Microbiol. Cult. Coll. 2（1）：135 ─ 20, 2009 16S rRNA gene sequences analysis of acetic acid isolated from Thailand

Yuki Muramatsu1)*, Pattaraporn Yukphan2), Mai Takahashi1), Mika Kaneyasu1), Taweesak Malimas2), Wanchern Potacharoen2), Yuzo Yamada2), Yasuyoshi Nakagawa1), Morakot Tanticharoen2) and Ken-ichiro Suzuki1)

1) Biological Resource Center (NBRC), Department of Biotechnology, National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan 2) BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC) Pathumthani 12120, Thailand

We determined the 16S rRNA gene sequences of 302 strains of acetic acid bacteria isolated from Thailand. The isolates were divided into 35 sequence groups based on differences in 16S rRNA gene sequences. A phylogenetic tree constructed from these sequences showed that 5 strains should be classified into new genera in the family . The other 297 strains were assigned to 33 sequence groups of 4 known genera Acetobacter, , Gluconacetobacter, and Gluconobacter. Seventeen strains belonging to Acetobacter were divided into 8 sequence groups (AB1-AB8). Seven groups except for AB2 were closely related to known Acetobacter species. AB2 was remote from known Acetobacter species. We sorted 150 strains of Asaia into 11 sequence groups (AS1-AS11). AS11 was distinct from all known Asaia species. Nine strains assigned to Gluconacetobacter showed 100% sequence similarity to Gluconacetobacter liquefaciens NBRC 12388T. Further, 121 strains of Gluconobacter were divided into 13 sequence groups (GB1-GB13). Of these, GB1 and GB6 seemed to constitute 2 distinct lineages in the genus. Based on these results, 155 Thai strains were deposited from the BIOTEC culture collection (BCC) in the NITE Biological Resource Center (NBRC) for public use.

Keywords: acetic acid bacteria, 16S rRNA gene sequence, Thailand

INTRODUCTION Acetic acid bacteria are important organisms in In accordance with the Convention on Biological food and beverage industries etc. It is known that Diversity (CBD), the National Institute of they adapt well to sugary and alcoholized fluid and Technology and Evaluation in Japan and the are isolated from vinegar, fruit juice, sap water, alco- National Center for Genetic Engineering and holic beverages and flowers. A common feature of Biotechnology in Thailand completed the this group is the aerobic oxidation of ethanol to ace- Memorandum of Understanding for conducting tic acid. The family Acetobacteraceae in the cooperative research to promote the conservation currently accommodates 10 gen- and sustainable use of biological resources in Japan era Acetobacter, Acidomonas, Asaia, and Thailand for academic, industrial, and other pur- Gluconacetobacter, Gluconobacter, Granulibacter, poses. It is expected that their utilization and com- , Neoasaia, Saccharibacter, and mercialization will provide benefits to both coun- Swaminathania. They have been isolated in various tries. The NITE Biological Resource Center (NBRC) countries in East and Southeast Asia, such as Japan, and the BIOTEC Culture Collection (BCC) started Indonesia, Thailand, and the Philippines since 1996 joint research projects on bacteria, yeast, and fungi (Kersters, et al., 2006; Sievers & Swings, 2005; in 2005 to increase microbiological resources in both Uchimura, 2007; Yamada & Yukphan, 2008). More collections. than 300 strains of acetic acid bacteria were isolated from various sources collected from various areas in *Corresponding author Thailand and maintained at the BCC (Yukphan et al., E-mail: [email protected] 2004c) to be identified at a later date. To publicize Accepted: January 28, 2009 these strains in our strain catalogues and to opti-

