Taxonomical and Physiological Comparisons of the Three Species of the Genus Amphibacillus

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 xylanus, the type species of the genus, grows well under both strictly anaerobic and aerobic conditions 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 isolates 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. fermentum 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 observed 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 observed 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 metabolism 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 phylogenetic 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 pathways (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 autoclaved 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. Catalase activity of cell-free for A. xylanus (Niimura et al., 1987, 1990). Neither iso- extract was measured spectroscopically at 240 nm prenoid quinones nor cytochromes were detected in (Aebi, 1983) at 37°C. Immunoblottings were done as the cells of A. fermentum DSM 13869T or A. trop- 158 ARAI et al. Vol. 55

Table 1. Cellular fatty acid composition of Amphibacillus species.

Fatty acid composition (% of total) Strain Straight-chain acids Iso-branched acids Anteiso-branched acids

A. xylanusa 37.2 35.4 23.7 (C14:0, C16:0) (C14:0, C15:0, C16:0, C17:0) (C15:0, C17:0) A. fermentumb 38.91 23.27 37.21 (C10:0, C12:0, C13:0, C14:0, C16:0, C18:0) (C11:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0) (C11:0, C13:0, C15:0, C17:0) A. tropicusb 35.73 30.58 32.21 (C12:0, C14:0, C16:0, C18:0) (C12:0, C13:0, C14:0, C15:0, C16:0, C17:0) (C13:0, C15:0, C17:0)

aAnaerobic culture; bstatic culture. icus DSM 13870T. In the typical catalase test, although the cells of A. fermentum and A. tropicus cultured at high concentra- tion of Na2CO3 sometimes generated small bubbles in 3% H2O2 after 2‒3 min following mixing, the observed bubbles were clearly distinguished from the bubbles generated by the catalase reaction, because in the au- thentic catalase reaction, catalase-positive control strains like Bacillus subtilis, rapidly produce a lot of bubbles just on mixing with H2O2 solution. Further- more, similar foaming by A. fermentum DSM 13869T Fig. 2. Catalase activity of cell-free extracts from Amphiba- and A. tropicus DSM 13870T was observed on modi- cillus xylanus Ep01T, Amphibacillus fermentum DSM 13869T, fied GYPFKMK agar plate without living bacterial cells. Amphibacillus tropicus DSM 13870T and Bacillus subtilis. These results suggest that the observed foaming was The reaction mixture (1 ml) contained 50 mM sodium phos- not due to the catalase reaction but probably due to phate buffer, pH 7.0, including 100 µg or 30 µg of cell-free ex- sodium carbonate contained in the medium. To con- tracts from Amphibacillus species or B. subtilis, respectively. firm the result, we prepared cell-free extracts of the The reactions were started at 37°C by addition of hydrogen per- both strains by French pressure and their catalase oxide (final 10 mM), as indicated by arrow. Catalase activity was activities were spectroscopically measured in terms of measured as the decrease in absorbance at 240 nm. decrease of H2O2. The spectroscopic analysis of catalase in the cell-free extracts showed that A. and ethanol equaled that of formate in the two species fermentum and A. tropicus clearly lacked catalase (Table 2). Accordingly, it was suggested that these activity, as did A. xylanus (Fig. 2). strains have two major metabolic systems as observed with A. xylanus. Different from A. xylanus, however, A. Analyses of the metabolites fermentum DSM 13869T and A. tropicus DSM 13870T The major metabolite under aerobic conditions of A. were suggested to have a side enzymatic pathway xylanus was acetate, and those under anaerobic con- producing lactate as it was detected as a metabolite of ditions were formate, acetate and ethanol. Character- both strains under both aerobic and anaerobic condi- istically, in the anaerobic pyruvate metabolism the total tions. molar amount of acetate and ethanol equals that of formate, because of the participation of pyruvate for- The enzymatic systems for oxygen metabolism and de- mate-lyase (Niimura et al., 1989). While in the cells of toxiﬁ cation of peroxides A. fermentum DSM 13869T and A. tropicus DSM We prepared resting cells from aerobic cultures of A. 13870T, glucose was aerobically metabolized to pro- fermentum DSM 13869T and A. tropicus DSM 13870T duce acetate as the main organic acid, under anaero- in the modified GYPFKMK medium. Strong oxygen bic conditions the major metabolites were formate, consumption with added glucose was resumed in the acetate and ethanol. The total molar amount of acetate resting cells of both strains regardless of the addition 2009 Physiology of the genus Amphibacillus 159

Table 2. Metabolites from glucose in aerobic and anaerobic cultivations with Amphibacillus species.

