タイ原産酢酸菌の16S rRNA遺伝子解析

誌名誌名 Microbiology and Culture Collections

ISSNISSN 13424041

著者著者

村松, 由貴Yukphan, P.高橋, 麻衣金安, 美香Malimas, T.Potacharoen, W.山田, 雄三中川, 恭好Tanticharoen, M.鈴木, 健一朗

巻/号巻/号 25巻1号

掲載ページ掲載ページ p. 13-20

発行年月発行年月 2009年6月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Microbiol. Cult. Coll. 25 (1): 13-20， 2009

16S rRNA gene sequences analysis of acetic acid bacteria isolated from Thailand

Yuki Muramatsu1)*， Pattaraporn Yukphan2)， Mai TakahashP)， Mika Kaneyasul)，

Taweesak Malimas2)， Wanchern Potacharoen2)， Yuzo Yamada2)， Yasuyoshi Nakagawa1

)， Morakot Tanticharoen2) and KerトichiroSuzuki1

)

1) Biological Resource Center (NBRC)， Department of Biotechnology， National Institute of Technology and

Evaluation (NITE)， 2-5-8 Kazusakamatari， Kisarazu， Chiba 292-0818， J apan

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 c10sely 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 12388'1、.Furth巴r，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 In accordance with the Convention on Biological

Diversity (CBD)， the National Institute of

Technology and Evaluation in Japan and the

National Center for Genetic Engineering and

Biotechnology in Thailand completed the

Memorandum of Understanding for conducting

cooperative research to promote the conservation

and sustainable use of biological resources in J apan

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.

'Corresponding author

E-mail: muramatsu回yuki@nite.go.jp

Accepted: January 28， 2009

-13

Acetic acid bacteria are important organisms in

food and beverage industries etc. It is known that

they adapt well to sugary and alcoholized f1uid and

are isolated from vinegar， fruit juice， sap water， alco-

holic beverages and f1owers. A common feature of

this group is the aerobic oxidation of ethanol to ace-

tic acid. The family Acetobacteraceae in the

Alphゆroteobacteriacurrently accommodates 10 gen-

era Acetobacter， Acidomonas， Asaia，

Gluconacetobacter， Gluconobacter， Granulibacter，

Kozakia， Neoasaia， Saccharibacter， and

Swaminathania. They have been isolated in various

countries in East and Southeast Asia， such as J apan，

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

th巴sestrains in our strain catalogues and to opti-

16S rRNA analysis of acetic acid bacteria

mize their use. we determined their 16S rRNA gene

sequences to understand th巴irtaxonomic 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 du1citol

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 30v

C in 5 ml of NBRC 804

broth (Polypepton 5 g (Wako Pure Chemical Ind.

Ltd.. Osaka. J apan). yeast extract 5 g. glucose 5 g.

MgS04・7H201 g. distilled water 1 1. pH 7.0) or

NBRC 350 broth (Polypepton 5 g. yeast extract 5 g.

glucose 5 g. mannitol 5 g. ethanol 5 ml. MgS04・7H20

1 g. and distilled water 1 1; 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

align巴dwith those of representative members of

Alph~ρroteobacteria 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/BioEditlbioedit.html). Phylogenetic trees were

constructed by the neighbor-joining method (Saitou

& Nei. 1987). with ca1culated 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.

Muramatsu et al

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 GAl 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 GAl.

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.

Acetobαcter

The genus Acetobacter currently accommodates

18 species. Based on the 16S rRNA gene sequence

analysis. 17 strains were classified in th巴 genus

Acetobacter and divided into 8 sequence groups

(ABl-AB8) (Fig. 2). ABl c1ustered with A. cerevisiae

LMG 16251'. A. malorum LMG 17461'. and A. orlean-

ensis NBRC 137521' . and was closely related to A.

cerevisiae. AB2 was relatively remote from all

known species. AB3. AB4. AB5. and AB6 were c1ose-ly related to A. indosinensis NRIC 03131'. A.

lovaniensis NBRC 137531'. A. orient，σlis. and A.ρer-oxydans NBRC 137551'. respectively. AB7 clustered

with A. ghanaensis 430A1'. A. syzygii 9H-21'

. and A.

lovaniensis. AB8 was c10sely related to A. tro.ρicalis NIRC 03121'. We are investigating their taxonomic

characteristics to reveal whether they constitute a

new species in the genus Acetobacter.