─ 13 ─ 16S rRNA analysis of acetic acid bacteria Muramatsu et al. mize their use, we determined their 16S rRNA gene RESULTS AND DISCUSSION sequences to understand their taxonomic positions. The analysis of the phylogenetic tree based on the It was also expected that some new taxa would be 16S rRNA gene sequences indicated that 297 strains found in these strains because many new acetic acid belonged to the genera Acetobacter, Asaia, bacteria have been isolated from tropical zones. Gluconacetobacter, and Gluconobacter. The other 5 strains (16S rRNA gene sequence groups GA1 and MATERIAL AND METHODS GA2) were not closely related to any known species Bacterial strains and cultivation conditions and occupied an independent position in the family We isolated 302 strains from various samples, Acetobacteraceae (Fig. 1). Their 16S rRNA gene including fruit, fermented fruit, flowers, seeds, and sequence similarities to all known species were mushrooms collected in various regions of Thailand, lower than 96.5%. We propose that the group GA1, such as Bangkok, Pak Kret, Ratchaburi, Suratthani, including the three strains BCC 15772 (=NBRC Samutsakorn, Ayutthaya, Chiang Mai, Sakaerat, and 103193), BCC 15773 (=NBRC 103194), and BCC 15774 Tong Pha Phum (Kanchanaburi). A glucose/ethanol/ (=NBRC 103195), be classified in a new genus acetic acid medium, a sorbitol medium, a dulcitol Tanticharoenia sakaeratensis (Yukphan et al., 2008). medium, and/or a sucrose medium were used for We will publish a new taxon elsewhere for the enrichment (Huong et al., 2007; Lisdiyanti et al., 2002; group GA2, consisting of the 2 strains BCC 15744 (= Yamada et al., 1976, 1999, 2000; Yukphan et al., 2004c, NBRC 103196) and BCC 15745 (=NBRC 103197). The 2005). To obtain cell mass for DNA extraction, the 302 isolates were divided into 35 groups based on strains were cultivated at 30℃ in 5 ml of NBRC 804 differences in the 16S rRNA gene sequence. Their broth (Polypepton 5 g (Wako Pure Chemical Ind. closely related species are summarized in Table 1. Ltd., Osaka, Japan), yeast extract 5 g, glucose 5 g,

MgSO4・7 H 2O 1 g, distilled water 1 l, pH 7.0) or Acetobacter NBRC 350 broth (Polypepton 5 g, yeast extract 5 g, The genus Acetobacter currently accommodates glucose 5 g, mannitol 5 g, ethanol 5 ml, MgSO4・7 H 2O 18 species. Based on the 16S rRNA gene sequence 1 g, and distilled water 1 l; pH 7.0). analysis, 17 strains were classified in the genus Acetobacter and divided into 8 sequence groups PCR amplification, sequencing, and phylogenetic (AB1-AB8) (Fig. 2). AB1 clustered with A. cerevisiae analysis of 16S rRNA genes LMG 1625T, A. malorum LMG 1746T, and A. orlean- Genomic DNA extraction and 16S rRNA sequenc- ensis NBRC 13752T , and was closely related to A. ing were performed as previously described cerevisiae. AB2 was relatively remote from all (Takahashi et al., 2006). Sequence data obtained were known species. AB3, AB4, AB5, and AB6 were close- aligned with those of representative members of ly related to A. indosinensis NRIC 0313T, A. Alphaproteobacteria by using ClustalX (Thompson et lovaniensis NBRC 13753T, A. orientalis, and A. per- al., 1997) and then modified manually by referring to oxydans NBRC 13755T, respectively. AB7 clustered the 16S rRNA secondary structure of Escherichia with A. ghanaensis 430AT, A. syzygii 9H-2T, and A. coli (Gutell et al., 1994), and using the BioEdit lovaniensis. AB8 was closely related to A. tropicalis sequence alignment editor (http://www.mbio.ncsu. NIRC 0312T. We are investigating their taxonomic edu/BioEdit/bioedit.html). Phylogenetic trees were characteristics to reveal whether they constitute a constructed by the neighbor-joining method (Saitou new species in the genus Acetobacter.

