Acetobacter Okinawensis Sp. Nov., Acetobacter Papayae Sp. Nov., and Acetobacter Persicus Sp

J. Gen. Appl. Microbiol., 58, 235‒243 (2012) Full Paper

Acetobacter okinawensis sp. nov., Acetobacter papayae sp. nov., and Acetobacter persicus sp. nov.; novel acetic acid bacteria isolated from stems of sugarcane, fruits, and a ﬂ ower in Japan

Takao Iino,1 Rei Suzuki,2 Yoshimasa Kosako,1 Moriya Ohkuma,1 Kazuo Komagata,2 and Tai Uchimura2

1 Japan Collection of Microorganisms, RIKEN BioResource Center, Wako, Saitama 351‒0198, Japan 2 Laboratory of General and Applied Microbiology, Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, Setagaya, Tokyo 156‒8502, Japan

(Received October 3, 2011; Accepted February 29, 2012)

Eleven strains of acetic acid bacteria were isolated from stems of sugarcane, fruits, and a ﬂ ower in Japan. The isolates were separated into three groups, Groups I, II, and III, in the genus Aceto- bacter according to phylogenetic analysis based on 16S rRNA sequences. The isolates had sequence similarities of 99.8‒100% within the Group, 99.3‒99.6% to those of the type strains of each related Acetobacter species, and less than 98.4% to those of the type strains of other Ac- etobacter species. Genomic DNA G+C contents of Groups I, II, and III were 59.2‒59.4, 60.5‒60.7, and 58.7‒58.9 mol%, respectively. The isolates in the Group showed high values of DNA-DNA relatedness to each other, but low values less than 46% to the type strains of related Acetobacter species. A good correlation was found between the three Groups and groups based on DNA G+C contents and DNA-DNA relatedness. All the strains had Q-9 as the main component, and Q-8 and Q-10 as minor components. The isolates in the three Groups did not completely match with any Acetobacter species on catalase reaction, the production of ketogluconic acids from D-glucose, growth on ammoniac nitrogen with ethanol (Hoyer-Frateur medium and Frateur mod- iﬁ ed Hoyer medium), growth on 30% (w/v) D-glucose, growth in 10% (v/v) ethanol, or DNA G+C contents. On the basis of phylogenetic relationships in the genus Acetobacter and chemosys- tematic and phenotypic characteristics, the three Groups were regarded as novel species in the genus Acetobacter. Acetobacter okinawensis sp. nov. is proposed for Group I, Acetobacter papayae sp. nov. for Group II, and Acetobacter persicus sp. nov. for Group III.

Key Words—acetic acid bacteria; Acetobacter; Acetobacter okinawensis; Acetobacter papayae; Ac- etobacter persicus

* Address reprint requests to: Dr. Takao Iino, Japan Collection Introduction of Microorganisms, RIKEN BioResource Center, 2‒1 Hirosawa, Wako, Saitama 351‒0198, Japan. Acetic acid bacteria are important for the industrial Tel: +81‒48‒467‒9564 Fax: +81‒48‒462‒4618 production of vinegar, cellulose, gluconic acid, and E-mail: [email protected] sorbose. Novel acetic acid bacteria are frequently iso- The DDBJ/EMBL/GenBank accession numbers for the 16S rRNA gene sequences of isolates 1-25T, 1-26, 1-34, 1-35T, 1-37, lated from sources in Southeast Asia and Europe, and T-120T, T-122, T-622, T-626, T-639, and T-640 are AB665066, more than 40 species of acetic acid bacteria have AB665067, AB665065, AB665068, AB665069, AB665070, AB665071, been described since 2000 (Sievers and Swings, AB665072, AB665073, AB665074, and AB665075, respectively. 2005). In Japan, many kinds of vinegar are produced 236 IINO et al. Vol. 58 from various Japan-originated materials such as rice, sake lees, and fruits. Recently, fi ve novel acetic acid bacteria, which are Saccharibacter fl oricola, Asaia as- tilbis, Asaia platycodi, Asaia prunellae, and Gluconac- Isolation year etobacter kakiaceti, were isolated from fl owers and

kaki vinegar in Japan (Iino et al., 2012; Jojima et al., EM6 2007

2004; Suzuki et al., 2010). These ﬁ ndings suggest not- Media used yet cultivated acetic acid bacteria inhabiting various Japan-originated materials. Recognition of novel acetic acid bacteria interesting in understanding the diver-

