Biosci. Biotechnol. Biochem., 73 (10), 2156–2162, 2009

Ameyamaea chiangmaiensis gen. nov., sp. nov., an Acetic Acid Bacterium in the -

Pattaraporn YUKPHAN,1 Taweesak MALIMAS,1 Yuki MURAMATSU,2 Mai TAKAHASHI,2 Mika KANEYASU,2 Wanchern POTACHAROEN,1 Somboon TANASUPAWAT,3 Yasuyoshi NAKAGAWA,2 Koei HAMANA,4 Yasutaka TAHARA,5 Ken-ichiro SUZUKI,2 y Morakot TANTICHAROEN,1 and Yuzo YAMADA1; ,*

1BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, Thailand 2Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation (NITE), Kisarazu 292-0818, Japan 3Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 4School of Health Sciences, Faculty of Medicine, Gunma University, Maebashi 371-8514, Japan 5Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

Received January 27, 2009; Accepted July 8, 2009; Online Publication, October 7, 2009 [doi:10.1271/bbb.90070]

Two isolates, AC04T and AC05, were isolated from Key words: Ameyamaea chiagmaiensis gen. nov., sp. the flowers of red ginger collected in Chiang Mai, nov.; acetic acid ; 16S rRNA gene Thailand. In phylogenetic trees based on 16S rRNA sequences; 16S rRNA gene restriction anal- gene sequences, the two isolates were included within a ysis; lineage comprised of the genera Acidomonas, Glucona- cetobacter, Asaia, , Swaminathania, Neoasaia, In acetic acid bacteria, several new genera have been Granulibacter, and Tanticharoenia, and they formed an reported for strains isolated from isolation sources independent cluster along with the type strain of obtained in Southeast Asia. The first was the genus Tanticharoenia sakaeratensis. The calculated pair-wise Asaia Yamada et al. 2000, with a single , Asaia sequence similarities of isolate AC04T were 97.8–92.5% bogorensis Yamada et al. 2000.2) Asaia siamensis to the type strains of the type species of the 11 genera Katsura et al. 2001,3) Asaia krungthepensis Yukphan of acetic acid bacteria. The DNA base composition et al. 2004,4) and Asaia lannensis Malimas et al. 20085,6) was 66.0–66.1 mol % G+C with a range of 0.1 mol %. were additionally described. The second and the third A single-stranded, labeled DNA from isolate AC04T were the genus Kozakia Lisdiyanti et al. 20027) and the presented levels of DNA-DNA hybridization of 100, 85, genus Neoasaia Yukphan et al. 2006,8,9) with Kozakia 4, and 3% respectively to DNAs from isolates AC04T baliensis Lisdiyanti et al. 2002 and Neoasaia chiang- and AC05 and the type strains of Tanticharoenia maiensis Yukphan et al. 2006 respectively. The fourth sakaeratensis and Gluconacetobacter liquefaciens. The was recently described as the genus Tanticharoenia two isolates were unique morphologically in polar Yukphan et al. 200810,11) with Tanticharoenia sakaer- flagellation and physiologically in intense acetate oxida- atensis Yukphan et al. 2008. tion to carbon dioxide and water and weak lactate In the course of studies on microbial diversity in the oxidation. The intensity in acetate oxidation almost natural environment of Thailand, a chance to isolate two equaled that of the type strain of Acetobacter aceti. The types of unique acetic acid bacteria was given on the two isolates had Q-10. Isolate AC04T was discriminated same day, September 20, 2002, from the same kind of from the type strains of the type species of the 11 genera isolation sources obtained at the same place, the campus by 16S rRNA gene restriction analysis using restriction of Chiang Mai University, Chiang Mai, in the northern endonucleases TaqI and Hin6I. The unique phylogenet- district of Thailand. One was the type strain of Neoasaia ic, genetic, morphological, physiological, and biochem- chiagmaiensis,8) and the others were two isolates ical characteristics obtained indicate that the two discussed below. isolates can be classified into a separate genus, and This paper proposes Ameyamaea chiangmaiensis Ameyamaea chiangmaiensis gen. nov., sp. nov. is pro- gen. nov., sp. nov. for the two isolates accommodated posed. The type strain is isolate AC04T (¼ BCC 15744T, to the family Acetobacteraceae Gillis and De Ley ¼ NBRC 103196T), which has a DNA G+C content of 1980.12) 66.0 mol %.