& Nei, 1987), with calculated Knuc values (Kimura, 1980). The topology of the trees was evaluated using Asaia Felsenstein's (1985) bootstrap method with 1000 rep- In the genus Asaia, we grouped 150 strains, and licates. Nearly complete sequences of the 16S rRNA they were divided into 11 sequence groups (AS1- genes from positions 28 through 1494 according to AS11) (Fig. 3). We found that AS4, including BCC the Escherichia coli numbering system (Brosius et 15733T (=NBRC 102526) and BCC 15734 (=NBRC al., 1978) were determined for all strains. The 16S 102527) constituted an independent species. We pro- rRNA gene sequences of type strains were obtained posed to classify them as Asaia lannensis (Malimas from Japan's DNA databank. et al., 2008). Other sequence groups (AS1-AS3 and AS5-AS11) showed high 16S rRNA gene sequence

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BCC 15905 (= NBRC 103590, AS1) 0.01 Knuc BCC 15911 (= NBRC 103528, AS11) BCC 15650 (= NBRC 103415, AS2) BCC15926 (= NBRC 103537, AS10) Asaia bogorensis 71T (AB025928) BCC 15641 (= NBRC 103412, AS9) BCC 15733 (= NBRC 102526, AS4, Asaia lannensis) BCC 15653 (= NBRC 103417, AS5) BCC 15677 (= NBRC 103426, AS3) 1000 BCC 15940 (= NBRC 103543, AS6) BCC 15645 (AS8) 783 BCC 15660 (= NBRC 103419, AS7) Swaminathania salitolerans PA 51 T (AF459454) Kozakia baliensis Yo-3 T (AB056321) T 850 Neoasaia chiangmaiensis BCC 15763 (AB208549) Granulibacter bethesdensis CGDNIH1T (AY788950) Gluconacetobacter liquefaciens NBRC 12388T (X75617) 920 1000 BCC 12457 (= NBRC 103411, GA3) 750 Acidomonas methanolica NRIC 0498T (AB110702) BCC 15744 (= NBRC 103196, GA2) 887 BCC 15772T (= NBRC 103193, GA1, Tanticharoenia sakaeratensis) 751 BCC 15812 (= NBRC 103481, AB5) BCC 15761 (= NBRC 103463, AB8) BCC 15756 (= NBRC 103578, AB1) BCC 15762 (= NBRC 103464, AB3) 924 704 BCC 15839 (= NBRC 103583, AB2) Acetobacter aceti JCM 7641T (D30768) 854 1000 BCC 15851 (= NBRC 103584, AB7) BCC 15845 (= NBRC 103497, AB4) 829 BCC 15884 (= NBRC 103585, AB6) BCC 15775 (= NBRC 103581, GB6) 868 BCC 15863 (= NBRC 103504, GB7) BCC 15764 (= NBRC 103465, GB11) 916 BCC 15643 (= NBRC 103413, GB12) BCC 15726 (= NBRC 103446, GB9) BCC 15832 (= NBRC 103488, GB8) 999 776 BCC 15680 (= NBRC 103428, GB10) BCC 15749 (= NBRC 103576, GB5) 898 Gluconobacter oxydans NBRC 14819T (AB178433) BCC 15750 (= NBRC 103577, GB4) 996 BCC 15889 (= NBRC 103587, GB1) BCC 15875 (= NBRC 103510, GB13) BCC 15872 (= NBRC 103508, GB3) 996 BCC 15874 (= NBRC 103509, GB2) 992 Saccharibacter floricola S-877T (AB110421) Fig. 1 A neighbor-joining tree showing the phylogenetic positions of the family Acetobacteraceae based on the 16S

