INTRODUCTION
In accordance with the Convention on Biological D i v e r s i t y ( C B D ) , t h e N a t i o n a l I n s t i t u t e o f Technology and Evaluation in Japan and the National Center for Genetic Engineering and B i o t e c h n o l o g y i n T h a i l a n d c o m p l e t e d t h e Memorandum of Understanding for conducting cooperative research to promote the conservation and sustainable use of biological resources in Japan and Thailand for academic, industrial, and other pur- poses. It is expected that their utilization and com- mercialization will provide benefits to both coun- tries. The NITE Biological Resource Center (NBRC) and the BIOTEC Culture Collection (BCC) started joint research projects on bacteria, yeast, and fungi in 2005 to increase microbiological resources in both collections.
Acetic acid bacteria are important organisms in food and beverage industries etc. It is known that they adapt well to sugary and alcoholized fluid and are isolated from vinegar, fruit juice, sap water, alco- holic beverages and flowers. A common feature of this group is the aerobic oxidation of ethanol to ace- tic acid. The family Acetobacteraceae in the Alphaproteobacteria currently accommodates 10 gen- e r a A c e t o b a c t e r , A c i d o m o n a s , A s a i a , Gluconacetobacter, Gluconobacter, Granulibacter, K o z a k i a , N e o a s a i a , S a c c h a r i b a c t e r , a n d Swaminathania. They have been isolated in various countries in East and Southeast Asia, such as Japan, Indonesia, Thailand, and the Philippines since 1996 (Kersters, et al., 2006; Sievers & Swings, 2005;
Uchimura, 2007; Yamada & Yukphan, 2008). More than 300 strains of acetic acid bacteria were isolated from various sources collected from various areas in Thailand and maintained at the BCC (Yukphan et al., 2004c) to be identified at a later date. To publicize these strains in our strain catalogues and to opti-
*Corresponding author
E-mail: [email protected] Accepted: January 28, 2009
16S rRNA gene sequences analysis of acetic acid bacteria isolated from Thailand
Yuki Muramatsu
1)*, Pattaraporn Yukphan
2), Mai Takahashi
1), Mika Kaneyasu
1), Taweesak Malimas
2), Wanchern Potacharoen
2), Yuzo Yamada
2), Yasuyoshi
Nakagawa
1), Morakot Tanticharoen
2)and Ken-ichiro Suzuki
1)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 Acetobacteraceae. The other 297 strains were assigned to 33 sequence groups of 4 known genera Acetobacter, Asaia, 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
mize their use, we determined their 16S rRNA gene sequences to understand their taxonomic positions.
It was also expected that some new taxa would be found in these strains because many new acetic acid bacteria have been isolated from tropical zones.
MATERIAL AND METHODS
Bacterial strains and cultivation conditions
We isolated 302 strains from various samples, including fruit, fermented fruit, flowers, seeds, and mushrooms collected in various regions of Thailand, such as Bangkok, Pak Kret, Ratchaburi, Suratthani, Samutsakorn, Ayutthaya, Chiang Mai, Sakaerat, and Tong Pha Phum (Kanchanaburi). A glucose/ethanol/
acetic acid medium, a sorbitol medium, a dulcitol medium, and/or a sucrose medium were used for enrichment (Huong et al., 2007; Lisdiyanti et al., 2002;
Yamada et al., 1976, 1999, 2000; Yukphan et al., 2004c, 2005). To obtain cell mass for DNA extraction, the strains were cultivated at 30℃ in 5 ml of NBRC 804 broth (Polypepton 5 g (Wako Pure Chemical Ind.
Ltd., Osaka, Japan), yeast extract 5 g, glucose 5 g, MgSO4・7H2O 1 g, distilled water 1 l, pH 7.0) or NBRC 350 broth (Polypepton 5 g, yeast extract 5 g, glucose 5 g, mannitol 5 g, ethanol 5 ml, MgSO4・7H2O 1 g, and distilled water 1 l; pH 7.0).