Products (mM) A. xylanusa A. fermentum A. tropicus

Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic Lactate N.D. N.D. 7.9 15.7 7.3 5.4 Pyruvate 0.7 0.9 22.1 N.D. 28.3 N.D. Acetate 25.4 14.0 78.7 38.8 71.3 45.1 Ethanol N.D. 16.3 N.D. 57.0 N.D. 53.0 Formate N.D. 34.0 8.6 96.9 7.1 104.3 Glucose consumed 14.7 17.0 47.8 54.4 48.9 57.6 Carbon recovery (%) 59.9 95.4 89.3 102.8 87.4 91.6

aNiimura et al. (1989); N.D., not detected.

Fig. 3. Oxygen consumption during the catalysis of cell-free extracts from Amphibacillus xylanus Ep01T (A), Amphibacillus fermentum DSM 13869T (B) and Amphibacillus tropicus DSM 13870T (C) in the absence of and presence of Prx from A. xylanus Ep01T. The reaction mixture (2.4 ml) contained 50 mM sodium phosphate buffer, pH 7.0, including 0.5 mM EDTA, 300 mM ammonium sulfate and 250 µM NADH, with or without 30 µM Prx. The reactions were started at 37°C by addition of 982 µg, 289 µg, or 277 µg of cell-free extracts from A. xylanus, A. fermentum or A. tropicus, respectively, as indicated by arrow a. During oxygen consumption, 20 µg of catalase was added, as indicated by arrow b. Straight line, in the presence of Prx (+Prx); dotted line, in the absence of Prx (-Prx).

of respiratory inhibitors such as NaN3 or KCN (data not consumption was made with glucose, pyruvate, or lac- shown). It was suggested that the observed oxygen tate. The addition of catalase to the reaction mixture consumption was not derived from respiratory sys- containing the cell-free extracts from A. fermentum tems, but due to other enzyme system such as that of DSM 13869T and A. tropicus DSM 13870T resulted in NADH oxidase as previously shown in A. xylanus (Ni- the regeneration of oxygen, of which the amount was imura et al., 1989). Significant oxygen consumption by half of the theoretically calculated amount that should cell-free extracts prepared from the aerobically cul- be consumed in the absence of catalase (Fig. 3). This T tured cells of A. fermentum DSM 13869 and A. tropi- suggested that the H2O2-forming NADH oxidase pri- cus DSM 13870T was observed after the addition of marily catalyzed the oxygen consumption in these re- NADH, while consumption by addition of NADPH was action mixtures. significantly lower (5%) than that of NADH, and no 160 ARAI et al. Vol. 55

We have found that addition of catalase did not pro- Discussion duce oxygen in the reaction mixture of A. xylanus in the presence of a 21-kDa disulfide-containing redox pro- The genus Amphibacillus is classified in the family tein (AhpC), now commonly referred to as peroxire- Bacillaceae, a large family for aerobic gram-positive doxin (Prx), indicating that A. xylanus NADH oxidase low G+C bacteria (Brenner et al., 2005). The genus scavenged hydrogen peroxide with Prx (Niimura et al., Amphibacillus constitutes an independent taxon of de- 2000). The production of oxygen was observed in the scent within the group composed of halophilic/halo- reaction mixture of A. fermentum DSM 13869T, but in tolerant/alkaliphilic and/or alkalitolerant members in contrast, the production of oxygen was not observed rRNA group 1 of the genus Bacillus and related bacte- in A. tropicus DSM 13870T as observed with A. xylanus ria based on the 16S rRNA gene sequences (Ash et (Fig. 3, see also DISCUSSION). The actual NADH per- al., 1991). The common features of the genus are: oxidase activity, i.e., NADH-depending reduction of spore-forming, facultatively anaerobic, isoprenoid qui- hydrogen peroxide with Prx, was detected spectro- none-lacking, cytochrome-negative, catalase-nega- scopically (at 340 nm) in the cell-free extracts of A. tive, and the anteiso-, iso-branched and straight chain tropicus with Prx, but in contrast the activity was not type of the cellular fatty acids. detected in that of A. fermentum DSM 13869T (not Amphibacillus fermentum and Amphibacillus tropi- shown). Furthermore, immunoblot analysis revealed cus were later proposed by Zhilina et al. (2001) for the that cell-free extracts from A. fermentum DSM 13869T isolates from a soda lake. However, the above-men- and A. tropicus DSM 13870T reacted with antibodies tioned phenotypic properties ―presence of isoprenoid raised against A. xylanus Prx and NADH oxidase (Fig. quinones, cytochrome, catalase and the constituents 4). These analytical results strongly support the pros- of the cellular fatty acids in the two species―are not pect based on the oxygen electrode experiments with clearly presented in previous study (Zhilina et al., Prx in the two species (see DISCUSSION). 2001). In this paper, we revealed that the two species also lack isoprenoid quinones, cytochrome and catalase and the constituents of the cellular fatty acids are of anteiso-, iso-branched and straight chain. Thus, those phenotypic properties clearly confirmed that Amphiba- cillus fermentum and Amphibacillus tropicus belong to the genus Amphibacillus (Table 3). Due to the presence of anaerobic and aerobic pathways producing a similar amount of ATP (Niimura et al., 1989, 2000), A. xylanus, grows well with the same growth rate and cell yield under strictly anaerobic and aerobic conditions (Niimura et al., 1990). A. fermentum and A. tropicus also grow well under both conditions, and their metabolite analysis indicated that although the two species have a side enzymatic pyruvate pathway to produce lactate under both conditions, they have the two major pyruvate metabolic pathways observed in A. xylanus (Fig. 5). In the anaerobic glycolytic pathway of Amphibacil- lus, NADH formed from NAD+ is re-oxidized by NAD- T Fig. 4. Immunoblotting of Amphibacillus xylanus Ep01 , linked aldehyde dehydrogenase and NAD-linked alco- T Amphibacillus fermentum DSM 13869 and Amphibacillus tropi- hol dehydrogenase. NADH formed both by the aerobic cus DSM 13870T. pyruvate pathway and by the glycolytic pathway Twenty micrograms proteins of the cell-free extracts from Am- phibacillus species were separated on 12.5% SDS-PAGE and should be re-oxidized by the NADH oxidase reaction, + immunoblotted using polyclonal anti-NADH oxidase antibody which forms NAD to provide an oxidation/reduction (A) or anti-Prx antibody (B) from A. xylanus. balance in the aerobic pathway in Amphibacillus lack- 2009 Physiology of the genus Amphibacillus 161