sity of acetic acid bacteria and development of the o, Japan; LMG, BCCM/LMG Bacteria, vinegar production. This paper deals with the isolation

of novel acetic acid bacteria from stems of sugarcane, Japan. kyo University of Agriculture, Tokyo,

fruits, and a fl ower in Japan and the proposal of three ), Okayama, Japan (2007) ), Okinawa, Japan (2004)), Okinawa, Japan (2004) EM1 EM1 2004 2004 novel species in the genus Acetobacter on the basis of ), Okinawa, Japan (2004) EM1 2004 ), Okayama, Japan (2007) EM6 2007 ), Tottori, Japan (2007)), Tottori, EM6 2007 morphological, biochemical, physiological, and phylo- Japan (2007)), Tottori, EM1 2007 makuwa genetic characteristics. var. var. ), Okinawa, Japan (2004) EM1 2004 ), Okinawa, Japan (2004) EM1 2004 Materials and Methods Japan (2007)), Tottori, EM1 2007 Vicia amoena Prunus salicina Punica granatum Cucumis melo Saccharum offi cinarum Saccharum offi Saccharum offi cinarum Saccharum offi Saccharum offi cinarum Saccharum offi Sources of isolation. Pieces of the stem of sugar- Japan (2007)), Tottori, EM6 2007 cane (Saccharum offi cinarum), fruits of grape (Vitis vin- Carica papaya Carica papaya ifera), Japanese plum (Prunus salicina), oriental melon Prunus persica (Cucumis melo var. makuwa), papaya (Carica papaya), peach (Prunus papaya), and pomegranate (Punica Vitis vinifera granatum), and a fl ower of broad-leaf vetch (Vicia Sugarcane (stem) ( Oriental melon (fruit) ( (fruit) ( Papaya (fruit) ( Peach

amoena) were collected in Okayama, Okinawa, and Isolates used in this study.

Tottori Prefectures in Japan from 2004 to 2007 T (Table 1).

Isolation of acetic acid bacteria. EM1 (Lisdiyanti et 1. Table al., 2003; Suzuki et al., 2010) and EM6 (Suzuki et al., T , NRIC 0658 , NRIC 0655 2010) media were used for the isolation of acetic acid T T bacteria. Serial decimal dilutions (10-1 to 10-10) of the samples were made with saline; 0.1 ml of each diluted , LMG 26458 , LMG 26457 , LMG 26456

