J. Gen. Appl. Microbiol., 55, 101‒110 (2009) Full Paper MxaF gene, a gene encoding alpha subunit of methanol dehydrogenase in and false growth of acetic acid bacteria on methanol Rei Suzuki, Puspita Lisdiyanti†, Kazuo Komagata, and Tai Uchimura* Laboratory of General and Applied Microbiology, Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156‒8502, Japan (Received September 30, 2008; Accepted November 21, 2008) MxaF gene, a gene encoding alpha subunit of methanol dehydrogenase, was investigated for acetic acid bacteria, and growth on methanol was examined for the bacteria by using various media. Of 21 strains of acetic acid bacteria studied, Acidomonas methanolica strains showed the presence of mxaF gene exclusively, and grew on a defi ned medium containing methanol. Further, none of the strains tested of which the growth on methanol had been previously reported, except for Acidomonas methanolica, showed the presence of mxaF gene or the growth on methanol. Precautions were taken against false growth on compounds used for identifi cation of bacteria. Key Words—acetic acid bacteria; Acidomonas; growth on methanol; mxaF gene Introduction grow on methanol (Gosselé et al., 1983b), but this fi nding was rejected by using the same strains as Acetic acid bacteria grow on a variety of sugars, theirs (Uhlig et al., 1986; Urakami et al., 1989). Further, sugar alcohols, and alcohols, and produce corre- Acetobacter pomorum LTH 2458T was described to sponding oxidation products by incomplete oxidation grow on methanol (Sokollek et al., 1998), but it was of the compounds. However, confl icting data have not recognized (Cleenwerck et al., 2002). The growth been reported on the growth on methanol. Several of Acetobacter malorum LMG 1746T (DSM 14337T) strains of Acetobacter pasteurianus were reported to and Acetobacter lovaniensis LMG 1617T (NCIB 8620T) on methanol was reported (Cleenwerck et al., 2002). In addition, assimilation of methanol and acid produc- * Address reprint requests to: Dr. Tai Uchimura, Laboratory of tion from methanol were reported on the strains of A. General and Applied Microbiology, Department of Applied Biol- pasteurianus group A which were isolated from rice ogy and Chemistry, Faculty of Applied Bioscience, Tokyo Uni- versity of Agriculture, Sakuragaoka 1‒1‒1, Setagaya-ku, Tokyo vinegar (komesu) and unpolished rice vinegar (kuro- 156‒8502, Japan. su) in Japan (Nanda et al., 2001). Tel: 03‒5477‒2327 Fax: 03‒5477‒2619 An acidophilic methylotroph, strain MB 58, was re- E-mail: [email protected] ported to grow on methanol (Steudel et al., 1980), and † Present address: Research Center for Biotechnology, Indo- this strain was described as Acetobacter methanolicus nesian Institute of Sciences, Jalan Raya Bogor Km. 46, Cibi- (Uhlig et al., 1986). Later, such methanol-assimilating nong 16911, Indonesia. strains were reidentifi ed as Acidomonas methanolica DDBJ accession numbers for the partial mxaF gene sequence (Urakami et al., 1989), and further this genus was of Acidomonas methanolica NRIC 0498T, LMG 1667, and NRIC 0554, Methylobacterium extorquens NRIC 0601T, Methylobacte- emended (Yamashita et al., 2004). The phylogenetic rium organophilum NRIC 0602T, and Ancylobacter aquaticus relationship of the genus Acidomonas to acetic acid IAM 12364T are AB455971, AB455972, AB455973, AB455974, bacteria (the family Acetobacteraceae) was fi rst re- AB455975, and AB455976, respectively. ported by 5S rRNA sequencing (Bulygina et al., 1992). 102 SUZUKI et al. Vol. 55 Further, the phylogenetic allocation of the genus Aci- shows their sources and references. In addition, Meth- domonas in the family Acetobacteraceae was con- ylobacterium extorquens NRIC 0601T, Methylobacterium fi rmed by 16S rRNA gene sequences (Boesch et al., organophilum NRIC 0602T, and Ancyclobacter aquati- 1998; Kersters et al., 2006; Sievers and Swings, 2005; cus IAM 12364T were used as reference strains. Since Yamada et al., 1997; Yamashita et al., 2004). the strains of A. pasteurianus group A (Nanda et al., Contradictory studies of the response of acetic acid 2001) were not available, it was not possible to check bacteria to methanol would be ascribed to the ambig- their growth on methanol. uous defi nition of the growth on methanol, the assimi- Cultivation of microorganisms. Medium YPM-glu- lation of methanol, and the acid production from meth- cose (YPM-G medium) was used as a basal medium, anol. In addition, various ingredients in media employed which contained 10 g of glucose, 3 g of yeast extract, and methods tested would make explanation diffi cult 5 g of peptone, 3 g of malt extract, and 1,000 ml of and interpretations confusing. Indistinct characteris- distilled water (pH 6.8). YPM-G medium supplemented tics would bring about a problematic misunderstand- with 10% glucose was used for the cultivation of Sac- ing to the identifi cation of acetic acid bacteria. charibacter fl oricola. If necessary, 2% agar was added Methanol dehydrogenases (MDH) of methylotrophs to the media. and methanotrophs are pyrroloquinoline-quinone DNA extraction, PCR, sequencing, and analysis