rRNA gene sequences. Bar, 0.01 Knuc. Bootstrap values greater than 700 are shown in 1000 replicates. Type spe- cies are highlighted in bold. similarities to known Asaia species. Because differ- the genus Gluconacetobacter showed an identical ences in the 16S rRNA gene sequences are extreme- 16S rRNA gene sequence with the type strain of ly small in the genus Asaia, as shown previously Gluconacetobacter liquefaciens NBRC 12388T (Fig. 1). (Yamada & Yukphan, 2008), we could not obtain reli- able phylogenetic relationships based on the low Gluconobacter bootstrap values. For example, the number of nucle- We divided 121 strains into 13 sequence groups otide differences between A. bogorensis (AB025928) (GB1-GB13) under the genus Gluconobacter (Fig. 4). and A. siamensis (AB035416) is merely 2 bases. Three sequence groups, namely GB2, GB3 and GB13, However, it was reported that they can be distin- formed a cluster with G. albidus and G. kondonii. guished from each other by DNA-DNA relatedness GB1 showed the highest 16S rRNA gene sequence and several phenotypes (Katsura et al. 2001). similarity with G. albidus. GB4 and GB5 were close- ly related to G. oxydans NBRC 14819T. GB6 was rel- Gluconacetobacter atively remote from all known species. GB7-11 Nine strains (sequence group GA3) belonging to formed a cluster with G. frateurii NBRC 3264T or

─ 15 ─ 16S rRNA analysis of acetic acid bacteria Muramatsu et al.

Table 1 List of sequence groups of Thai isolates Number of Closest species based on 16S rRNA Phylogenetic group strains sequence similaritiesa New genus GA1 3 Proposed as Tanticharoenia sakaeratensis GA2 2 Tanticharoenia sakaeratensis (97.8) Acetobacter AB1 1 A. cerevisiae (99.6) AB2 1 A. orientalis (97.9) AB3 1 A. indonesiensis (99.3) AB4 6 A. lovaniensis (99.4) AB5 5 A. orientalis (99.9) AB6 1 A. peroxydans (99.4) AB7 1 A. syzygii (99.7) AB8 1 A. tropicalis (99.8) Asaia AS1 1 A. bogorensis (99.7) AS2 2 A. siamensis (99.9) AS3 6 A. lannensis (99.5) AS4 7 Proposed as Asaia lannensis AS5 12 A. lannensis (99.5) AS6 1 A. krungthepensis (99.5) AS7 13 A. krungthepensis (99.7) AS8 3 A. krungthepensis (99.9) AS9 95 A. bogorensis (100) AS10 6 A. krungthepensis (99.5) AS11 4 A. bogorensis (99.6) Gluconacetobacter GA3 9 G. liquefaciens (100) Gluconobacter GB1 2 G. albidus (99.5) GB2 6 G. albidus (100) GB3 2 G. albidus (99.9) GB4 1 G. oxydans (99.9) GB5 3 G. oxydans (99.7) GB6 2 G. frateurii (99.1) GB7 1 G. frateurii (99.8) GB8 1 G. frateurii (99.9) GB9 1 G. frateurii (99.9) GB10 41 G. frateurii (99.9) GB11 20 G. frateurii (99.9) GB12 39 G. frateurii (100) GB13 2 G. kondonii (100) a 16S rRNA sequence similarities (%) are shown in parenthesis.

G. japonicus NBRC 3271T (Malimas et al., 2009). restriction endonucleases was used to identify Intrageneric relationships of the genus Gluconobacter species (Huong et al., 2007; Malimas et Gluconobacter based on 16S rRNA gene sequences al., 2006). More information through several analyses, were not reliable because the bootstrap values were such as restriction analysis of 16S-23S ITS and phe- not high, as shown in Fig. 4. It was reported that notypic characteristics, is required to identify iso- the analysis of the 16S-23S ITS sequence analysis lates of the genus Gluconobacter. was useful for the identification of Gluconobacter species (Takahashi et al., 2006; Yukphan et al., 2004a, CONCLUSION 2004b). Restriction analysis of 16S-23S ITS using 6 As described above, since the 302 isolates from