sample was spread on EM1 and EM6 agar plates, and T T T cultivated at 30°C for 2 weeks. Visible colonies grown on agar plates were picked up, and transferred to fresh EM1 or EM6 agar plates. After several purifi cations, 11 JCM 25146 JCM 25143 JCM 25330 pure cultures were obtained (Table 1). The isolates T were maintained on agar slants of GYP medium (Su- T T T T-640 JCM 25402ower) ( Broad-leaf vetch (fl 1-25 1-26 JCM 25144, NRIC 0656T-122 JCM 25332 (fruit) ( Papaya (fruit) ( Pomegranate T-120 1-37T-622 JCM 25147, NRIC 0659 JCM 25387 Sugarcane (stem) ( Grape ( T-626T-639 JCM 25390 JCM 25401 Japanese plum (fruit) ( zuki et al., 2010). Acetobacter aceti JCM 7641 (De 1-341-35 JCM 15829 Sugarcane (stem) ( Ley and Frateur, 1974), Acetobacter cerevisiae JCM 17273T (Cleenwerck et al., 2002), Acetobacter fabarum LMG 24244T (Cleenwerck et al., 2008), Acetobacter ghanensis LMG 23848T (Cleenwerck et al., 2007), Ac- etobacter lovaniensis JCM 17121T (De Ley and Fra- teur, 1974; Lisdiyanti et al., 2000), Acetobacter malo- Species Strain No. Other designations Sources rum JCM 17274T (Cleenwerck et al., 2002), Acetobacter , Type strain. Abbreviations of the culture collections: JCM, RIKEN BioResource Center, Japan Collection of Microorganisms, Wak strain. Abbreviations of the culture collections: JCM, RIKEN BioResource Center, , Type T T Acetobacter papayae Acetobacter persicus Acetobacter okinawensis orleanensis JCM 7639 (De Ley and Frateur, 1974; Lis- To Research Institute Culture Collection Center, Laboratorium voor Microbiologie, Universiteit Gent, Belgium; NRIC, NODAI 2012 Three novel Acetobacter species (31 characteristics) 237 diyanti et al., 2000), Acetobacter peroxydans JCM 25077T (De Ley and Frateur, 1974), and Acetobacter syzygii JCM 11197T (Lisdiyanti et al., 2001) were used as reference strains. Phylogenetic analysis based on 16S rRNA gene sequences. The 16S rRNA gene was amplifi ed by PCR with the two primers as described in a previous paper (Iino et al., 2007). Purifi ed PCR products were se- quenced directly as described previously (Iino et al., 2012). After alignment of the sequences obtained and those of related species in public DNA databases with ARB software (Ludwig et al., 2004), phylogenetic trees were constructed by the neighbor-joining method (NJ) (Saitou and Nei, 1987) with the CLUSTAL_X program (Thompson et al., 1997), and by the maximum-likelihood method (ML) with the MOLPHY software version 2.3b3 (Adachi and Hasegawa, 1995; Felsenstein, 1981; Hasegawa et al., 1985). Determination of the genomic DNA G+C content and DNA-DNA relatedness. Cells of the isolates culti- Fig. 1. Phylogenetic tree based on 16S rRNA gene sequ- vated on the GYP agar plate at 30°C for 2 days were ences of 11 isolates and Acetobacter species. used for the determination of the genomic DNA G+C The tree was based on an alignment of 1,318 bp of 16S rRNA content. The genomic DNA was extracted and purifi ed gene sequences, and constructed by the neighbor-joining by the method of Saito and Miura (1963). The DNA method. Numbers at nodes indicate bootstrap percentages, derived from 1,000 bootstrap replications (numbers before the G+C content of the isolates was determined with slash are determined by the neighbor-joining analysis, and HPLC model LC-20AD equipped with spectrophotom- those after the slash are by the maximum-likelihood analysis); eter detector model SPD-M20A (Shimadzu) and Cos- values of 70% or more are shown. Dots indicate that the corre- mosil 5C18-MS-II column (Nacalai Tesque) as de- sponding nodes were recovered in the tree generated with the scribed by Tamaoka and Komagata (1984). DNA-DNA maximum-likelihood algorithm. ‒, values of 70% or less. Bar, relatedness was determined by fl uorometric DNA-DNA 0.002 substitutions per nucleotide position. hybridization by using a photobiotin-labeled DNA probe (Ezaki et al., 1989). Reference strains used were selected on the basis of the phylogenetic analysis Suzuki (1987). based on 16S rRNA gene sequences. More recently, Characterization of phenotypic characteristics. Acetobacter farinalis has been validated (Tanaspawat Phenotypic characterization was carried out by the et al., 2011). However, the DNA-DNA hybridization was methods described by Lisdiyanti et al. (2000, 2001). not performed because all the isolates signifi cantly dif- Growth temperatures were tested at 5, 10, 15, 20, 25, fered from A. farinalis in the phylogenetic position 30, 37, and 42°C. The production of cellulose was con- based on 16S rRNA gene sequences (Fig. 1) and fi rmed by the method described by Navarro et al. many phenotypic characteristics (Table 3). (1999). L-Arabinose and 13 other sugars, dulcitol and Quinone analysis. Cells of the isolates cultivated two other sugar alcohols, and ethanol and three other on the GYP agar plate at 30°C for 2 days were used alcohols were used for acid production (Table 3), refer- for the determination of the major quinone. Quinones ring to the description by Lisdiyanti et al. (2000, were extracted with chloroform-methanol (2：1, v/v), 2001). Acid production was determined for the four and purifi ed by thin layer chromatography (TLC). The reference strains on fi ve sugars because the produc- quinones were determined with HPLC model LC-10AD tion from the sugars has not been reported yet equipped with spectrophotometer detector model (Table 3).

SPD-M10Avp (Shimadzu) and a Cosmosil 5C18 column (Nacalai Tesque) described by Komagata and 238 IINO et al. Vol. 58

Table 2. Phenotypic characteristics of the isolates.