y To whom correspondence should be addressed. Tel/Fax: +81-54-635-2316; E-mail: [email protected] * JICA Senior Overseas Volunteer, Japan International Cooperation Agency (JICA), Shibuya-ku, Tokyo 151-8558, Japan; Professor Emeritus, Shizuoka University, Suruga-ku, Shizuoka 422-8529, Japan The DDBJ accession numbers of the 16S rRNA gene sequences are AB303366 and AB303367 for isolates AC04T and AC05 respectively.1) Ameyamaea chiangmaiensis gen. nov., sp. nov. 2157 Materials and Methods ever, the phylogenetic distance between the isolates and the type strain of the Tanticharoenia species was much Isolates AC04T and AC05 were examined. They were isolated from longer than that between the type strains of the Asaia the flowers of red ginger (khing daeng in Thai, Alpinia purpurea) and Swaminathania species. In addition, the type strains collected in Chiang Mai, Thailand by an enrichment culture approach of and Neoasaia chiangmaiensis gave using a sorbitol medium, which contained 2.0% D-sorbitol, 0.3% peptone, and 0.3% yeast extract (all by w/v) and was adjusted to a similar cluster, with a bootstrap value of 85%, to that pH 3.5, among the four media used.2–5,7,8,10,13,14) Acetobacter aceti of the isolates and the type strain of the Tanticharoenia NBRC 14818T (NBRC, NITE Biological Resource Center, Department species. In both clusters, the phylogenetic distances were of Biotechnology, National Institute of Technology and Evaluation, almost identical to each other. The cluster comprised of Kisarazu, Chiba, Japan), Gluconobacter oxydans NBRC 14819T, the two isolates and the type strain of the Tanticharoenia T Acidomonas methanolica NRIC 0498 (NRIC, NODAI Research species was independently connected to a large cluster Institute Culture Collection Center, Tokyo University of Agriculture, of the genera Asaia, Swaminathania, Kozakia, Neoasaia, Tokyo, Japan), Gluconacetobacter liquefaciens NBRC 12388T, Asaia bogorensis NBRC 16594T, Kozakia baliensis NBRC 16664T, Swami- Acidomonas, Granulibacter, and Gluconacetobacter, nathania salitolerans LMG 21291T (LMG, Laboratorium voor Micro- with a bootstrap value of 85%. biologie, Universiteit Gent, Ghent, Belgium), Saccharibacter floricola In a phylogenetic tree constructed by the maximum BCC 16445T (BCC, BIOTEC Culture Collection, National Center parsimony method, the cluster of the two isolates and the for Genetic Engineering and Biotechnology, National Science and type strain of the Tanticharoenia species had a bootstrap Technology Development Agency, Pathumthani, Thailand), Neoasaia value of 75%, connected itself with the cluster com- chiangmaiensis T Granulibacter bethesdensis BCC 15763 , ATCC BAA prised of the genera Kozakia, Neoasaia, Acidomonas, 1260T (ATCC, American Type Culture Collection, Rockville, MD, USA), and Tanticharoenia sakaeratensis BCC 15772T were used as Granulibacter, and Gluconacetobacter, with a bootstrap reference strains. value below 50%, and formed a large cluster together PCR amplification of the 16S rRNA genes was done, and the with the cluster of the genera Asaia and Swaminathania, amplified 16S rRNA genes were sequenced and analyzed as described with a bootstrap value of 53% (Fig. 1B). The phyloge- previously.4,8,15) Multiple sequence alignment was done with the netic distance between the two isolates and the type 16) program CLUSTAL X (version 1.8). Alignment gaps and unidenti- strain of the Tanticharoenia species was almost identical fied bases were eliminated. Distance matrices for the aligned sequences were calculated by the two-parameter method of Kimura.17) Phyloge- to that between the type strains of the Kozakia and netic trees were constructed for 1,374 bases by the neighbor-joining,18) Neosasaia species and much longer than that between maximum parsimony,19) and maximum likelihood20) methods. The the type strains of the Asaia and Swaminathania species, robustness of individual branches was estimated by bootstrapping21) as found in the phylogenetic tree constructed by the with the program MEGA (version 4.0).22) Bootstrap values below 50% neighbor-joining method. were not shown. In constructing the phylogenetic tree by the maximum In the phylogenetic tree constructed by the maximum likelihood method, the program PHYLIP (version 3.6; J. Felsenstein, likelihood method, the cluster of the two isolates and the University of Washington) was used. Pair-wise sequence similarities were calculated in 16S rRNA gene sequences of 1,390 bases. type strain of the Tanticharoenia species was connected A computerized 16S rRNA gene restriction analysis was done using to a cluster comprised of the genera Gluconacetobacter, NEBcutter (version 2.0; New England BioLabs, Ipswich, MA, USA) Acidomonas, and Granulibacter with a bootstrap value for isolate AC04T and the type strains of the type species of the 11 below 50%, and the resulting cluster formed a large genera of acetic acid bacteria. The 16S rRNA gene PCR products of cluster with the cluster of the genera Asaia, Swamina- isolate AC04T and the type strains were prepared and digested with 23) thania, Kozakia, and Neoasaia, with a bootstrap value restriction endonucleases as described previously. below 50% (Fig. 1C). In all the three phylogenetic trees Chromosomal DNA was prepared as described previously.4,8,10) The DNA base composition was determined by the method of Tamaoka and obtained, the phylogenetic distance between the isolates Komagata.24) Levels of DNA-DNA hybridization (%) were determined and the type strain of Tanticharoenia sakaeratensis was by fluorometric DNA-DNA hybridization in microplate wells as almost identical to that between the genera Kozakia and described by Ezaki et al.25) Isolated, single-stranded, and labeled Neoasaia, and was much longer than that between the DNAs were hybridized with DNAs from test strains in 2 SSC genera Asaia and Swaminathania. containing 50% formamide at 50.0 C for 3 h. Fluorescence intensity Pair-wise sequence similarities of isolate AC04T in was measured with a Fluoroskan Ascent (Thermo Labsystems, Helsinki, Finland) at wavelengths of 335 nm for excitation and 16S rRNA gene sequences of 1,390 bases were 460 nm for emission. The highest and lowest values obtained in each calculated to be 96.1, 95.3, 95.3, 96.6, 96.9, 96.4, sample were excluded, and the mean of the remaining three values was 96.8, 92.8, 92.5, 94.6, and 97.8% respectively to the type taken as the DNA-DNA relatedness value. strains of Acetobacter aceti, Gluconobacter oxydans, Phenotypic characteristics were determined by the methods of Asai Acidomonas methanolica, Gluconacetobacter liquefa- 26) 3) 27) 7) et al., Katsura et al., Kersters et al., Lisdiyanti et al., Yamada ciens Asaia bogorensis Kozakia baliensis Swamina- 2,13) 4,8,10) , , , et al., and Yukphan et al. A major isoprenoid quinone was thania salitolerans, Saccharibacter floricola, Neoasaia determined by the method of Yamada et al.28) chiangmaiensis, Granulibacter bethesdensis, and Tanti- charoenia sakaeratensis. The sequence similarity was Results and Discussion 100% between the two isolates. The pair-wise sequence similarity of 97.8% obtained above was almost identical In a phylogenetic tree constructed by the neighbor- to that between the genera Asaia and Kozakia (97.4%),7) joining method, isolates AC04T and AC05 formed a and was much lower than that between the genera Asaia cluster along with the type strain of Tanticharoenia and Swaminathania (98.6%).29) sakaeratensis with a bootstrap value of 92% when Stella The combination of the two restriction endonucleases, humosa DSM 5900T was used as the outgroup (Fig. 1A). TaqI and Hin6I theoretically discriminated the isolate The type strain of Swaminathania salitolerans formed a and the type strains of the type species of the 11 genera different cluster along with the type strains of the four of acetic acid bacteria from one another in 16S rRNA Asaia species, with a bootstrap value of 100%. How- gene restriction analysis using NEBcutter (version 2.0) 2158 P. YUKPHAN et al.

