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J. Gen. Appl. Microbiol., 59, 153-166 J. Gen. Appl. Microbiol., 59, 153‒166 (2013) Short Communication Nguyenibacter vanlangensis gen. nov., sp. nov., an unusual acetic acid bacterium in the α-Proteobacteria Huong Thi Lan Vu,1 Pattaraporn Yukphan,2 Winai Chaipitakchonlatarn,2 Taweesak Malimas,2 Yuki Muramatsu,3 Uyen Thi Tu Bui,1 Somboon Tanasupawat,4 Kien Cong Duong,1 Yasuyoshi Nakagawa,3 Ho Thanh Pham,1 and Yuzo Yamada2,*,** 1 Department of Microbiology, Faculty of Biology, University of Science, Vietnam National University-HCM City, 227 Nguyen Van Cu Street, Ward 4, District 5, Hochiminh City, Vietnam 2 BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phaholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand 3 NITE Biological Resource Center, National Institute of Technology and Evaluation, Kisarazu, Chiba 292‒0818, Japan 4 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Wangmai, Pathumwan, Bangkok 10330, Thailand (Received October 4, 2012; Accepted January 17, 2013) Key Words—acetic acid bacteria; Acetobacteraceae; Acidomonas; Gluconacetobacter; Komaga- taeibacter; Nguyenibacter vanlangensis gen. nov., sp. nov. The genus Gluconacetobacter corrig. Yamada et al. In the systematic and ecological studies of acetic 1998 was recently divided into two genera (Yamada acid bacteria, the present authors found that a certain and Yukphan, 2008; Yamada et al., 2012a, b, 2013). strain isolated from the natural environment of Vietnam One is the genus Gluconacetobacter, in which the was located in a unique phylogenetic position, name- type species is retained as Gluconacetobacter liquefa- ly, outside of the Gluconacetobacter cluster (Yamada ciens (Asai 1935) Yamada et al. 1998, and the other is et al., 2012b). the genus Komagataeibacter Yamada et al. 2013, in This paper describes Nguyenibacter vanlangensis which the type species is newly designated as Komaga- gen. nov., sp. nov., for isolate TN01LGIT as well as iso- taeibacter xylinus (Brown 1898) Yamada et al. 2013. In late VTH-AC01. The former was isolated from the rhi- the genus Gluconacetobacter, seven species are now- zosphere of Asian rice collected at Long Thanh Trung adays accommodated (Franke et al., 1999; Fuentes- Commune, Hoa Thanh District, Tay Ninh Province, Ramírez et al., 2001; Gillis et al., 1989; Tazato et al., Vietnam on March 6, 2011, and the latter was isolated 2012; Yamada and Yukphan, 2008). from the root of Asian rice collected at Hoc Mon Dis- trict, Ho Chi Minh City, Vietnam on November 12, 2011. * Corresponding author: Dr. Yuzo Yamada, 2‒3‒21 Seinan-cho, The two isolates were obtained by an enrichment Fujieda, Shizuoka 426‒0063, Japan. culture approach using nitrogen-free LGI medium that Tel/Fax: +81‒54‒635‒2316 contained 10.0% sucrose w/v, 0.06% KH PO w/v, E-mail: [email protected] 2 4 ** JICA Senior Overseas Volunteer, Japan International Co- 0.02% K2HPO4 w/v, 0.02% MgSO4 w/v, 0.002% CaCl2 operation Agency, Shibuya-ku, Tokyo 151‒8558, Japan; Profes- w/v. 0.001% FeCl3 w/v, and 0.0002% Na2MoO4 w/v, sor Emeritus, Shizuoka University, Suruga-ku, Shizuoka, Shi- pH 6.0 (Cavalcante and Döbereiner, 1988). When mi- zuoka 422‒8529, Japan. crobial growth was seen in the LGI medium, the cul- 154 VU et al. Vol. 59 ture was transferred to pH-3.5 medium that contained were calculated by use of the bootstrap analysis of 1.0% D-glucose w/v, 1.0% ethanol v/v, 0.3% peptone Felsenstein (1985) based on 1,000 replications. The w/v, and 0.2% yeast extract w/v. One loop of the pH- pair-wise 16S rRNA gene sequence similarities were 3.5 medium showing microbial growth was streaked calculated for 1,403 bases. onto an agar plate comprised of 2.0% D-glucose w/v, In a 16S rRNA gene sequence phylogenetic tree 0.5% ethanol v/v, 0.3% peptone w/v, 0.3% yeast extract constructed by the neighbor-joining method, the two w/v, 0.7% calcium carbonate w/v, and 1.5% agar w/v, isolates that formed a small cluster with a bootstrap and the resulting colonies that dissolved calcium car- value of 100% were located on the outside but not on bonate on the agar plate were picked up and exam- the inside of the Gluconacetobacter cluster, and the ined again for growth at pH 3.5 (Yamada and Yukphan, resulting cluster formed a large cluster along with the 2008). type strains of Komagataeibacter species (Fig. 1). The Gluconacetobacter liquefaciens NBRC 12388T, Glu- large cluster was then connected to the type strain of conacetobacter diazotrophicus LMG 7603T, Komaga- Acidomonas methanolica. The calculated bootstrap taeibacter xylinus NBRC 15237T, Komagataeibacter han- value at the branching point of the two clusters was senii NBRC 14820T, Acidomonas methanolica NRIC 65%. The data obtained indicated that the two isolates 0498T, and Acetobacter aceti NBRC 14818T were used were not very tightly coupled to the type strains of Glu- as reference strains. conacetobacter species phylogenetically. The 16S rRNA gene sequences of the two