1-35T 1-34 1-37 T-622 T-626 T-639 T-640 1-25T 1-26 T-120T T-122 Motility + + + + + + + ---- Growth on glutamate agar ------mannitol agar - + - w - + ---++ Growth on ammoniac with (Hoyer-Frateur medium) D-glucose ------++ D-mannitol ------++ ethanol ------Growth on ammoniac nitrogen with (Frateur modiﬁ ed Hoyer medium) D-glucose --w --ww--++ D-mannitol ------++ ethanol ------Oxidation of acetate + + + + + w + + + + + lactate + + + + + + + + + + + ethanol + + + + + + + + + + + Production of gluconic and ketogluconic acids gluconic acid + + + + + + + + + + + 2-ketogluconic acid ------++ 5-ketogluconic acid ------++ 2,5-diketogluconic acid ------Acid production from L-arabinose --w ----++++ D-ribose ------wwww D-xylose + + w w - +w++++ D-fructose ------D-galactose ------++++ D-glucose + + + + + + + + + + + D-mannose w ------++++ D-sorbose ------lactose ------maltose ------sucrose ------trehalose ------D-rafﬁ nose ------starch ------dulcitol ------D-mannitol ------D-sorbitol ------ethanol + + + + + + + + + + + 1-propanol + + + + + + + + + + + 1-butanol + + + + + + + + + + + 2-butanol ------Quinone (%) Q-8 6.5 9.1 3.9 4.9 7.8 5.9 6.9 7.8 7.1 7.7 6.1 Q-9 86.3 84.2 89.2 86.6 87.6 84.6 86.1 87.6 87.8 87.1 85.6 Q-10 7.3 6.7 6.9 8.5 4.7 9.5 7.1 4.7 5.1 5.3 8.3 DNA G+C content (mol%) 59.3 59.2 59.3 59.3 59.3 59.3 59.4 60.5 60.7 58.7 58.9 2012 Three novel Acetobacter species (31 characteristics) 239

Table 3. Differential characteristics of Acetobacter okinawensis, Acetobacter papayae, Acetobacter persicus and related Acetobacter species.

Characteristics 1 2 3 45678910111213

Catalase + +++++- ++++++ Production of ketogluconic acids from D-glucose: 2-ketogluconic acid ------++++++ 5-ketogluconic acid ------+ ----+ Growth on ammoniac nitrogen with ethanol - v(+) - + --+ --w --- Growth on 30% (w/v) on D-glucose --+ ------+ -- Growth in 10% (v/v) ethanol v(+) v(-)v --+ - +nd- + -- Acid production from: a a a a L-arabinose -- + v(+) v + - + - + - v(-)+ a a a D-galactose -- - v(+) - + - ++ ++a v(-)+ a a a a D-glucose + + + +++- ++ ++ ++ a a a a D-mannose --+ v(+) v + - ++ + - v(+) + a a a a D-xylose + + + v(+) v + - ++ - + v(+) + DNA G+C content (mol%) 59.2‒59.4 56.8‒58.0 56.9‒57.3 57.1‒58.9 54.3‒55.4 60.5‒60.7 59.7‒60.7 58.7‒58.9 56.0‒57.6 56.3‒56.5 57.2 56.5‒58.7 56.2‒57.2

Taxa (n, number of strains tested) are: 1, A. okinawensis (n = 7); 2, A. fabarum (Cleenwerck et al., 2008); 3, A. ghanensis (Cleen- werck et al., 2007); 4, A. lovaniensis (Cleenwerck et al., 2002; Lisdiyanti et al., 2000, 2001); 5, A. syzygii (Lisdiyanti et al., 2001; Ndoye et al., 2007); 6, A. papayae (n = 2); 7, A. peroxydans (Cleenwerck et al., 2002; Lisdiyanti et al., 2000, 2001); 8, A. persicus (n = 2); 9, A. cerevisiae (Cleenwerck et al., 2002); 10, A. farinalis (Tanaspawat et al., 2011); 11, A. malorum (Cleenwerck et al., 2002; Ndoye et al., 2007); 12, A. orleanensis (Cleenwerck et al., 2002; Lisdiyanti et al., 2000, 2001); and 13, A. aceti (Cleenwerck et al., 2002; Lisdiyanti et al., 2000, 2001). Species were grouped in the light of similarities of 16S rRNA sequences. Symbols; +, growth; -, no growth; v, variable; nd, no data available. Characteristics of the type strains are given in parentheses. aData from our study.