A Asaia bogorensis isolate 71T (AB025928) Asaia siamensis isolate S60-1T (AB035416) Asaia krungthepensis isolate AA08T (AB102953) 100 Asaia lannensis isolate AB92T (AB286050) 88 Swaminathania salitolerans NBRC 104436T (AB445099) Kozakia baliensis isolate Yo-3T (AB056321) T 85 Neoasaia chiangmaiensis isolate AC28 (AB208549) 75 Gluconacetobacter liquefaciens IFO 12388T (X75617) 73 Gluconacetobacter xylinus NCIB 11664T (X75619) 85 Granulibacter bethesdensis CGDNIH1T (AY788950) 56 T Acidomonas methanolica LMG 1668 (X77468) 100 Ameyamaea chiangmaiensis isolate AC04T (AB303366) 85 92 Ameyamaea chiangmaiensis isolate AC05 (AB303367) Tanticharoenia sakaeratensis strain AC37T (AB304087) T 93 94 Acetobacter aceti NCIB 8621 (X74066) Acetobacter pasteurianus LMD 22.1T (X71863) 100 Gluconobacter cerinus IFO 3267T (AB063286) 99 Gluconobacter frateurii IFO 3264T (X82290) Gluconobacter oxydans ATCC 19357T (X73820) Saccharibacter floricola strain S-877T (AB110421) Stella humosa DSM 5900T (AJ535710) Knuc 0.01

