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Neokomagataea Gen. Nov., with Descriptions of Neokomagataea Thailandica Sp

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Neokomagataea Gen. Nov., with Descriptions of Neokomagataea Thailandica Sp Biosci. Biotechnol. Biochem., 75 (3), 419–426, 2011 Neokomagataea gen. nov., with Descriptions of Neokomagataea thailandica sp. nov. and Neokomagataea tanensis sp. nov., Osmotolerant Acetic Acid Bacteria of the -Proteobacteria Pattaraporn YUKPHAN,1 Taweesak MALIMAS,1 Yuki MURAMATSU,2 Wanchern POTACHAROEN,1 y Somboon TANASUPAWAT,3 Yasuyoshi NAKAGAWA,2 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 Received May 7, 2010; Accepted November 29, 2010; Online Publication, March 7, 2011 [doi:10.1271/bbb.100344] Isolates AH11T and AH13T were isolated from Of the 12 genera of acetic acid bacteria described to flowers of lantana and candle bush respectively collected date,1–5) the genus Saccharibacter Jojima et al. 20046) in Thailand. In phylogenetic trees based on 16S rRNA is unique from the osmophilic point of view and does gene sequences, the two isolates formed an independent not show any growth on 1.0% D-glucose w/v, differing cluster, which was then connected to the type strain of from other acetic acid bacteria. In addition, the genus Saccharibacter floricola. The calculated pair-wise 16S Saccharibacter is characterized by no oxidation of rRNA gene sequence similarities of isolate AH11T were acetate and weak oxidation of lactate, and no growth is 95.7–92.3% to the type strains of the type species of the shown in the presence of 0.35% acetic acid w/v.6) 12 genera of acetic acid bacteria. The DNA base During the course of studies on microbial diversity in composition was from 51.2 to 56.8 mol % G+C, with a the natural environment of Thailand, two isolates were range of 5.6 mol %. When isolate AH11T was labeled, obtained, related phylogenetically to strains of Saccha- DNA-DNA similarities were 100, 12, 4, 5, and 4% ribacter floricola Jojima et al. 2004,6) but the two respectively to isolates AH11T and AH13T and the isolates were not osmophilic but osmotolerant, being type strains of Saccharibacter floricola, Gluconobacter different in this respect from strains of Saccharibacter oxydans, and Acetobacter aceti. The two isolates were floricola. non-motile and did not oxidize either acetate or lactate. This paper proposes Neokomagataea gen. nov., along No growth was found in the presence of 0.35% acetic with descriptions of Neokomagataea thailandica sp. acid w/v. The two isolates were not osmophilic but nov. and Neokomagataea tanensis sp. nov., for the two osmotolerant, produced 2,5-diketo-D-gluconate from isolates, which were isolated at Tan Island, Hat D-glucose, and did not oxidize lactate, thus differing Khanom-Mu Ko Thale Tai National Park, Nakhon-Si- from strains of Saccharibacter floricola, which showed Thammarat, Thailand on March 13, 2007. weak lactate oxidation. The two isolates contained unsaturated C18:1!7c fatty acid as the major fatty acid, Materials and Methods and were unique in the presence of a considerable amount of straight-chain C18:12OH fatty acid. Q-10 Isolation of acetic acid bacteria and reference strains. Two isolates, was present as the major isoprenoid quinone. Neo- designated AH11T and AH13T, were isolated by an enrichment culture T komagataea gen. nov. was proposed with the two species, approach: isolate AH11 was isolated from a flower of lantana Neokomagataea thailandica sp. nov. for isolate AH11T (Lantana camera; hedge flower; pha-ka-krong in Thai) by the use of a T T glucose/ethanol medium containing 2.0% D-glucose w/v, 0.5% (¼ BCC 25710 ¼ NBRC 106555 ), which has 56.8 ethanol w/v, 0.5% peptone w/v, and 0.3% yeast extract w/v, and mol % G+C, and Neokomagataea tanensis sp. nov. was adjusted to pH 3.5, and isolate AH13T was isolated from a flower T T for isolate AH13T (¼ BCC 25711 ¼ NBRC 106556 ), of candle bush (Senna alata; ringworm bush; chum-het-thet in Thai) by which has 51.2 mol % G+C. the use of a glucose medium containing 10.0% D-glucose w/v, 0.5% peptone w/v, and 0.3% yeast extract w/v, and was adjusted to 7,8) Key words: Neokomagataea gen. nov.; Neokomagataea pH 3.5. Neither of the enrichment culture media contained any acetic acid, and they differed from that of Yamada et al.9) Acetobacter thailandica sp. nov.; Neokomagataea aceti NBRC 14818T (NBRC, NITE Biological Resource Center, tanensis sp. nov.; acetic acid bacteria; Department of Biotechnology, National Institute of Technology and Acetobacteraceae Evaluation, Kisarazu, Japan), Gluconobacter oxydans NBRC 14819T, Gluconobacter cerinus NBRC 3267T, Gluconobactr frateurii NBRC The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences are AB513364 for Neokomagataea thailandica isolate AH11T and AB513363 for Neokomagataea tanensis isolate AH13T. y To whom correspondence should be addressed. 