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 se- quence 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 pa- payae 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 ace- tic 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. T 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 se- quences. 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-likeli- hood 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).