Results III formed a distinct subline with A. cerevisiae, A. farinalis, A. malorum, and A. orleanensis. The 16S rRNA Almost complete 16S rRNA gene sequences (ap- gene sequence of the two isolates had sequence sim- proximately 1,400 bases) were determined for the ilarities of 99.9% with each other, 99.3‒99.6% to those eleven isolates. The phylogenetic analysis showed of the type strains of the above-mentioned four spe- that the isolates were separated into three groups, cies (The similarity to strain T-120T was 99.6, 99.4, Groups I, II, and III, in the genus Acetobacter (Fig. 1). 99.4, and 99.3%, respectively), and less than 98.4% to The topologies of the trees by NJ and ML (data not those of other Acetobacter species. shown) were almost identical to each other. The seven Genomic DNA G+C contents of Groups I, II, and III isolates 1-35T, 1-34, 1-37, T-622, T-626, T-639, and were 59.2‒59.4, 60.5‒60.7, and 58.7‒58.9 mol%, re- T-640 in Group I formed a distinct subline with A. faba- spectively (Table 3). DNA-DNA hybridization was car- rum, A. ghanensis, A. lovaniensis, and A. syzygii. The ried out on the isolates with the type strains of Aceto- 16S rRNA gene sequence of the seven isolates had bacter species, which were selected in the light of the sequence similarities of 99.8‒100% to each other, phylogenetic relationship with 16S rRNA sequences. 99.3‒99.6% to those of the type strains of the above- Strains 1-35T, 1-34, 1-37, T-622, T-626, T-639, and T-640 mentioned four species (The similarity to strain 1-35T in Group I showed high values of DNA-DNA related- was 99.5, 99.6, 99.4, and 99.7%, respectively), and ness ranging from 72 to 100% between them, but less than 97.7% to those of other Acetobacter species. showed low values less than 24% with A. fabarum LMG Strains 1-25T and 1-26 in Group II formed a distinct 24244T, A. ghanensis LMG 23848T, A. lovaniensis JCM subline with A. peroxydans. The 16S rRNA gene se- 17121T, and A. syzygii JCM 11197T (Table 4). Strains quence of the two isolates had sequence similarities of 1-25T and 1-26 in Group II showed high values of DNA- 100% with each other, 99.4% to that of A. peroxydans DNA relatedness of 100% to each other, but showed JCM 25077T, and less than 97.4% to those of other low values less than 41% to A. peroxydans JCM 25077T. Acetobacter species. Strains T-120T and T-122 in Group Strains T-120T and T-122 in Group III showed high val- 240 IINO et al. Vol. 58

Table 4. DNA-DNA relatedness (%) between the isolates and the type strains of related Acetobacter species.

Value of DNA-DNA relatedness(%) to the strain

Strains 1-35T 1-25T T-120T

1-35T 100 1-34 93 1-37 100 T-622 72 T-626 78 T-639 81 T-640 85 A. fabarum LMG 24244T 21 A. ghanensis LMG 23848T 24 A. lovaniensis JCM 17121T 17 A. syzygii JCM 11197T 23 1-25T 100 1-26 100 A. peroxydans JCM 25077T 41 T-120T 100 T-122 86 A. cerevisiae JCM 17273T 45 A. malorum JCM 17274T 28 A. orleanensis JCM7639T 46