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; Tel/Fax: +81-54-635-2316; E-mail: [email protected] 420 P. YUKPHAN et al. 3264T, Gluconobacter thailandicus BCC 14116T (BCC, BIOTEC bootstrap value of 66% (Fig. 1). Between the two Culture Collection, National Center for Genetic Engineering and isolates, the calculated bootstrap value was 100%. Biotechnology, National Science and Technology Development In a phylogenetic tree based on 16S rRNA gene Agency, Pathumthani, Thailand), Gluconobacter japonicus NBRC 3271T, Gluconobacter wancherniae BCC 15775T, Acidomonas meth- sequences constructed by the maximum parsimony 16) anolica NRIC 0498T (NRIC, NODAI Research Institute Center, Tokyo method, almost the same clustering was found in the University of Agriculture, Tokyo, Japan), Gluconacetobacter liquefa- isolates and the genera Saccharibacter and Glucono- ciens NBRC 12388T, Asaia bogorensis NBRC 16594T, Kozakia bacter (Fig. 2). The calculated bootstrap values were 50, baliensis NBRC 16664T, Swaminathania salitolerans LMG 21291T 66, and 99% respectively. In a phylogenetic tree based (LMG, Laboratorium voor Microbiologie, Universiteit Gent, Ghent, T on 16S-rRNA gene sequences constructed by the Belgium), Saccharibacter floricola BCC 16445 , Neoasaia chiang- maximum likelihood method,17) similar small clusters maiensis BCC 15763T, Granulibacter bethesdensis ATCC BAA 1260T (ATCC, American Type Culture Collection, Rockville, MD, USA), and a similar large cluster were found, with bootstrap Tanticharoenia sakaeratensis BCC 15772T, and Ameyamea chiag- values respectively of 74, 80, and 100% (Fig. 3). maiensis BCC 15744T were used as reference strains. The calculated pair-wise sequence similarities of isolate AH11T were 94.2, 95.7, 95.3, 95.0, 95.0, 94.5, Sequencing of 16S rRNA genes and phylogenetic analyses. PCR 94.5, 94.1, 94.1, 93.9, 93.9, and 92.3% respectively to the amplification of 16S rRNA genes was performed, and amplified type strains of the type species of the respective genera, 16S rRNA genes were sequenced and analyzed as described previ- Saccharibacter floricola, Gluconobacter oxydans, Asaia ously.7,8,10–12) Multiple sequence alignment was performed with the program CLUSTAL X (version 1.8) (Thompson et al.13)). Alignment bogorensis, Tanticharoenia sakaeratensis, Ameyamaea gaps and unidentified bases were eliminated. Distance matrices for the chiangmaiensis, Acetobacter aceti, Swaminathania sal- aligned sequences were calculated by Kimura’s two-parameter itolerans, Neoasaia chiangmaiensis, Kozakia baliensis, method.14) Phylogenetic trees for 1,356 bases of 16S rRNA genes Acidomonas methanolica, Gluconacetobacter liquefa- were constructed by the neighbor-joining method,15) the maximum ciens, and Granulibacter bethesdensis. The sequence parsimony method,16) and the maximum likelihood method.17) Robust- 18) similarity was 99.3% between the two isolates. Since the ness for individual branches was estimated by bootstrapping with two isolates were closely related to Gluconobacter 1,000 replications by means of the program MEGA (version 4.0).19) Bootstrap values below 50% were not shown. To construct the species phylogenetically (Fig. 1), the pair-wise sequence phylogenetic tree by the maximum likelihood method,17) the program similarities of the isolates were additionally calculated PHYLIP (version 3.6; J. Felsenstein, University of Washington) was to representative strains of the genus Gluconobacter. used. Pair-wise sequence similarities were calculated in 16S rRNA Isolates AH11T and AH13T had 96.0–97.1% similarity gene sequences of 1,382 bases. A computerized 16S rRNA gene to the type strains of Gluconobacter albidus, Glucono- restriction analysis was made using NEBcutter (version 2.0; New bacter cerinus, Gluconobacter frateurii, Gluconobacter England BioLabs, Ipswich, MA, USA) for isolates AH11T and AH13T. The 16S rRNA gene PCR products of the isolates were prepared and thailandicus, Gluconobacter japonicus, and Glucono- digested with restriction endonucleases TaqI and BccI.5,8,10,11,20) bacter wancherniae. 16S rRNA gene restriction analysis using NEBcutter DNA base composition determination and DNA-DNA hybridization. (version 2.0) theoretically discriminated the two isolates Chromosomal DNA was prepared as described previously.4,5,7,8) The from the type strains of the type species of the 12 DNA base composition was determined by the
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