T B 100 Ameyamaea chiangmaiensis isolate AC04 (AB303366) 75 Ameyamaea chiangmaiensis isolate AC05 (AB303367) Tanticharoenia sakaeratensis strain AC37T (AB304087) Granulibacter bethesdensis CGDNIH1T (AY788950) Gluconacetobacter liquefaciens IFO 12388T (X75617) Gluconacetobacter xylinus NCIB 11664T (X75619) Acidomonas methanolica LMG 1668 T (X77468) Neoasaia chiangmaiensis isolate AC28T (AB208549) Kozakia baliensis isolate Yo-3T (AB056321) 53 Asaia siamensis isolate S60-1T (AB035416)

Asaia krungthepensis isolate AA08T (AB102953) Asaia bogorensis isolate 71T (AB025928) 87 Asaia lannensis isolate AB92T (AB286050) 90 Swaminathania salitolerans NBRC 104436T (AB445099) 92 83 Acetobacter aceti NCIB 8621T (X74066) Acetobacter pasteurianus LMD 22.1T (X71863) T 94 Gluconobacter oxydans ATCC 19357 (X73820) Gluconobacter cerinus IFO 3267T (AB063286) 95 Gluconobacter frateurii IFO 3264T (X82290) Saccharibacter floricola strain S-877T (AB110421) Stella humosa DSM 5900T (AJ535710) Nucleotides changed 20

C Gluconacetobacter liquefaciens IFO 12388T (X75617) Gluconacetobacter xylinus NCIB 11664T (X75619) Acidomonas methanolica LMG 1668T (X77468) Granulibacter bethesdensis CGDNIH1T (AY788950) 68 Ameyamaea chiangmaiensis isolate AC04 T (AB303366) 50 Ameyamaea chiangmaiensis isolate AC05 (AB303367) Tanticharoenia sakaeratensis strain AC37T (AB304087) Neoasaia chiangmaiensis isolate AC28T (AB208549) Kozakia baliensis isolate Yo-3T (AB056321) Asaia siamensis isolate S60-1T (AB035416) T 61 Asaia krungthepensis isolate AA08 (AB102953) Asaia bogorensis isolate 71T (AB025928) 60 Asaia lannensis isolate AB92 T (AB286050) Swaminathania salitolerans NBRC 104436T (AB445099) 61 T 62 Acetobacter aceti NCIB 8621 (X74066) Acetobacter pasteurianus LMD 22.1T (X71863) T 61 Gluconobacter frateurii IFO 3264 (X82290) 57 Gluconobacter cerinus IFO 3267T (AB063286) Gluconobacter oxydans ATCC 19357T (X73820) Saccharibacter floricola strain S-877T (AB110421) Stella humosa DSM 5900T (AJ535710)

Substitution/site 0.01

Fig. 1. Phylogenetic Relationships of Ameyamaea chiangmaiensis Isolates AC04T and AC05 Based on 16S rRNA Gene Sequences. The phylogenetic trees were constructed by the neighbor-joining (A), maximum parsimony (B), and maximum likelihood (C) methods. Stella humosa DSM 5900T was used as an outgroup. Numerals at nodes indicate bootstrap values (%) derived from 1,000 replications. Numerals below 50% are not shown. Ameyamaea chiangmaiensis gen. nov., sp. nov. 2159 A

B

Fig. 3. Transmission Electron Micrograph of Cells of Ameyamaea chiangmaiensis Isolate AC04T. Cells were cultivated at 18 C for 16 h on glucose/ethanol/ peptone/yeast extract/calcium carbonate agar slant. Motility was observed by the hanging-drop method, and the cells were stained by negative staining. Bar, 500 nm.