ues of DNA-DNA relatedness ranging from 86 to 100% medium (De Ley et al., 1984) containing D-glucose, D- to each other, but showed low values less than 46% to mannitol or ethanol was variable with strains as well. A. cerevisiae JCM 17273T, A. malorum JCM 17274T, Gluconic acid was produced from D-glucose by all the and A orleanensis JCM 7639T. A good correlation was isolates, but 2-ketogluconic acid and 5-ketogluconic found between Groups I, II, and III and groups based acid were produced only by the strains in Group III. on the DNA G+C content and the value of DNA-DNA 2,5-Diketogluconic acid was not produced by any of relatedness. the isolates. No isolates produced cellulose from glu- The major quinone of all the isolates was identiﬁ ed cose or dihydroxyacetone from glycerol. Acid produc- as ubiquinone Q-9 (84.2‒89.2%) (Table 2). Ubiquino- tion from sugars and sugar alcohols were variable with nes Q-8 (3.9‒9.1%) and Q-10 (4.7‒9.5%) were also the isolate (Table 2). They grew without acetic acid or detected from all the isolates as minor components. with 0.35% (v/v) acetic acid. All the isolates grew at 10 No correlation was found between the Groups and to 37°C, but not at 4°C or 42°C. They grew at pH 3.5 to quinone compositions. 8.0, but not at pH 3.0 or 9.0. Strains 1-35T, 1-34, and Cells of all the isolates were Gram-negative rods 1-37 grew at pH 8.5. Growth occurred between 1 and and aerobic. Spores were not formed. Strains in Group 20% (w/v) of D-glucose, and no growth was observed I showed motility, but those in Groups II and III did not on 30% (w/v) of D-glucose. Growth occurred between indicate motility. All the isolates were catalase positive 0.5 and 10% (v/v) of ethanol, and no growth was ob- and oxidase negative, and oxidized acetate, lactate, served on 15% (v/v) ethanol. and ethanol after 7 days’ cultivation. Colonies of the isolates were entire, smooth, butyrous, and cream- Discussion colored on the GYP agar plate. The growth on glutamate and mannitol agars was variable with strains All the eleven isolates showed phenotypical charac- (Table 2). The growth on Hoyer-Frateur medium (De teristics of the genus Acetobacter, which are the pro- Ley and Frateur; 1974) and Frateur modiﬁ ed Hoyer duction of acetic acid from ethanol, the growth on 2012 Three novel Acetobacter species (31 characteristics) 241

