J. Gen. Appl. Microbiol., 55, 155‒162 (2009) Full Paper Taxonomical and physiological comparisons of the three species of the genus Amphibacillus Toshiaki Arai,1,† Shuhei Yanahashi,1,† Junichi Sato,1 Takumi Sato,1 Morio Ishikawa,2 Yukimichi Koizumi,2 Shinji Kawasaki,1 Youichi Niimura,1,* and Junichi Nakagawa3 1 Department of Bioscience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156‒8502, Japan 2 Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156‒8502, Japan 3 Department of Food Science and Technology, Tokyo University of Agriculture, Abashiri, Hokkaido 099‒2493, Japan (Received December 2, 2008; Accepted December 22, 2008) Amphibacillus is a genus for Gram-positive, spore-forming, rod-shaped, facultatively anaerobic bacteria with low-G+C content of DNA, established by Niimura et al. in 1990. Amphibacillus xy- lanus, the type species of the genus, grows well under both strictly anaerobic and aerobic condi- tions in spite of lacking any isoprenoid quinones, cytochromes, and catalase. Amphibacillus fermentum and Amphibacillus tropicus were later proposed by Zhilina et al. in 2001 for the iso- lates from a soda lake. In this paper, we revealed the latter two species also lacked isoprenoid quinones, cytochrome and catalase, and that they grew well under strictly anaerobic and aerobic conditions. The consistent growth of A. xylanus under both conditions is due to the presence of anaerobic and aerobic pathways for glucose metabolism in the organism. Although A. fermen- tum and A. tropicus are supposed to have a side enzymatic pyruvate pathway to produce lactate under both conditions, the two species have two major pyruvate metabolic pathways as ob- served in A. xylanus. Analysis data indicated that NADH formed both by the aerobic pyruvate pathway and by the glycolytic pathway was re-oxidized by the NADH oxidase in A. fermentum and A. tropicus as well as A. xylanus, and furthermore that the NADH oxidase-Prx (AhpC) system, i.e., NADH oxidase scavenging hydrogen peroxide with Prx, also functions in A. tropicus as ob- served with A. xylanus. Not only the taxonomical character of the genus Amphibacillus but also the growth characterization based on the two metabolic pathways and unique oxygen metabo- lism are distinctive in those traits from other facultative anaerobes. Key Words—Amphibacillus; NADH oxidase Introduction nus Amphibacillus, which was established by Niimura et al. (1990) as a Gram-positive, spore-forming, rod- Amphibacillus xylanus is the first species of the ge- shaped, facultatively anaerobic bacteria isolated from alkaline compost. Since then, halophilic/halotolerant/ alkaliphilic and/or alkalitolerant bacilli have been clas- * Address reprint requests to: Dr. Youichi Niimura, Depart- sified in the HA group (Ishikawa et al., 2002) and their ment of Bioscience, Tokyo University of Agriculture, 1‒1‒1 physiology studied extensively. The HA group consist Sakuragaoka, Setagaya-ku, Tokyo 156‒8502, Japan. of 82 species of 19 genera including A. xylanus which Tel: +81‒3‒5477‒2761 Fax: +81‒3‒5477‒2668 E-mail: [email protected] are phylogenetically closely related to each other † Toshiaki Arai and Shuhei Yanahashi contributed equally to based on 16S rRNA gene sequences. A. xylanus is this research and are co-first authors. classified as an independent taxon in the group and 156 ARAI et al. Vol. 55 Fig. 1. Phylogenetic relationships among Amphibacillus species and other related bacteria, based on 16S rRNA gene sequences. Alicyclobacillus acidoterrestris DSM 3922T (AJ133631) was used as an outgroup. The 16S rRNA gene sequences re- trieved from public databases were aligned using the CLUSTAL X program (version 1.8) (Thompson et al., 1997). The phy- logenetic tree, constructed by using the neighbor-joining method, is based on a comparison of approximately 1,400 nucle- otides. In the phylogenetic analysis, hypervariable regions were omitted. Bar, 0.01 Knuc in nucleotide sequences. Bootstrap values, expressed as a percentage of 1,000 replications, are given at branching points; only values above 50% are shown. closely related to 3 genera and 11 species (Paralioba- taxonomic properties. In this report we provide de- cillus, Gracilibacillus, Halolactibacillus, Fig. 1) (Ishika- tailed taxonomical and physiological characterization wa et al., 2002). A. xylanus, which lacks a respiratory of the three species of the genus Amphibacillus to system and heme proteins, including catalase, grows show the unique behavior to oxygen of the genus. well and displays the same growth rate and cell yield under strictly anaerobic and aerobic conditions. This Materials and Methods is due to the presence of anaerobic and aerobic path- ways (Niimura et al., 1989), comprising a unique oxy- Bacterial strains, media and growth conditions. The gen metabolic system (Niimura et al., 1995, 2000). strains studied were A. xylanus Ep01T (=DSM 6626T Zhilina et al. (2001) subsequently isolated alkaliphilic =JCM 7361T=NBRC 15112T), A. fermentum Z-7984T rod-shaped, Gram-positive anaerobic