isolates In a 16S rRNA gene sequence phylogenetic tree were determined, as described previously (Murama- constructed by the maximum parsimony method, the tsu et al., 2009; Yamada et al., 2000; Yukphan et al., two isolates constituted a cluster first along with the 2004, 2011). Gene fragments for 16S rRNA gene- type strain of Acidomonas methanolica, and then the encoding regions were amplified by PCR with the follow- resulting cluster was connected to the Gluconaceto- ing two primers; 20F (5′-GAGTTTGATCCTGGCTCAG-3′, bacter cluster (Fig. 2). The calculated bootstrap values positions 9‒27) and 1500R (5′-GTTACCTTGTTACGACT- at the respective branching points were 41 and 16%. T- 3 ′, positions 1509‒1492) (the numbering of positions The data obtained indicated that the two isolates were was based on the Escherichia coli numbering system, related phylogenetically to the Acidomonas cluster rather Brosius et al., 1978, 1981; accession number V00348). than the Gluconacetobacter cluster. Amplified 16S rRNA genes were sequenced with the four In a 16S rRNA gene sequence phylogenetic tree primers, 27F (5′-AGAGTTTGATCMTGGCTCAG-3′, posi- constructed by the maximum likelihood method, the two tions 8‒27), 1492R (5′- TACGGYTACCTTGTTACGACT- isolates were located on the outside but not on the inside T- 3 ′, positions 1513-1492), 518F (5′-CCAGCAGCCGC- of the Gluconacetobacter cluster, as found in the phylo- GGTAATACG-3′, positions 518‒537), and 800R (5′-TAC- genetic tree constructed by the neighbor-joining method CAGGGTATCTAATCC-3′, positions 802‒785). Other 16S (Fig. 3). The calculated bootstrap value was 27%. rRNA gene sequences were cited from the GenBank/ The calculated pair-wise 16S rRNA gene sequence EMBL/DDBJ databases. similarities of isolate TN01LGIT were 97.9, 97.7, 96.5, Multiple sequence alignments were made with the 96.9, 95.5, 94.1, 96.2, 96.0, 95.6, 92.0, 96.3, 95.1, 96.2, program Clustal X (version 1.81) (Thompson et al., 97.1, and 94.0% respectively to the type strains of Glu- 1997). Sequence gaps and ambiguous bases were conacetobacter liquefaciens, Gluconacetobacter diaz- excluded. Distance matrices were calculated by the otrophicus, Acidomonas methanolica, Komagataeibacter two-parameter method of Kimura (1980). Phylogenetic xylinus, Acetobacter aceti, Gluconobacter oxydans, Asaia trees based on 16S rRNA gene sequences of 1,216 bogorensis, Kozakia baliensis, Swaminathania salitoler- bases were constructed by the neighbor-joining meth- ans, Saccharibacter floricola, Neoasaia chiangmaiensis, od (Saitou and Nei, 1987), the maximum parsimony Granulibacter bethesdensis, Tanticharoenia sakaeraten- method (Felsenstein, 1983), and the maximum likeli- sis, Ameyamaea chiangmaiensis, and Neokomagataea hood method (Felsenstein, 1981) using the program thailandica. Between the two isolates, the 16S rRNA gene MEGA 5 (version 5.05, Tamura et al., 2011). In the phylo- sequence similarity was 99.9%. On the other hand, the genetic trees constructed, the type strain of Acidocella 16S rRNA gene sequence similarity was 98.5% between facilis was designated as an outgroup. The confidence the type strains of Gluconacetobacter liquefaciens and values of individual branches in the phylogenetic trees Gluconacetobacter diazotrophicus. 2013 Nguyenibacter vanlangensis gen. nov., sp. nov. 155 Fig. 1. Phylogenetic relationships of Nguyenibacter vanlangensis gen. nov., sp. nov. (1). The phylogenetic tree based on 16S rRNA gene sequences was construct- ed by the neighbor-joining method. The type strain of Acidocella facilis was used as an outgroup. The numer- als at the nodes of the respective branches indicate bootstrap values (%) derived from 1,000 replications. 156 VU et al. Vol. 59 Fig. 2. Phylogenetic relation- ships of Nguyenibacter vanlan- gensis gen. nov., sp. nov. (2). The phylogenetic tree based on 16S rRNA gene sequences was constructed by the maxi- mum parsimony method. The type strain of Acidocella facilis was used as an outgroup. The phylogenetic relationships were represented by tree #1 out of 28 most parsimonious trees. The fi- nal data set had a total of 1,216 positions (bases), of which 660 were parsimony informative. Consistency index=0.328, re- tention index=0.804, composite index=0.264, homoplasy index =0.672. The numerals at the nodes of the respective branches indicate bootstrap values (%) de- rived from 1,000 replications. 2013 Nguyenibacter vanlangensis gen. nov., sp. nov. 157 Fig. 3. Phylogenetic relationships of Nguy- enibacter vanlangensis gen. nov., sp. nov. (3). The phylogenetic tree based on 16S rRNA gene sequences was constructed by the maxi- mum likelihood method. The type strain of Ac- idocella facilis was used as an outgroup. The numerals at the nodes of the respective branch- es indicate bootstrap values (%) derived from 1,000 replications. 158 VU et al. Vol. 59 A phylogenetic tree based on the so-called partial methanolica, and Komagataeibacter xylinus.
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