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0.002 Knuc 1000 Acetobacter indonesiensis NRIC 0313T (AB032356) BCC 15762 (= NBRC 103464, AB3) BCC 15756 (= NBRC 103578, AB1) T 997 Acetobacter cerevisiae LMG 1625 (AJ419843) T 728 Acetobacter malorum LMG 1746 (AJ419844) Acetobacter orleanensis NBRC 13752T (AB032350) Acetobacter senegalensis CWBI B-418T (AY883036) 940 Acetobacter tropicalis NRIC 0312T (AB032355) 996 BCC 15761 (= NBRC 103463, AB8) Acetobacter cibinongensis 4H-1T (AB052710) 835 BCC 15812 (= NBRC 103481, AB5) 991 Acetobacter orientalis 21F-2T (AB052706) BCC 15839 (= NBRC 103583, AB2) Acetobacter aceti JCM 7641T (D30768) Acetobacter nitrogenifigens RG1T (AY669513) Acetobacter oeni B13T (AY829472) 721 Acetobacter estunensis NBRC 13751T (AB032349) 917 1000 Acetobacter pasteurianus LMD 22.1T (X71863) Acetobacter pomorum LMG 18848T (AJ419835) Acetobacter peroxydans NBRC 13755T (AB032352) 1000 BCC 15884 (= NBRC 103585, AB6) Acetobacter ghanaensis 430AT (EF030713) Acetobacter syzygii 9H-2T (AB052712) 1000 BCC 15851 (= NBRC 103584, AB7) Acetobacter lovaniensis NBRC 13753T (AB032351) 917 BCC 15845 (= NBRC 103497, AB4) Fig. 2 A neighbor-joining tree showing the phylogenetic positions of the genus Acetobacter

based on the 16S rRNA gene sequences. Bar, 0.002 Knuc. Bootstrap values greater than 700 are shown in 1000 replicates.

0.001 Knuc BCC 15911 (= NBRC 103528, AS11)

BCC 15940 (= NBRC 103543, AS6)

BCC 15660 (= NBRC 103419, AS7) 812 BCC 15645 (AS8) 850 Asaia krungthepensis AA08T (AB102953)

BCC 15926 (= NBRC 103537, AS10)

Asaia bogorensis 71T (AB025928)

BCC 15905 (= NBRC 103590, AS1)

BCC 15650 (= NBRC 103415, AS2)

BCC 15641 (= NBRC 103412, AS9)

Asaia siamensis S60-1T (AB035416) Asaia lannensis BCC 15733T (= NBRC 102526, AS4)

BCC 15653 (= NBRC 103417, AS5)

BCC 15677 (= NBRC 103426, AS3)

Swaminathania salitolerans PA 51 T (AF459454)

Fig. 3 A neighbor-joining tree showing the phylogenetic positions of the genus

Asaia based on the 16S rRNA gene sequences. Bar, 0.001 Knuc. Bootstrap values greater than 700 are shown in 1000 replicates.

─ 17 ─ 16S rRNA analysis of acetic acid bacteria Muramatsu et al.

0.002 Knuc BCC 15775 (= NBRC 103581, GB6) BCC 15863 (= NBRC 103504, GB7)

BCC 15832 (= NBRC 103488, GB8) BCC 15680 (= NBRC 103428, GB10) BCC 15764 (= NBRC 103465, GB11) BCC 15276 (= NBRC 103446, GB9) BCC 15643 (= NBRC 103413, GB12) 858 Gluconobacter japonicus NBRC 3271T (AB253435) 833 Gluconobacter frateurii NBRC 3264T (AB178403) 1000 Gluconobacter thailandicus F149-1T (AB128050) 809 Gluconobacter cerinus NBRC 3267T (AB178406) Gluconobacter oxydans NBRC 14819T (AB178433) 766 BCC 15749 (= NBRC 103576, GB5) BCC 15750 (= NBRC 103577, GB4)

BCC 15889 (= NBRC 103587, GB1)

T 779 Gluconobacter kondonii NBRC 3266 (AB178405) 745 BCC 15875 (= NBRC 103510, GB13)

981 Gluconobacter albidus NBRC 3250T (AB178392) BCC 15872 (= NBRC 103508, GB3) BCC 15874 (= NBRC 103509, GB2) Fig. 4 A neighbor-joining tree showing the phylogenetic positions of the genus

Gluconobacter based on the 16S rRNA gene sequences. Bar, 0.002 Knuc. Bootstrap values greater than 700 are shown in 1000 replicates.