bacteria from a (=DSM 13869T=UNIQEM 210T) and A. tropicus Z- soda lake in Africa, and proposed two new species of 7792T (=DSM 13870T =UNIQEM 212T). A. xylanus was the genus Amphibacillus, namely A. fermentum and A. grown aerobically or anaerobically in a glucose-con- tropicus for the strains. However, physiological com- taining medium as previously described (Niimura et parisons of the three Amphibacillus species have not al., 1987, 1990). For static culture of A. fermentum and been studied in detail yet in consideration with their A. tropicus, we used a modified GYPF medium (Ishika- 2009 Physiology of the genus Amphibacillus 157 wa et al., 2005) named GYPFKMK medium in this previously described (Nishiyama et al., 1997). Briefly, report, composed of 10 g glucose, 5 g yeast extract cell-free extracts of genus Amphibacillus were pre- (Difco), 5 g Polypeptone (Nihon Seiyaku), 5 g Extract pared from aerobically grown cells. Equal amounts of Bonito (fish extract; Wako Pure Chemical), 1 g K2HPO4, protein (20 µg) were subjected to 12.5% polyacry- 0.2 g KCl, 0.1 g MgCl2, 50.4 g NaHCO3 and 63.6 g lamide gels and electrophoresed under denaturing Na2CO3 (per 1,000 ml). Glucose and NaHCO3-Na2CO3 conditions, then transferred onto polyvinylidene difluo- were autoclaved separately at 121°C, 15 min. After ride (PVDF) membranes. Immunoblottings were incu- autoclaving, each component of the medium was bated with a rabbit polyclonal anti-NADH oxidase anti- mixed (final pH 9.5). For aerobic and anaerobic culture body or anti-Prx antibody, followed by a horseradish of A. fermentum and A. tropicus, we used a modified peroxidase-conjugated anti-rabbit secondary antibody GYPFKMK, composed of 10 g glucose, 5 g yeast and visualized with Immunostaining HRP-1000 kit extract (Difco), 5 g Polypeptone (Nihon Seiyaku), 5 g (KONICA MINOLTA). Purified Prx (BAA33808) or NADH Extract Bonito (fish extract; Wako Pure Chemical), 1 g oxidase (BAA33809) from A. xylanus was used as a -1 K2HPO4, 0.2 g KCl, 5 ml salt solution [(ml ): 40 mg control. MgSO4･7H2O, 2 mg MnSO4･4H2O, 2 mg FeSO4･7H2O], 50.4 g NaHCO3 and 63.6 g Na2CO3 (per 1,000 ml). Results Glucose, salt solution, and NaHCO3-Na2CO3 were au- toclaved separately at 121°C, 15 min. Oxygen-free N2 Optimization of the culture media for growth of Amphi- gas was used for preparation of anaerobic media as bacillus fermentum and Amphibacillus tropicus previously described (Niimura et al., 1987). After auto- In anaerobic or static culture, A. fermentum DSM claving, each component of medium was mixed (final 13869T and A. tropicus DSM 13870T grew well in GYP- pH 9.5). The cells were cultivated at 37°C with aerobic FKMK medium in which NaCl and salt solution were shaking or under anaerobic conditions (Niimura et al., removed from GYPF medium. On the other hand, the 1987, 1990), harvested at late log phase, washed with two species did not show good growth in GYPFKMK 50 mM sodium phosphate buffer (pH 8.0), and then medium under aerobic conditions with shaking. Aero- stored at -80°C until use, or incubated with shaking at bic growth was rescued by supplementing with MnSO4 37°C for 1 h to prepare resting cells (Niimura et al., and FeSO4 to the medium (named modified GYPFKMK 1989). medium) as observed with A. xylanus (Niimura et al., Chemotaxonomic and biochemical characteristics. 1990). The cell yield of these strains in the modified Cellular fatty acid composition and quinone systems GYPFKMK medium was good enough to use as the were determined as previously described (Komagata starting biomass for further study on their physiology and Suzuki, 1987). Cytochrome systems were deter- and chemotaxonomy. mined as previously described (Niimura et al., 1987). The fermentation products were analyzed as previ- Chemotaxonomic characterization of Amphibacillus ously described (Ishikawa et al., 2002). fermentum and Amphibacillus tropicus Cell-free extract was prepared as previously de- Cellular fatty acid composition of A. fermentum DSM scribed (Nishiyama et al., 2001) without ultracentrifu- 13869T and A. tropicus DSM 13870T was analyzed by gation and dialysis process. NADH oxidase activity using the cells cultivated in GYPFKMK broth for static was measured as previously described (Niimura et al., culture. The constituents of the cellular fatty acids of A. 1989). Oxygen consumptions in the presence of AhpC fermentum DSM 13869T and A. tropicus DSM 13870T (Prx) were measured as previously described (Niimura were anteiso-, iso-branched and straight chain, as et al., 2000) with cell-free extracts instead of purified those of A. xylanus (Table 1). NADH oxidase. NADH dependent hydrogen peroxide Subsequently measurement of isoprenoid qui- reductase activities in the presence of Prx were mea- nones, cytochromes and catalase activity was carried sured as previously described (Niimura et al., 1995) in out by using the cells obtained by aerobic culture in anaerobic conditions with cell-free extracts instead of the modified GYPFKMK medium as previously done purified NADH oxidase.