167, 154, 137, and 35-bp fragments. Tanticharoenia sakaeratensis BCC 15772T showed quite different Fig. 2. Restriction of 16S rRNA Gene PCR Products of Ameyamaea restriction patterns, designated the Ts types, comprising chiangmaiensis Isolate AC04T. fragments of 362, 217, 194, 183, 156, 104, 86, 66, and The restriction fragments theoretically produced by TaqI (A) and 43 bp and 385, 210, 184, 174, 167, 154, and 137 bp Hin6I (B) digestion respectively are shown within parentheses. 1, Ameyamaea chiangmaiensis isolate AC04T (497, 362, 183, 170, respectively. The 16S rRNA gene restriction analysis 156, and 43 bp; 385, 341, 210, 184, 154, and 137 bp); 2, using the two restriction endonucleases completely Tanticharoenia sakaeratensis BCC 15772T (362, 217, 194, 183, discriminated the type strains of the type species of 156, 104, 86, 66, and 43 bp; 385, 210, 184, 174, 167, 154, and T the 12 genera of acetic acid bacteria from one another 137 bp); 3, Gluconacetobacter liquefaciens NBRC 12388 (497, molecular-phylogenetically. 362, 183, 172, 156, and 43 bp; 352, 210, 184, 174, 167, 154, 137, T and 35 bp); 4, Acetobacter aceti NBRC 14818T (879, 361, and Isolates AC04 and AC05 had DNA base composi- 170 bp; 525, 385, 210, 154, and 136 bp); 5, Gluconobacter oxydans tions of 66.0 and 66.1 mol % G+C respectively with a NBRC 14819T (357, 194, 183, 156, 112, 105, 104, 86, 66, and 43 bp; range of 0.1 mol %. The calculated DNA G+C content 385, 341, 184, 154, 132, 106, and 104 bp); 6, Acidomonas was the highest of the acetic acid bacteria tested methanolica NRIC 0498T (532, 194, 183, 156, 112, 105, 86, and 43 bp; 385, 341, 184, 137, 106, 104, 79, and 75 bp); 7, Asaia (Table 1). T bogorensis NBRC 16594T (392, 362, 183, 170, 156, 105, and 43 bp; The labeled DNA of isolate AC04 presented levels 385, 341, 184, 154, 137, 106, and 104 bp); 8, Kozakia baliensis of DNA-DNA hybridization of 100, 85, 4, and 3% to NBRC 16664T (497, 362, 183, 156, 104, 70, and 43 bp; 354, 184, DNAs from isolates AC04T and AC05, Tanticharoenia 174, 167, 154, 137, 106, 104, and 35 bp); 9, Swaminathania T T sakaeratensis BCC 15772 , and Gluconacetobacter salitolerans LMG 21291 (390, 365, 183, 171, 157, 105, and 43 bp; T 386, 356, 244, 168, 154, and 106 bp); 10, Saccharibacter floricola liquefaciens NBRC 12388 respectively, which were BCC 16445T (774, 357, 170, and 105 bp; 495, 350, 210, 184, 132, used as reference strains. When isolate AC05 was and 35 bp); 11, Neoasaia chiangmaiensis BCC 15763T (392, 362, labeled, the calculated levels of DNA-DNA hybrid- 201, 156, 105, 104, 84, and 43 bp; 513, 403, 184, 137, 106, and ization were 84 and 100% respectively to isolates T 104 bp); 12, Granulibacter bethesdensis ATCC BAA 1260 (653, AC04T and AC05. The genetic data obtained indicate 360, 187, 172, and 43 bp; 335, 210, 184, 174, 167, 158, 135, and 52 bp); M, 50-bp DNA marker. that the two isolates constituted a single species and were quite distant genetically from the type strains of Tanticharoenia sakaeratensis and Gluconacetobacter (Fig. 2). In TaqI digestion, isolate AC04T represented an liquefaciens. almost identical restriction pattern, designated the Gal The phenotypic characteristics of isolates AC04T and type, comprising 497, 362, 183, 170, 156, and 43-bp AC05 are described in the genus and the species fragments, to Gluconacetobacter liquefaciens NBRC descriptions. 12388T, which showed restriction fragments comprising In particular, the two isolates were unique morpho- 497, 362, 183, 172, 156, and 43 bp. However, the isolate logically in having polar flagellation (Fig. 3) and was distinguished in Hin6I digestion. The former physiologically in intense acetate oxidation to carbon showed a restriction pattern, designated the Amc type, dioxide and water and weak lactate oxidation. The comprising 385, 341, 210, 184, 154, and 137-bp intensity observed in acetate oxidation almost equaled fragments, while the latter showed a restriction pattern, that of the type strain of Acetobacter aceti.30) The two designated Gal type, comprising 352, 210, 184, 174, isolates had Q-10 as a major isoprenoid quinone as 2160 P. YUKPHAN et al. Table 1. Characteristics Differentiating the Genus Ameyamaea from the 11 Genera of Acetic Acid Bacteria