PCR amplification, sequencing, and phylogenetic analysis of 16S rRNA genes
Genomic DNA extraction and 16S rRNA sequenc- ing were performed as previously described (Takahashi et al., 2006). Sequence data obtained were aligned with those of representative members of Alphaproteobacteria by using ClustalX (Thompson et al., 1997) and then modified manually by referring to the 16S rRNA secondary structure of Escherichia coli (Gutell et al., 1994), and using the BioEdit sequence alignment editor (http://www.mbio.ncsu.
edu/BioEdit/bioedit.html). Phylogenetic trees were constructed by the neighbor-joining method (Saitou
& Nei, 1987), with calculated Knuc values (Kimura, 1980). The topology of the trees was evaluated using Felsenstein's (1985) bootstrap method with 1000 rep- licates. Nearly complete sequences of the 16S rRNA genes from positions 28 through 1494 according to the Escherichia coli numbering system (Brosius et al., 1978) were determined for all strains. The 16S rRNA gene sequences of type strains were obtained from Japan's DNA databank.
RESULTS AND DISCUSSION
The analysis of the phylogenetic tree based on the 16S rRNA gene sequences indicated that 297 strains belonged to the genera Acetobacter, Asaia, Gluconacetobacter, and Gluconobacter. The other 5 strains (16S rRNA gene sequence groups GA1 and GA2) were not closely related to any known species and occupied an independent position in the family Acetobacteraceae (Fig. 1). Their 16S rRNA gene sequence similarities to all known species were lower than 96.5%. We propose that the group GA1, including the three strains BCC 15772 (=NBRC 103193), BCC 15773 (=NBRC 103194), and BCC 15774 (=NBRC 103195), be classified in a new genus Tanticharoenia sakaeratensis (Yukphan et al., 2008).
We will publish a new taxon elsewhere for the group GA2, consisting of the 2 strains BCC 15744 (=
NBRC 103196) and BCC 15745 (=NBRC 103197). The 302 isolates were divided into 35 groups based on differences in the 16S rRNA gene sequence. Their closely related species are summarized in Table 1.
Acetobacter
The genus Acetobacter currently accommodates 18 species. Based on the 16S rRNA gene sequence analysis, 17 strains were classified in the genus Acetobacter and divided into 8 sequence groups (AB1-AB8) (Fig. 2). AB1 clustered with A. cerevisiae LMG 1625T, A. malorum LMG 1746T, and A. orlean- ensis NBRC 13752T , and was closely related to A.
cerevisiae. AB2 was relatively remote from all known species. AB3, AB4, AB5, and AB6 were close- ly related to A. indosinensis NRIC 0313T, A.
lovaniensis NBRC 13753T, A. orientalis, and A. per- oxydans NBRC 13755T, respectively. AB7 clustered with A. ghanaensis 430AT, A. syzygii 9H-2T, and A.
lovaniensis. AB8 was closely related to A. tropicalis NIRC 0312T. We are investigating their taxonomic characteristics to reveal whether they constitute a new species in the genus Acetobacter.
Asaia
In the genus Asaia, we grouped 150 strains, and they were divided into 11 sequence groups (AS1- AS11) (Fig. 3). We found that AS4, including BCC 15733T (=NBRC 102526) and BCC 15734 (=NBRC 102527) constituted an independent species. We pro- posed to classify them as Asaia lannensis (Malimas et al., 2008). Other sequence groups (AS1-AS3 and AS5-AS11) showed high 16S rRNA gene sequence
similarities to known Asaia species. Because differ- ences in the 16S rRNA gene sequences are extreme- ly small in the genus Asaia, as shown previously (Yamada & Yukphan, 2008), we could not obtain reli- able phylogenetic relationships based on the low bootstrap values. For example, the number of nucle- otide differences between A. bogorensis (AB025928) and A. siamensis (AB035416) is merely 2 bases.