Thailand constituted a variety of a phylogenetic CBD. We will maintain and, in fact, strengthen this group in the genus Acetobacter, Asaia, and partnership to explore and use microbiological Gluconobacter, it is expected that they will possess resources both in Japan and Thailand. some new and/or useful properties. In addition, we found several new sequence groups that will be clas- REFERENCES sified into new species or genera. We are investigat- Brosius, J., Palmer, M.L., Kennedy, P.J. & Noller, H.F. ing their taxonomic characteristics to confirm (1978). Complete nucleotide sequence of a 16S ribo- whether they constitute new taxa, and the results somal RNA gene from Escherichia coli. Proc. Natl. will be published elsewhere. After the CBD is vali- Acad. Sci. U.S.A. 75: 4801-4805. dated, access to biological resources in foreign coun- Felsenstein, J. (1985). Confidence limits on phyloge- tries becomes difficult. Establishment of techniques nies: an approach using the bootstrap. Evolution for classification and identification of microorganisms 39: 783-791. has become important for performing microbiologi- Gutell, R.R., Larsen, N. & Woese, C.R. (1994). Lessons cal studies in compliance with the CBD. This study from an evolving rRNA; 16S and 23S rRNA struc- resulted in depositing 155 Thai strains from the tures from a comparative perspective. Microbiol. BCC into the NBRC and making them available to Rev. 58: 10-26. the public. Accordingly, cooperative research Huong, V.T.L., Malimas, T., Yukphan, P., through culture collections in different countries, Potacharoen, W., Tanasupawat, S., Loan, L.T.T., such as those from the NBRC and the BCC in this Tanticharoen, M. & Yamada, Y. (2007). study, should be useful to promote the conservation Identification of Thai isolates assigned to the and sustainable use of biological resources under the genus Gluconobacter based on 16S-23S rRNA ITS

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タイ原産酢酸菌の 16S rRNA 遺伝子解析

村松由貴 1), Pattaraporn Yukphan2), 高橋麻衣 1), 金安美香 1), Taweesak Malimas2), Wanchern Potacharoen2), 山田雄三 2), 中川恭好 1), Morakot Tanticharoen2), 鈴木健一朗 1)

1) 独立行政法人製品評価技術基盤機構（NITE） バイオテクノロジー本部 生物遺伝資源部門（NBRC） 2) BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC)

タイ国で分離された酢酸菌 302 株の 16S rRNA 遺伝子塩基配列を決定し，系統解析を行った．5 株は Acetobacteraceae 科 の新属に分類すべきと考えられ，残りの 297 株は Acetobacter，Asaia，Gluconacetobacter あるいは Gluconobacter 属のいず れかに含まれた．Acetobacter 属には 17 株が含まれ，8 グループ（AB1-AB8）に分かれた．グループ AB2 以外の 7 グルー プは Acetobacter 属の既知種に近縁であった．Asaia 属には 150 株が属し，11 グループ（AS1-AS11）に分かれた．グループ AS11 は Asaia 属の既知種から独立していた．Gluconacetobacter 属に含まれた 9 株は，Gluconacetobacter liquefaciens NBRC 12388T と 100% の 16S rRNA 塩基配列相同性を示した．残りの 121 株は Gluconobacter 属に含まれ，13 グループ （GB1-GB13）に分かれた．2 つのグループ GB1 と GB6 は Gluconobacter 属の既知種から独立していた． 新種あるいは新属に分類すべきと考えられたいくつかのグループが見つかり，タイの酢酸菌は系統的に多様であると考え られた．日本国内のユーザーが利用できるよう，今回研究した株のうち 155 株を BIOTEC Culture Collection (BCC) から NITE Biological Resource Center (NBRC) に寄託して公開した． （担当編集委員：河村好章）

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