of (PQQ)-containing enzymes (quinoproteins), and cata- mxaF gene. Chromosomal DNAs were extracted by lyze oxidation of methanol to formaldehyde (Anthony, the method described previously (Saito and Miura, 1986; Duine and Frank, 1981; Duine et al., 1980). MDH 1963). Amplifi cation of the mxaF gene from chromo- of Methylobacterium extorquens AM1 was reported to somal DNA was performed by using mxaF-specifi c consist of a large subunit α (62 kDa) and a small sub- primers, mxa f1003 (5′-GCGGCACCAACTGGGGCT- unit β (8.5 kDa) and arrange in an α2β2 confi guration GGT-3′) and mxa r1561 (5′-GGGCAGCATGAAGGGC- (Nunn et al., 1989). MDH of Methylobacillus glycogens TCCC-3′), as described previously (McDonald and showed an α2β2 confi guration as well (Adachi et al., Murrell, 1997). PCR was carried out with a thermocy- 1990). Genes mxaF and mxaI encode the large (α) and cler under the conditions described previously (Mc- small (β) subunits of MDH (Chistoserdova et al., 2003; Donald and Murrell, 1997). Purifi ed PCR products were Lidstrom, 1992; Lidstrom et al., 1994). The mxaF gene sequenced directly by using an ABI PRISMTM BigDye has been described to contain the active site of the Terminator Cycle sequencing Ready Reaction Kit, and enzyme and to be highly conserved in the methylo- ABI PRISMTM model 3130 Genetic analyzer. The prim- trophs and methanotrophs of α-Proteobacteria (Mc- ers mxa f1003 and mxa r1561 were used for the se- Donald and Murrell, 1997). quencing reaction. The amplifi cation of the mxaF gene has been used The sequence of the mxaF gene was determined for for the identifi cation of Hyphomicrobium spp. (Fesefeldt Acidomonas methanolica NRIC 0498T, LMG 1667, and and Gliesche, 1997), and for the detection of methyl- NRIC 0554, Methylobacterium extorquens NRIC 0601T, otrophs and methanotrophs in a blanket bog peat core Methylobacterium organophilum NRIC 0602T, and An- sample (McDonald and Murrell, 1997), rice fi eld soil cylobacter aquaticus IAM 12364T in this study. The se- (Henckel et al., 1999), roots of submerged rice plants quences of the mxaF gene of Paracoccus denitrifi cans (Horz et al., 2001), a peat soil microcosm (Morris et al., PD1207, Methylomonas methanica S1, and Methylo- 2002), and marine environments (Inagaki et al., coccus capsulatus Bath were obtained from EMBL/ 2004). GenBank/DDBJ. This study aims to make it clear whether the acetic Growth on methanol as a sole source of carbon. acid bacteria have the mxaF gene for their oxidation of Media for the growth on methanol were selected from methanol and whether the bacteria grow on methanol those widely used for the identifi cation of acetic acid as a sole source of carbon. bacteria: They were Asai’s medium (Asai et al., 1964), Hoyer-Frateur medium (De Ley and Frateur, 1974), Materials and Methods Frateur’s modifi ed Hoyer medium (De Ley et al., 1984), Cleenwerck’s medium (Cleenwerck et al., 2002), YPM- Bacterial strains. Acetic acid bacteria used in this Me medium (Urakami and Komagata, 1986; Urakami study were obtained from culture collections. Table 1 et al., 1989), Medium C (Urakami et al., 1985; Yamashi- 2009 mxaF gene in acetic acid bacteria 103 Table 1. Strains used in this study. Strains Strain numbers Sources References Acetobacter aceti IFO 14818T beech-wood shavings of a vinegar plant Lisdiyanti et al. (2000) Acetobacter LMG 1262T beer, Netherlands =LMD 22.1, Gosselé et al. (1983b) pasteurianus Acetobacter LMG 1698 brewery, UK =Martin 1, Gosselé et al. (1983b) pasteurianus Acetobacter LMG 1701 beerwort, Alberton, South Africa =VV3, Gosselé et al. (1983b) pasteurianus Acetobacter LMG 1803 Malus sylvestris (crab apple), UK =NCPPB 461, Gosselé et al. (1983b) pasteurianus Acetobacter LMG 1805 Malus sylvestris (crab apple), UK =NCPPB 463, Gosselé et al. (1983b) pasteurianus Acetobacter DSM 11825T submerged cider vinegar fermentation fac- =LTH 2458T, Sokollek et al. (1998); LMG pomorum tory, Germany 18848T, Cleenwerck et al. (2002) Acetobacter IFO 13753T sewage on soil, Belgium Lisdiyanti et al. (2000) lovaniensis Acetobacter AJ 2914 Lisdiyanti et al. (2000) lovaniensis Acetobacter DSM 14337T rotting apple, Belgium =LMG 1746T, Cleenwerck et al. (2002) malorum Gluconobacter IFO 14819T beer =NCIB 9013T oxydans Acidomonas NRIC 0498T methanol fermentation with Candida sp. =MB 58, Steudel et al. (1980); Uhlig et al. methanolica (not sterilized), Germany (1986); Yamashita et al. (2004) Acidomonas LMG 1667 sludge, Germany =MB 57, Uhlig et al. (1986) methanolica Acidomonas NRIC 0554 activated sludge, Japan Yamashita et al. (2004) methanolica Gluconacetobacter IFO 12388T Diospyros sp. (dried persimmon), Japan Yamada et al. (1997) liquefaciens Asaia bogorensis NBRC 16594T Bauhinia purpurea (fl ower of orchid), Indo- Yamada et al. (2000) nesia Kozakia baliensis NBRC 16664T palm brown sugar, Indonesia Lisdiyanti et al. (2002) Swaminathania LMG 21291T Porteresia coarctata (wild rice), India Loganathan & Nair (2004) salitolerans Saccharibacter JCM 12116T pollen, Japan Jojima et al. (2004) fl oricola Neoasaia chiang- NBRC 101099T Alpinia purpurata (fl ower), Thailand Yukphan et al.