However, it was reported that they can be distin- guished from each other by DNA-DNA relatedness and several phenotypes (Katsura et al. 2001).
Gluconacetobacter
Nine strains (sequence group GA3) belonging to
the genus Gluconacetobacter showed an identical 16S rRNA gene sequence with the type strain of Gluconacetobacter liquefaciens NBRC 12388T (Fig. 1).
Gluconobacter
We divided 121 strains into 13 sequence groups (GB1-GB13) under the genus Gluconobacter (Fig. 4).
Three sequence groups, namely GB2, GB3 and GB13, formed a cluster with G. albidus and G. kondonii.
GB1 showed the highest 16S rRNA gene sequence similarity with G. albidus. GB4 and GB5 were close- ly related to G. oxydans NBRC 14819T. GB6 was rel- atively remote from all known species. GB7-11 formed a cluster with G. frateurii NBRC 3264T or Saccharibacter floricola S-877T (AB110421)
BCC 15874 (= NBRC 103509, GB2)BCC 15872 (= NBRC 103508, GB3) BCC 15875 (= NBRC 103510, GB13) 992
BCC 15889 (= NBRC 103587, GB1) 996
BCC 15750 (= NBRC 103577, GB4)
Gluconobacter oxydans NBRC 14819BCC 15749 (= NBRC 103576, GB5)T (AB178433) 898
BCC 15680 (= NBRC 103428, GB10) BCC 15832 (= NBRC 103488, GB8) BCC 15726 (= NBRC 103446, GB9) BCC 15643 (= NBRC 103413, GB12) BCC 15764 (= NBRC 103465, GB11)
776
BCC 15863 (= NBRC 103504, GB7)BCC 15775 (= NBRC 103581, GB6) 916
996 868
BCC 15884 (= NBRC 103585, AB6) BCC 15845 (= NBRC 103497, AB4)BCC 15851 (= NBRC 103584, AB7) 1000
Acetobacter aceti JCM 7641T (D30768) BCC 15839 (= NBRC 103583, AB2) BCC 15762 (= NBRC 103464, AB3)BCC 15756 (= NBRC 103578, AB1) BCC 15761 (= NBRC 103463, AB8) 704
BCC 15812 (= NBRC 103481, AB5)
829 924
BCC 15772T (= NBRC 103193, GA1, Tanticharoenia sakaeratensis) BCC 15744 (= NBRC 103196, GA2)Acidomonas methanolica NRIC 0498T (AB110702)
BCC 12457 (= NBRC 103411, GA3)
Gluconacetobacter liquefaciens NBRC 12388Granulibacter bethesdensis CGDNIH1T (X75617) T (AY788950) 1000
Neoasaia chiangmaiensis BCC 15763T (AB208549) Kozakia baliensis Yo-3Swaminathania salitolerans PA 51T (AB056321)T (AF459454) BCC 15660 (= NBRC 103419, AS7)
BCC 15645 (AS8)BCC 15940 (= NBRC 103543, AS6) BCC 15677 (= NBRC 103426, AS3)BCC 15653 (= NBRC 103417, AS5)
BCC 15733 (= NBRC 102526, AS4, Asaia lannensis) BCC 15641 (= NBRC 103412, AS9)
Asaia bogorensis 71T (AB025928) BCC15926 (= NBRC 103537, AS10)BCC 15650 (= NBRC 103415, AS2) BCC 15911 (= NBRC 103528, AS11) BCC 15905 (= NBRC 103590, AS1)
850 1000
750 783
887
854 920
999 751 0.01 Knuc
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.
G. japonicus NBRC 3271T (Malimas et al., 2009).
I n t r a g e n e r i c r e l a t i o n s h i p s o f t h e g e n u s Gluconobacter based on 16S rRNA gene sequences were not reliable because the bootstrap values were not high, as shown in Fig. 4. It was reported that the analysis of the 16S-23S ITS sequence analysis was useful for the identification of Gluconobacter species (Takahashi et al., 2006; Yukphan et al., 2004a, 2004b). Restriction analysis of 16S-23S ITS using 6
restriction endonucleases was used to identify Gluconobacter species (Huong et al., 2007; Malimas et al., 2006). More information through several analyses, such as restriction analysis of 16S-23S ITS and phe- notypic characteristics, is required to identify iso- lates of the genus Gluconobacter.