Asαiα

In the genus Asaia. we grouped 150 strains. and

they were divided into 11 sequence groups (ASl-

AS11) (Fig. 3). We found that AS4. including BCC

157331' (=NBRC 102526) and BCC 15734 (=NBRC

102527) constituted an independent species. We pro-

posed to c1assify them as Asaia lannensis (Malimas

et al.. 2008). Other sequence groups (ASI-AS3 and

AS5-AS11) showed high 16S rRNA gene sequence

-14一

Microbiol. Cult. Coll. June 2009

0.01 KI1I1C

751

999

BCC 15905 (=NBRC 103590，AS1) BCC 15911 (=NBRC 103528， AS11) BCC 15650 (= NBRC 103415， AS2) BCCI5926 (=NBRC 103537，ASIO)

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) BCC 15940 (= NBRC 103543， AS6)

BCC 15645 (AS8) BCC 15660 (= NBRC 103419， AS7)

Swαminathania salitolerans PA 51 T (AF459454) Kozakia baliensis Yo・3T(AB056321)Neoasαia chiangmaiensis BCC 15763T (AB208549)

Vol. 25. NO.1

Granlllibacter bethesdensis CGDNIHIT (AY788950) Glllconacetobacter liqllξfaciens NBRC 12388T (X75617) BCC 12457 (口NBRC103411， GA3)

Acidomonas methanolica NRIC 0498T (ABll0702) BCC 15744 (=NBRC 103196， GA2)

BCC 15772T (= NBRC 103193， GA1， Tanticharoenia sakαeratellsis) BCC 15812 (=NBRC 103481， AB5) BCC 15761 (=NBRC 103463，AB8)

BCC 15756 (= NBRC 103578， AB1) BCC 15762 (= NBRC 103464， AB3)

BCC 15839 (=NBRC 103583，AB2) Acetobacter aceti JCM 7641T (D30768) BCC 15851 (= NBRC 103584， AB7)

BCC 15845 (= NBRC 103497， AB4) BCC 15884 (= NBRC 103585， AB6)

BCC 15775 (= NBRC 103581， GB6) BCC 15863 (=NBRC 103504， GB7) BCC 15764 (=NBRC 103465， GB11) BCC 15643 (=NBRC 103413， GBI2) BCC 15726 (=NBRC 103446， GB9) BCC 15832 (= NBRC 103488， GB8)

77θBCC 15680 (= NBRC 103428， GBIO) _ BCC 15749 (= NBRC 103576， GB5) Gluconobacter o.λ:ydans NBRC 14819T (ABI78433) BCC 15750 (= NBRC 103577， GB4) BCC 15889 (= NBRC 103587， GB1)

BCC 15875 (=NBRC 103510， GB13) 996γBCC 15872 (=NBRC 103508， GB3)

!BCC 15874 (= NBRC 103509， GB2) 992 Sacchat必acterfloricola S-877T (ABll0421)

Fig. 1 A neighbor幽joiningtree showing the phylogenetic positions of the family Acetobαcter，αceαe based on the 16S

rRNA gene sequences. Bar. 0.01 J;丸山 Bootstrapvalues greater than 700 are shown in 1000 replicat巴s.Type spe-

cies are highlighted in bold.

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 m巴rely2 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 sequ巴ncewith the type strain of

Gluconacetobacter liquefaciens NBRC 12388T (Fig. 1).

Gluconobαcter

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. j均 teuriiNBRC 3264T or

-15一

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

Table 1 List of sequence groups of Thai isolates

Phylogenetic group Number of Closest species based on 16S rRNA strams sequence similaritiesa

New genus GA1 3 Proposed as Tanticharoenia sakaeratensis

GA2 2 Tanticharoenia sakaeratensis (97.8)

Acetobacter AB1 A. cerevisiae (99.6)

AB2 A. orientalis (97.9)

AB3 A. indonesiensis (99.3)

AB4 6 A. lovaniensis (99のAB5 5 A. orientalis (99.9)

AB6 A.ρeroxydans (99.4)

AB7 1 A. syzygii (99.7)

AB8 1 A. troρicalis (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 A. krungtheρensis (99.5)

AS7 13 A. krungtheρensis (99.7)

AS8 3 A. krungtheρensis (99劫

AS9 95 A. bogorensis (100)

ASlO 6 A. krungthepensis (99.5)

ASll 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 G. oxydans (99.9)

GB5 3 G. oxydans (99.7)

GB6 2 G.j示。teurii(99.1)

GB7 G.j示。teurii(99.8)

GB8 G. frateurii (99.9)

GB9 G. frateurii (99.9)

GBlO 41 G.j均teurii(99.9)

GBll 20 G.J争ateurii(99.9)

GB12 39 G.j均teurii(100)

GB13 2 G. kondonii (100)

a 16S rRNA sequence similarities (%) are shown in parenthesis.