Am T A G Ac Ga As K S Sa N Gr Characteristic 12345678 9a 10b 11c 12d Flagellation po n pe po poe pe pe n pe n n n Oxidation of Acetate þþþþww w w Lactate w þþwwwwþ Growth on 30% D-Glucose (w/v) þþþw þþþnd Glutamate agar w w þþ þ þþþ Mannitol agar þþvw þ w þþþþþþw Methanol w þ þ Growth in the presence of 0.35% Acetic acid (w/v) þþþþþþþ þ þnd a 1% KNO3 þ þ nd nd Production of acetic acid from ethanol þþþþþþþ þ w/- þ vw Water-soluble brown pigment production þþ þ nd Production of dihydroxyacetone from glycerol w þþþþww þw Production of levan-like polysaccharide þ nd nd Assimilation of ammoniac nitrogen on D-Glucose vw þw þþ nd þ D-Mannitol vw þw þþ nd wnd Ethanol vw w w nd nd Production of 2-Keto-D-gluconate þþþþnd þþþ nd þþnd 5-Keto-D-gluconate þþþþnd þþþ nd þþnd 2,5-Diketo-D-gluconate þnd þ nd nd nd Acid production from D-Mannitol þw þþw D-Sorbitol þþ(d) þþ(d) Dulcitol w þ(d) v w Glycerol w þþþþþþþ þ þw/ Ethanol þþþþþþþ þ þþ 16S rRNA gene restriction pattern withj TaqI Gal Ts Aa Go Acm Gal Asb Kb Asb Saf Nc Grb Hin6I Amc Ts Aa Go Acm Gal Asb Kb Ss Saf Nc Grb Major isoprenoid quinone Q-10 Q-10 Q-9 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10 Q-10j DNA base composition (mol% G+C) 66.0 65.6 f 58.6g 60.6g 62e 64.5g 60.2h 57.2i 57.6–59.9 52.3 63.1 59.1