0.35% (v/v) of acetic acid, the oxidation of acetate and positive and oxidase negative. Colonies are entire, lactate, no acid production from dulcitol and sorbitol, smooth, butyrous, and cream-colored on GYP agar. and major ubiquinone Q-9 (Yamada et al., 2000). Fur- Some strains grow on mannitol agar, but not on gluta- ther, the strains were separated into three groups by mate agar. No growth on Hoyer-Frateur medium or phylogenetic analysis and chemotaxonomic charac- Frateur modifi ed Hoyer medium containing D-glucose, teristics. However, the phenotypic and chemotaxo- mannitol, or ethanol. Oxidizes acetate, lactate, and nomic characteristics of the three groups did not com- ethanol. No production of cellulose from D-glucose or pletely match with those of the related Acetobacter dihydroxyacetone from glycerol. Produces gluconic species (Table 3). acid from D-glucose, but not 2-ketogluconic, 5-keto- The seven isolates in Group I differed from A. fabar- gluconic, or 2, 5-diketogluconic acids. Acid is produc- um, A. ghanensis, A. lovaniensis, and A. syzygii by the ed from D-xylose, D-glucose, ethanol, 1-propanol, and growth on ammoniac nitrogen with ethanol, the growth 1-butanol, but not from L-arabinose, D-ribose, D-fruc- on 30% (w/v) of D-glucose, acid production from sug- tose, D-galactose, D-mannose, D-sorbose, lactose, ars, or the DNA G+C content. The two isolates in maltose, sucrose, trehalose, D-raffi nose, starch, dulci- Group II differed from A. peroxydans by catalase reac- tol, D-mannitol, D-sorbitol, or 2-butanol. Growth occurs tion, the growth on ammoniac nitrogen with ethanol, at 10 and 37°C with optimum growth at 30°C, but not at the growth in 10% (v/v) of ethanol, and acid production 4 or 45°C. Growth occurs at pH 3.0 and 8.5, but not at from sugars. The two isolates in Group III differed from pH 2.5 or 9.0. Growth occurs on 20% (w/v) of D-glu- A. cerevisiae, A. farinalis, A. malorum, and A. orleanen- cose. Growth occurs on 10.0% (w/v) of ethanol. Growth sis by the production of ketogluconic acids from D- occurs without acetic acid or with 0.35% (w/v) acetic glucose, the growth on 30% (w/v) of D-glucose, the acid. The major isoprenoid quinone is Q-9. The ge- growth in 10% (v/v) of ethanol, acid production from nomic DNA G+C content is 59.2‒59.4 mol% (as deter- sugars, or the DNA G+C content. mined with HPLC). The similarity of 16S rRNA gene sequences and The type strain is Acetobacter okinawensis 1-35T (= DNA-DNA relatedness are a reliable way to identify the JCM 25146T = NRIC 0658T = LMG 26457T), which species. All the isolates in the three groups could be was isolated from a piece of the stem of sugarcane distinguished from the related Acetobacter species collected in Okinawa Prefecture in Japan on August 4, with the DNA-DNA relatedness. Furthermore, the iso- 2004. The genomic DNA G+C content of the type lates are separated from other Acetobacter species strain is 59.3 mol%. because the sequence similarity of the 16S rRNA gene is low enough for species differentiation of 98.7‒99.0% Description of Acetobacter papayae sp. nov. recommended previously (Stackebrandt and Ebers, Acetobacter papayae (pa.pa’yae. N.L. fem. adj. pa- 2006). payae of papaya, from which the type strain was iso- In conclusion, the eleven strains differed from the lated.) known species in the genus Acetobacter on the basis Cells are Gram-negative rods, 0.4‒0.8 × 1.8‒3.2 μm of morphology, biochemical and physiological charac- in size, aerobic, non-motile, and non-sporulating. Cat- teristics, and phylogenetic position. Consequently, Ac- alase positive and oxidase negative. Colonies are en- etobacter okinawensis sp. nov. is proposed for Group tire, smooth, butyrous, and cream-colored on GYP I, Acetobacter papayae sp. nov. for Group II, and Ace- agar. No growth on glutamate or mannitol agar. No tobacter persicus sp. nov. for Group III. growth on Hoyer-Frateur medium or Frateur modifi ed Hoyer medium containing D-glucose, mannitol, or eth- Descriptions anol. Oxidizes acetate, lactate, and ethanol. No production of cellulose from D-glucose or dihydroxyace- Description of Acetobacter okinawensis sp. nov. tone from glycerol. Produces gluconic acid from Acetobacter okinawensis (o.ki.na.wen’sis. N.L. fem. D-glucose, but not 2-ketogluconic, 5-ketogluconic, or adj. okinawensis from Okinawa, from where the type 2,5-diketogluconic acids. Acid is produced from L-ara- strain was isolated.) binose, D-ribose (weak), D-xylose, D-galactose, D-glu- Cells are Gram-negative rods, 0.6‒1.0 × 1.8‒4.0 μm cose, D-mannose, ethanol, 1-propanol, and 1-butanol, in size, aerobic, motile, and non-sporulating. Catalase but not from D-fructose, D-sorbose, lactose, maltose, 242 IINO et al. Vol. 58 sucrose, trehalose, D-raffi nose, starch, dulcitol, D-man- July 23, 2007. The genomic DNA G+C content of the nitol, D-sorbitol, or 2-butanol. Growth occurs at 10 and type strain is 58.7 mol%. 