia strains, which showed weak growth, and pro- Description of Neoasaia chiangmaiensis sp. nov. duction of acetic acid from ethanol and growth in the Neoasaia chiangmaiensis (chi.ang.ma.i.en’sis. N. L. presence of 0.35% acetic acid (w/v), differing from fem. adj. chiangmaiensis of or pertaining to Chiang Asaia strains. No oxidation of acetate and lactate dis- Mai, Thailand, where the type strain was isolated). tinguished the isolate from Asaia and Kozakia strains, Characteristics are identical with those described for which showed weak oxidation. The isolate did not pro- the genus. Restriction patterns of 16S-23S rDNA ITS duce a levan-like polysaccharide on a sucrose regions are the Neoasaia chiangmaiensis types by di- medium, differing from Kozakia strains. On the basis of gestion with Tsp509I, MboII, BccI, and Hin6I. The type the above descriptions, it is proposed that a new strain is isolate AC28T (BCC 15763TNBRC genus can be introduced for isolate AC28T (Table 1). 101099T), which was isolated from a flower of red gin- ger (khing daeng in Thai; Alpinia purpurata) collected Description of Neoasaia gen. nov. in Chiang Mai, Thailand, in September 2002. Neoasaia (Ne.o.a.sa’i.a. Gr. pref. neo- new; N. L. fem. n. Asaia derived from Toshinobu Asai, a Japa- nese bacteriologist who contributed to the systematics 310 YUKPHAN et al. Vol. 51

Acknowledgments coarctata Tateoka). Int. J. Syst. Evol. Microbiol., 54, 1185–1190. The authors wish to express their thanks to Dr. Hiroshi Ku- Marmur, J. (1961) A procedure for isolation of deoxyribonucleic raishi, Professor Emeritus, Tokyo University of Agriculture and acid from microorganisms. J. Mol. Biol., 3, 208–218. Technology, Fuchu, Tokyo, Japan, for his comments in naming Saito, H. and Miura, K. (1963) Preparation of transforming de- the generic name of Neoasaia. Thanks are also due to Miss oxyribonucleic acid by phenol treatment. Biochim. Biophys. Watcharee Chittrong and Dr. Saisamorn Lumyong, Associate Acta, 72, 619–629. Professor, Department of Biology, Faculty of Science, Chiang Saitou, N. and Nei, M. (1987) The neighbor-joining method: A Mai University, Chiang Mai, Thailand, for their help in collecting new method for reconstructing phylogenetic trees. Mol. isolation sources. This study was supported in part by the Biodi- Biol. Evol., 4, 406–425. versity Research and Training Program, Bangkok, Thailand. Stackebrandt, E. and Goebel, B. M. (1994) Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence References analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol., 44, 846–849. Asai, T., Iizuka, H., and Komagata, K. (1964) The flagellation Tamaoka, J. and Komagata, K. (1984) Determination of DNA and of genera Gluconobacter and Acetobacter base composition by reversed-phase high-performance liq- with reference to the existence of intermediate strains. J. uid chromatography. FEMS Microbiol. Lett., 25, 125–128. Gen. Appl. Microbiol., 10, 95–126. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., Brosius, J., Dull, T. J., Sleeter, D. D., and Noller, H. F. (1981) and Higgins, D. G. (1997) The CLUSTAL X windows inter- Gene organization and primary structure of a ribosomal face: Flexible strategies for multiple sequence alignment RNA operon from Escherichia coli. J. Mol. Biol., 148, aided by quality analysis tools. Nucleic Acids Res., 25, 107–127. 4876–4882. Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric Trcˇek, J. and Teuber, M. (2002) Genetic and restriction analysis deoxyribonucleic acid-deoxyribonucleic acid hybridization of the 16S-23S rDNA internal transcribed spacer regions of in microdilution wells as an alternative to membrane filter the acetic acid bacteria. FEMS Microbiol. Lett., 208, 69–75. hybridization in which radioisotopes are used to determine Verlander, C. P. (1992) Detection of horseradish peroxidase by genetic relatedness among bacterial strains. Int. J. Syst. colorimetry. In Nonisotopic DNA Probe Techniques, ed. by Bacteriol., 39, 224–229. Kricka, L. J., Academic Press, New York, pp. 