po, polar; pe, peritrichous; n, none; þ, positive; , negative; w, weakly positive; vw, very weakly positive; d, delayed; v, variable; nd, not determined; Amc, Ameyamaea chiangmaiensis; Ts, Tanticharoenia skaeratensis; Aa, Acetobacter aceti; Go, Gluconobacter oxydans; Acm, Acidomonas methanolica; Gal, Gluconacetobacter liquefaciens; Asb, Asaia bogorensis; Kb, Kozakia baliensis; Ss, Swaminathania salitolerans; Saf, Saccharibacter floricola; Nc, Neoasaia chiangmaiensis; Grb, Granulibacter bethesdensis Cited from aLoganathan and Nair,29) except for the data on Neoasaia chiangmaiensis strain AC28T, Tanticharoenia sakaeratensis strain AC37T and Ameyamaea chiagmaiensis isolate AC04T, bJojima et al.,35) cYukphan et al.,8) d Greenberg et al.,31) eYamashita et al.,36) f Yukphan et al.,10) gYamada et al.,37) hYamada et al.,2) iLisdiyanti et al.,7) and jthe present study. Abbreviations: Am, Ameyamaea; T, Tanticharoenia; A, Acetobacter; G, Gluconobacter; Ac, Acidomonas; Ga, Gluconacetobacter; As, Asaia; K, Kozakia; S, Swaminathania; Sa, Saccharibacter; N, Neoasaia; Gr, Granulibacter;1,Ameyamaea chiangmaiensis isolate AC04T;2,Tanticharoenia sakaeratensis strain AC37T; 3, Acetobacter aceti NBRC 14818T;4,Gluconobacter oxydans NBRC 14819T;5,Acidomonas methanolica NRIC 0498T;6,Gluconacetobacter liquefaciens NBRC 12388T;7,Asaia bogorensis NBRC 16594T;8,Kozakia baliensis NBRC 16664T;9,Swaminathania salitolerans strain PA51T; 10, Saccharibacter floricola strain S-877T; 11, Neoasaia chiangmaiensis strain AC28T; 12, Granulibacter bethesdensis CGDNIH1T examined by reversed phase paper chromatography.28) observed distinguishes the two isolates from strains of The type strain of Granulibacter bethesdensis31) had a the genus Acetobacter. major isoprenoid quione comprised of Q-10 (Table 1). The two isolates constituted a cluster with the type The acetic acid bacteria are at present classified into strain of Tanticharoenia sakaeratensis in the three 11 genera.11,27,30,32) The two isolates, AC04T and AC05, phylogenetic trees based on 16S rRNA gene sequences, that are characterized by polar flagellation are morpho- but the phylogenetic distance between the isolates and logically distinguished from peritrichous-flagellated and the type strain of Tanticharoenia sakaeratensis was from non-motile strains accommodated to the genera almost identical to that between the type strains of Acetobacter, Gluconacetobacter, Asaia, Kozakia, Swa- Kozakia baliensis and Neoasaia chiangmaiensis and that minathania, Saccharibacter, Neoasaia, Granulibacter, between the type strains of Asaia bogorensis and and Tanticharoenia, except for strains of the genera Kozakia baliensis, and much longer than that between Gluconobacter and Acidomonas, which are equipped the type strains of Asaia bogorensis and Swaminathania with polar flagella (Table 1). Physiologically, the two salitolerans (Fig. 1). isolates are especially distinguished from strains of the The phylogenetic data obtained to the effect that 11 genera only by acetate and lactate oxidization. The the calculated pair-wise 16S rRNA gene sequence intense acetate oxidation observed distinguishes the two similarity was 97.8% suggests that the isolates are to isolates from strains of other genera, except for strains of be classified, in spite of showing a much longer distance the genus Acetobacter, and the weak lactate oxidation than that between the genera Asaia and Swaminathania Ameyamaea chiangmaiensis gen. nov., sp. nov. 2161 (98.6%),29) into the genus Tanticharoenia. However, the Acetobacter aceti (2%)34) and that of the type strains of isolates were too different in phenotypic characteristics Tanticharoenia sakaeratensis and Gluconacetobacter to be accommodated to either the genus Tanticharoenia liquefaciens (2%).10) or the genus Gluconacetobacter. The two isolates, AC04T and AC05, are distinguished Several phenotypic characteristics that discriminated at the generic level phylogenetically, genetically, mor- isolate AC04T from the type strain of Gluconacetobacter phologically, physiologically, and biochemically from liquefaciens, which is one of the phylogenetically most the type strains of the type species of the 11 genera closely related strains, can be seen (Table 1): i) The of acetic acid bacteria, especially of the genus Tanti- former has polar flagellation but the latter has peritri- charoenia, and can be classified into a separate genus chous flagellation. ii) Extremely intense acetate oxida- with a single species (Table 1). The name Ameyamaea tion is found in the former but not in the latter. iii) The chiangmaiensis gen. nov., sp. nov. is proposed. former oxidizes lactate weakly, but the intensity of lactate oxidation is not weak in the latter. iv) Growth on Description of Ameyamaea gen. nov. methanol is weakly positive in the former but negative Ameyamaea (A.me.ya.ma’e.a. N. L. fem. n. Ameya- in the latter. v) Production of 2,5-diketo-D-gluconate and maea derived from the late Dr. Minoru Ameyama, Pro- a water-soluble brown pigment from D-glucose are not fessor Emeritus of Yamaguchi University, Yamaguchi, found