G. jσρonicus NBRC 3271T (Malimas et al.， 2009).

Intrageneric relationships of the genus

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回23SITS 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

一時一

Microbiol. Cult. Coll. J une 2009 Vol. 25. NO.1

0ω.β0∞0ω2Kι"刷2山U

ト一一→ Acetobacter indonesiensis NRIC 0313T (AB032356)

BCC 15762 (=NBRC 103464，AB3) BCC 15756 (= NBRC 103578， AB1)

Acetobacter cerevisiae LMG 1625T (AJ419843)

Acetobacter malorum LMG 1746T (AJ419844)

Acetobacter orleanensis NBRC 13752T (AB032350) Acetobacter senegalensis CWBI B-418T (AY883036)

Acetobacter tropicalis NRIC 0312T (AB032355) 9%lBCC 15761 (=NBRC 103463，AB8)

Acetobacter cibinongensis 4H-IT (AB052710)

BCC 15812 (=NBRC 103481，AB5) Acetobacter orientalis 21F-2T (AB052706)

BCC 15839 (= NBRC 103583， AB2) Acetobacter aceti JCM 7641T (D30768)

Acetobacter nitrogenifigens RG lT (AY669513)

Acetobacter oeni B13T (AY829472) Acetobacter回 tunensisNBRC 13751T (AB032349)

Acetobacter past，日IrianusLMD 22.lT (X71863) Acetobacter pomorum L乱1018848T (AJ419835)

Acetobacter peroλ:ydans NBRC 13755T (AB032352) BCC 15884 (= NBRC 103585， AB6)

Acetobacter ghanaensis 430AT (EF030713) Acetobacter syzygii 9H-2T (AB052712)

BCC 15851 (=NBRC 103584，AB7) Acetobacter lovaniensis NBRC 13753T (AB032351)

現ナ"LBCC15845 (=NBRC 103497，AB4)

Fig. 2 A neighbor幽joiningtree showing the phylogenetic positions of the genus Acetobαcter

based on the 16S rRNA gene sequences. Bar， 0.002 Knuc. Bootstrap values greater than

700 are shown in 1000 replicates.

917 1000

ハu

nU

AU

I

0.001 K"uc トーーベ BCC 15911 (= NBRC 103528， ASll)

BCC 15940 (= NBRC 103543， AS6)

BCC 15660 (= NBRC 103419， AS7)

BCC 15926 (= NBRC 103537， AS1O)

Asαia bogorensお71T (AB025928)

BCC 15905 (= NBRC 103590，AS1)

BCC 15650 (口NBRC103415，AS2)

BCC 15641 (=NBRC 103412， AS9)

Asaia siamensis S60-1 T (AB035416)

Asaialαnnensis BCC 15733T (= NBRC 102526， AS4)

BCC 15653 (=NBRC 103417，AS5)

Swaminathania salitolerans PA 51 T (AF459454)

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

Asαtαbased on the 16S rRNA gene sequences. Bar， 0.001 Knuc. Bootstrap values

greater than 700 are shown in 1000 replicates

-17一

BCC 15775 (=NBRC 103581， GB6)

BCC 15863 (= NBRC 103504， GB7)

BCC 15832 (= NBRC 103488， GB8)

BCC 15680 (= NBRC 103428， GB 1 0)

BCC 15764 (=NBRC 103465，GBll)

BCC 15276 (=NBRC 103446，GB9)

BCC 15643 (= NBRC 103413， GBI2)

85別n Gluconobacter japonicus NBRC 3271T (AB253435) 833 I I I 'Gluconobacter ftロteuriiNBRC 3264T (AB 178403)

Gluconobacter thailandicus F 149-1 T (AB 128050)

Gluconobacter cerinus NBRC 3267T (AB 178406)

Gluconobαcter 0砂dansNBRC 14819T (ABI78433)

BCC 15749 (= NBRC 103576， GB5)

16S rRNA analysis of acetic acid bacteria

0.002 K.

ト一一一1

1000

Muramatsu et al.

BCC 15889 (=NBRC 103587，GB1)

7剥Glucon伽 cteバondoniiNBRC 3266T (AB 1冗405)

BCC 15875 (=NBRC 103510， GB13)

BCC 15872 (= NBRC 103508， GB3)

Fig.4 A neighbor醐joiningtree showing the phylogenetic positions of the genus

Gluconobαcter based on the 16S rRNA gene sequences. Bar， 0.002 Knuc. Bootstrap values great巴rthan 700 are shown in 1000 replicates

Thailand constituted a variety of a phylogen巴tic

group in the genus Acetobacter， Asaia， and

Gluconobacter， it is expect巴dthat 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 g巴nera.We are investigat同

ing their taxonomic characteristics to confirm

whether thεy 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

result巴din 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

r巴sourcesboth in J apan and Thailand.

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676

タイ原産酢酸菌の 16SrRNA 遺伝子解析

村松由貿 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) バイオテクノロジ一本部 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 16SrRNA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 (ABI-AB8)に分かれた.グループAB2以外の 7グルー

プは Acetobacter 腐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腐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16SrRNA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

(GBl-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.

新種あるいは新潟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. 日本留内のユーザーが利用できるよう，今宙研究した株のうち 155株を BIOTECCulture Collection (BCC)から

NITE Biological Resource Center (NBRC)に寄託して公開した.

(担当編集委員:河村好意)

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