185–201. Ezaki, T., Yamamoto, N., Ninomiya, K., Suzuki, S., and Yabu- Yamada, Y., Aida, K., and Uemura, T. (1969) Enzymatic studies uchi, E. (1983) Transfer of Peptococcus indolicus, Pepto- on the oxidation of sugar and sugar alcohol. V. Ubiquinone coccus asaccharolyticus, Peptococcus prevotii, and Pepto- of acetic acid bacteria and its relation to classification of coccus magnus to the genus Peptostreptococcus and pro- Gluconobacter and Acetobacter, especially of the so-called posal of Peptostreptococcus tetradius sp. nov. Int. J. Syst. intermediate strains. J. Gen. Appl. Microbiol., 15, 186–196. Bacteriol., 33, 683–698. Yamada, Y., Hosono, R., Lisdiyanti, P., Widyastuti, Y., Saono, Felsenstein, J. (1985) Confidence limits on phylogenies: An ap- S., Uchimura, T., and Komagata, K. (1999) Identification of proach using the bootstrap. Evolution, 39, 783–791. acetic acid bacteria isolated from Indonesian sources, es- Jojima, Y., Mihara, Y., Suzuki, S., Yokozeki, K., Yamanaka, S., pecially of isolates classified in the genus Gluconobacter. and Fudou, R. (2004) Saccharibacter floricola gen. nov., J. Gen. Appl. Microbiol., 45, 23–28. sp. nov., a novel osmophilic acetic acid bacterium isolated Yamada, Y., Itakura, N., Yamashita, M., and Tahara, Y. (1984) from pollen. Int. J. Syst. Evol. Microbiol., 54, 2263–2267. Deoxyribonucleic acid homologies in strains of Glu- Katsura, K., Kawasaki, H., Potacharoen, W., Saono, S., Seki, conobacter species. J. Ferment. Technol., 62, 595–600. T., Yamada, Y., Uchimura, T., and Komagata, K. (2001) Yamada, Y., Katsura, K., Kawasaki, H., Widyastuti, Y., Saono, Asaia siamensis sp. nov., an acetic acid bacterium in the a- S., Seki, T., Uchimura, T., and Komagata, K. (2000) Asaia Proteobacteria. Int. J. Syst. Evol. Microbiol., 51, 559–563. bogorensis gen. nov., sp. nov., an unusual acetic acid bac- Kimura, M. (1980) A simple method for estimating evolutionary terium in the a-Proteobacteria. Int. J. Syst. Evol. Microbiol., rates of base substitutions through comparative studies of 50, 823–829. nucleotide sequences. J. Mol. Evol., 16, 111–120. Yamada, Y., Okada, Y., and Kondo, K. (1976) Isolation and Lisdiyanti, P., Kawasaki, H., Widyastuti, Y., Saono, S., Seki, T., characterization of “polarly flagellated intermediate strains” Yamada, Y., Uchimura, T., and Komagata, K. (2002) Koza- in acetic acid bacteria. J. Gen. Appl. Microbiol., 22, kia baliensis gen. nov., sp. nov., a novel acetic acid bac- 237–245. terium in the a-Proteobacteria. Int. J. Syst. Evol. Microbiol., Yamashita, S., Uchimura, T., and Komagata, K. (2004) Emen- 52, 813–818. dation of the genus Acidomonas Urakami, Tamaoka, Loganathan, P. and Nair, S. (2004) Swaminathania salitolerans Suzuki, and Komagata 1989. Int. J. Syst. Evol. Microbiol., gen. nov., sp. nov., a salt-tolerant, nitrogen-fixing and phos- 54, 865–870. phate-solubilizing bacterium from wild rice (Porteresia Yukphan, P., Malimas, T., Takahashi, M., Potacharoen, W., Bus- 2005 Neoasaia chiangmaiensis gen. nov., sp. nov. 311

abun, T., Tanasupawat, S., Nakagawa, Y., Tanticharoen, Microbiol., 50, 9–15. M., and Yamada, Y. (2004a) Re-identification of Glu- Yukphan, P., Potacharoen, W., Tanasupawat, S., Tanticharoen, conobacter strains based on the restriction analysis of 16S- M., and Yamada, Y. (2004c) Asaia krungthepensis sp. nov., 23S rDNA internal transcribed spacer regions. J. Gen. an acetic acid bacterium in the a-Proteobacteria. Int. J. Appl. Microbiol., 50, 189–195. Syst. Evol. Microbiol., 54, 313–316. Yukphan, P., Potacharoen, W., Nakagawa, Y., Tanticharoen, M., Yukphan, P., Takahashi, M., Potacharoen, W., Tanasupawat, S., and Yamada, Y. (2004b) Identification of strains assigned to Nakagawa, Y., Tanticharoen, M., and Yamada, Y. (2004d) the genus Gluconobacter Asai 1935 based on the se- Gluconobacter albidus (ex Kondo and Ameyama 1958) sp. quence and the restriction analyses of the 16S-23S rDNA nov., nom. rev., an acetic acid bacterium in the a-Pro- internal transcribed spacer regions. J. Gen. Appl. teobacteria. J. Gen. Appl. Microbiol., 50, 235–242.