37°C with optimum growth at 30°C, but not at 4 or 45°C. Growth occurs at pH 3.0 and 8.0, but not at pH Acknowledgments 2.5 or 8.5. Growth occurs on 20% (w/v) of D-glucose. Growth occurs on 10.0% (w/v) of ethanol. Growth oc- The authors thank Prof. J. Sugiyama for his kind curs without acetic acid or with 0.35% (w/v) acetic suggestions about Latin grammar. We also thank Dr. T. acid. The major isoprenoid quinone is Q-9. The ge- Iida and Dr. M. Sakamoto for technical support. This nomic DNA G+C content is 60.5‒60.7 mol% (as deter- work was supported in part by a research grant (20003- mined with HPLC). 2005) from the Institute for Fermentation, Osaka, Ja- The type strain is Acetobacter papayae 1-25T (= pan, and an incentive research grant (FY2010) from JCM 25143T = NRIC 0655T = LMG 26456T), which the RIKEN, Japan. was isolated from a papaya fruit collected in Okinawa Prefecture in Japan on August 4, 2004. The genomic References DNA G+C content of the type strain is 60.5 mol%. Adachi, J. and Hasegawa, M. (1995) Improved dating of the hu- Description of Acetobacter persicus sp. nov. man/chimpanzee separation in the mitochondrial DNA tree: Heterogeneity among amino acid sites. J. Mol. Evol., Acetobacter persicus (per’si.cus. L. fem. adj. persi- 40, 622‒ 628. cus of persian (=peach), from which the type strain Cleenwerck, I., Camu, N., Engelbeen, K., De Winter, T., Vande- was isolated.) meulebroecke, K., De Vos, P., and De Vuyst, L. (2007) Ac- Cells are Gram-negative rods, 1.0‒1.4 × 1.6‒2.8 μm etobacter ghanensis sp. nov., a novel acetic acid bacterium in size, aerobic, non-motile, and non-sporulating. Cat- isolated from traditional heap fermentations of Ghanaian alase positive and oxidase negative. Colonies are en- cocoa beans. Int. J. Syst. Evol. Microbiol., 57, 1647‒ 1652. tire, smooth, butyrous, and cream-colored on GYP Cleenwerck, I., Gonzales, Á., Camu, N., Engelbeen, K., De Vos, agar. Grows on mannitol agar, but not on glutamate P., and De Vuyst, L. (2008) Acetobacter fabarum sp. nov., agar. Grows on Hoyer-Frateur medium and Frateur an acetic acid bacterium from a Ghanaian cocoa bean heap fermentation. Int. J. Syst. Evol. Microbiol., 58, 2180‒ modifi ed Hoyer medium containing D-glucose or man- 2185. nitol, but not on either medium containing ethanol. Cleenwerck, I., Vandemeulebroecke, K., Janssens, K., and Oxidizes acetate, lactate, or ethanol. No production of Swings, J. (2002) Re-examination of the genus Aceto- cellulose from D-glucose or dihydroxyacetone from bacter, with descriptions of Acetobacter cerevisiae sp. nov. glycerol. Produces gluconic, 2-ketogluconic, and 5-ke- and Acetobacter malorum sp. nov. Int. J. Syst. Evol. Micro- togluconic acids from D-glucose, but not 2,5-diketo- biol., 52, 1551‒ 1558. gluconic acid. Acid is produced from L-arabinose, D- De Ley, J. and Frateur, J. (1974) Genus Acetobacter Beijerinck 1898, 215. In Bergey s Manual of Determinative Bacteriol- ribose (weak), D-xylose, D-galactose, D-glucose, ’ ogy, 8th ed., ed. by Buchanan, R. E. and Gibbons, N. E., D-mannose, ethanol, 1-propanol, and 1-butanol, but Williams and Wilkins, Baltimore, pp. 276‒ 278. not from D-fructose, D-sorbose, lactose, maltose, su- De Ley, J., Swings, J., and Gosselé, F. (1984) Genus I. Aceto- crose, trehalose, D-raffi nose, starch, dulcitol, D-manni- bacter Beijerinck 1898 215AL. In Bergey’s Manual of Sys- tol, D-sorbitol, or 2-butanol. Growth occurs at 10 and tematic Bacteriology, Vol. 1, ed. by Krieg, N. R. and Holt, J. 37°C with optimum growth at 30°C, but not at 4 or G., Williams and Wilkins, Baltimore, pp. 268‒ 274. 45°C. Growth occurs at pH 3.0 and 8.0, but not at pH Ezaki, T., Hashimoto, Y., and Yabuuchi, E. (1989) Fluorometric 2.5 or 8.5. Growth occurs on 20% (w/v) of D-glucose. deoxyribonucleic acid-deoxyribonucleic acid hybridization Growth occurs on 10.0% (w/v) of ethanol. Growth oc- in microdilution wells as an alternative to membrane fi lter curs without acetic acid or with 0.35% (w/v) acetic hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. acid. The major isoprenoid quinone is Q-9. The ge- Bacteriol., 39, 224‒ 229. nomic DNA G+C content is 58.7‒58.9 mol% (as deter- Felsenstein, J. (1981) Evolutionary trees from DNA sequences: mined with HPLC). A maximum likelihood approach. J. Mol. Evol., 17, 368‒ T The type strain is Acetobacter persicus T-120 (= 376. JCM 25330T = LMG 26458T), which was isolated from Hasegawa, M., Kishino, H., and Yano, T. (1985) Dating of the a peach fruit collected in Tottori Prefecture in Japan on human-ape splitting by a molecular clock of mitochondrial 2012 Three novel Acetobacter species (31 characteristics) 243

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