in the former but are in the latter. Japan, who contributed to studies of acetic acid bacteria, Concerning the genus Tanticharoenia, the phyloge- especially their biochemical and systematic studies). netically most closely related, isolate AC04T is largely Gram-negative rods and motile with polar flagella, distinguished phenotypically from the type strain of measuring 0.6–0:8 1:0–1.8 mm. Colonies are creamy Tanticharoenia sakaeratensis (Table 1): i) The former is and smooth with entire margin when grown on glucose/ motile with polar flagella, but the latter is non motile. ii) ethanol/peptone/yeast extract/calcium carbonate agar. The former has intense acetate oxidation that almost Produces acetic acid from ethanol. Intensely oxidizes equals that of the type strain of Acetobacter aceti,as acetate to carbon dioxide and water, but weakly oxidizes well as weak lactate oxidation, but oxidation of acetate lactate. Grows on glutamate agar (weakly positive) and lactate is never seen in the latter. iii) The former and mannitol agar. Growth on 30% D-glucose (w/v) is does not grow on 30% D-glucose, but the latter does. iv) negative. Grows in the presence of 0.35% acetic acid Weak growth on methanol is found in the former but not (w/v). Grows on methanol weakly. Ammoniac nitrogen in the latter. v) Production of 2,5-diketo-D-gluconate and is very weakly assimilated in the presence of D-glucose, a water-soluble brown pigment from D-glucose is not D-mannitol, or ethanol as a carbon source. Production of found in the former, but is in the latter.10) dihydroxyacetone from glycerol is weakly positive. Of the phenotypic characteristics determined, the Produces 5-keto-D-gluconate and 2-keto-D-gluconate oxidation of acetate and lactate is utilized especially from D-glucose. Does not produce a water-soluble for classifying acetic acid bacteria at the generic brown pigment on glucose/peptone/yeast extract/ level.26,27,30) For example, the genera Acetobacter and calcium carbonate agar. Acid is produced from D- Gluconobacter are discriminated from each other by glucose, D-mannose, D-galactose, D-xylose, D-arabinose acetate and lactate oxidation. The former has the (weakly positive), L-arabinose, L-rhamnose, meso-eryth- capability of oxidizing acetate and lactate to carbon ritol, glycerol (weakly positive), melibiose, or ethanol, dioxide and water by way of the TCA cycle, and the latter but not from D-fructose, L-sorbose, D-mannitol, D- has no capability of oxidizing acetate and lactate due sorbitol, dulcitol, maltose, lactose, sucrose, or raffinose. to lack of the TCA cycle. And there is neither exception Grows on D-glucose, D-mannose (very weakly positive), nor confusion taxonomically in acetate and lactate D-galactose, D-xylose, L-arabinose, L-rhamnose, D- oxidation of test strains.30) fructose, L-sorbose, D-mannitol, D-sorbitol, dulcitol, The two isolates are distinguished from the type strain meso-erythritol, glycerol, or melibiose (very weakly of the type species of the genus Tanticharoenia by positive), but not on D-arabinose, maltose, lactose, acetate and lactate oxidation. The former has intense sucrose, raffinose, or ethanol. A major isoprenoid oxidation of acetate, as found in strains of the genus quinone is Q-10. The DNA base composition is 66.0– Acetobacter, and weak oxidation of lactate, while the 66.1 mol % G+C, with a range of 0.1 mol %. The type latter has no oxidation of acetate and lactate, as found species is Ameyamaea chiangmaiensis sp. nov. in strains of the genus Gluconobacter. In conclusion, the isolates and the type strain of Tanticharoenia Description of Ameyamaea chiangmaiensis sp. nov. sakaeratensis are quite different from each other in Ameyamaea chiangmaiensis (chi.ang.ma.i.en’sis. N. L. one of the most important and basic catabolic pathways, fem. adj. chiangmaiensis, of or pertaining to Chiang viz., the presence or absence, for example, of the ‘active’ Mai, Thailand, where the type strain was isolated). TCA cycle. Characteristics are the same as described for the genus. According to Madigan and Martinko,33) ‘‘Hybridiza- The type strain is isolate AC04T, which has a DNA G+C tion of DNAs from more definitely related organisms, content of 66.0 mol % and was isolated from a flower of for example, Clostridium (gram-positive) and Salmo- red ginger (khing daeng in Thai, Alpinia purpurea), and nella (gram-negative), will hybridize at only background deposited as BCC 15744T at BIOTEC Culture Collection levels, 10% or less.’’ In the present study, the calculated (BCC), National Center for Genetic Engineering and Bio- DNA-DNA hybridization levels, at 4 and 3%, were too technology (BIOTEC), Pathumthani, Thailand, and as low to accommodate the two isolates to the genus NBRC 103196T at Biological Resource Center (NBRC), Tanticharoenia, as found, for example, in the combina- Department of Biotechnology, National Institute of tion of the type strains of Gluconobacter sphaericus and Technology and Evaluation (NITE), Kisarazu, Japan. 2162 P. YUKPHAN et al. 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