CONCLUSION
As described above, since the 302 isolates from Table 1 List of sequence groups of Thai isolates
Phylogenetic group Number of
strains Closest species based on 16S rRNA 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.
BCC 15845 (= NBRC 103497, AB4)
Acetobacter lovaniensis NBRC 13753T (AB032351) BCC 15851 (= NBRC 103584, AB7)
917
Acetobacter syzygii 9H-2T (AB052712) Acetobacter ghanaensis 430AT (EF030713)
BCC 15884 (= NBRC 103585, AB6) Acetobacter peroxydans NBRC 13755T (AB032352) Acetobacter pomorum LMG 18848T (AJ419835)
Acetobacter pasteurianus LMD 22.1T (X71863)
1000 1000
1000
Acetobacter estunensis NBRC 13751T (AB032349) Acetobacter oeni B13T (AY829472)
Acetobacter nitrogenifigens RG1T (AY669513) Acetobacter aceti JCM 7641T (D30768)
BCC 15839 (= NBRC 103583, AB2) Acetobacter orientalis 21F-2T (AB052706) BCC 15812 (= NBRC 103481, AB5)
Acetobacter cibinongensis 4H-1T (AB052710) 991
BCC 15761 (= NBRC 103463, AB8)
Acetobacter tropicalis NRIC 0312T (AB032355)
Acetobacter senegalensis CWBI B-418T (AY883036) 996
Acetobacter orleanensis NBRC 13752T (AB032350) Acetobacter malorum LMG 1746T (AJ419844) Acetobacter cerevisiae LMG 1625T (AJ419843)
BCC 15756 (= NBRC 103578, AB1)
728
BCC 15762 (= NBRC 103464, AB3)
Acetobacter indonesiensis NRIC 0313T (AB032356)
997 1000
940
835
917 721 0.002 Knuc
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.
Swaminathania salitolerans PA 51T (AF459454) BCC 15677 (= NBRC 103426, AS3)
BCC 15653 (= NBRC 103417, AS5) Asaia lannensis BCC 15733T (= NBRC 102526, AS4) Asaia siamensis S60-1T (AB035416)
BCC 15641 (= NBRC 103412, AS9) BCC 15650 (= NBRC 103415, AS2)
BCC 15905 (= NBRC 103590, AS1) Asaia bogorensis 71T (AB025928)
BCC 15926 (= NBRC 103537, AS10)
Asaia krungthepensis AA08T (AB102953) BCC 15645 (AS8)
BCC 15660 (= NBRC 103419, AS7) BCC 15940 (= NBRC 103543, AS6)
850 812
BCC 15911 (= NBRC 103528, AS11) 0.001 Knuc
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.
Thailand constituted a variety of a phylogenetic group in the genus Acetobacter, Asaia, and Gluconobacter, it is expected that they will possess some new and/or useful properties. In addition, we found several new sequence groups that will be clas- sified into new species or genera. We are investigat- ing their taxonomic characteristics to confirm whether they constitute new taxa, and the results will be published elsewhere. After the CBD is vali- dated, access to biological resources in foreign coun- tries becomes difficult. Establishment of techniques for classification and identification of microorganisms has become important for performing microbiologi- cal studies in compliance with the CBD. This study resulted in depositing 155 Thai strains from the BCC into the NBRC and making them available to the public. Accordingly, cooperative research through culture collections in different countries, such as those from the NBRC and the BCC in this study, should be useful to promote the conservation and sustainable use of biological resources under the
CBD. We will maintain and, in fact, strengthen this partnership to explore and use microbiological resources both in Japan and Thailand.
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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）