Table 3. Characteristics that distinguish the Amphibacillus species from other members of the HA group in Bacillus rRNA group 1. g f e b g d d c b

Characteristic a Amphibacillus xylanus Amphibacillus fermentum Amphibacillus tropicus Amphibacillus sediminis Halolactibacillus halophilus Halolactibacillus miurensis Halolactibacillus alkaliphilus Paraliobacillus ryukyuensis Gracilibacillus dipsosauri Gracillibacillus halotolerans

Spore formation ++++---+++ Anaerobic growth ++++++w+-- Aerobic growth ++++++++++ Catalase ------+++ Cytochromes ------N.T. + + + Quinones ------+++ Products in anaerobic growth Lactate - ++N.T.++++-- Acetate + + + N.T. + + N.T. + -- Formate + + + N.T. + + N.T. + -- Ethanol + + + N.T. + + N.T. + -- Pyruvate + --N.T. --N.T. ---

aNiimura et al. (1990); bZhilina et al. (2001); cAn et al. (2007); dIshikawa et al. (2005); eCao et al. (2008); fIshikawa et al. (2002); gWainø et al. (1999); +, positive; -, negative; N.T., not tested; w, weak.

The scavenging reaction of hydrogen peroxide is important in the strains, because hydrogen peroxide was formed during the reaction of those NADH oxi- dases. In the presence of Prx, A. xylanus NADH oxi-

dase showed extremely high turnover and low Km values for peroxides (named the NADH oxdase-Prx system, Niimura et al., 1995, 1996, 2000). The high

turnover numbers and low Km values of the enzyme system are unusual for known peroxide-reducing en- zymes (Niimura and Massey, 1996). Thus, the A. xylanus NADH oxidase-Prx system plays an important role in removing peroxides in the bacterium. Additional enzyme assays and immunoblot analysis revealed that the NADH oxidase-Prx system also functions in A. tropicus as well as in A. xylanus. Although understand- ing of the detailed metabolism for removing peroxide in A. fermentum needs further investigation, three species belong to the genus Amphibacillus, capable of Fig. 5. Predicted metabolic pathway of Amphibacillus species. growing well under strictly anaerobic and aerobic con- a, in Amphibacillus fermentum DSM 13869Tand Amphibacil- ditions due to the presence of its common anaerobic lus tropicus DSM 13870T; b, NADH oxidase-AhpC (Prx) system and aerobic pathway, comprising a unique oxygen in Amphibacillus xylanus Ep01T and Amphibacillus tropicus metabolic system, NADH oxidase. DSM 13870T. An et al., recently isolated A. sediminis from sediment from Lake Hamana (An et al., 2007). The species ing respiratory chain. Actually, enzyme analysis indi- of A. sediminis share phylogenetic analysis data based cated the two species have the NADH oxidase as ob- on 16S rRNA gene sequence analysis (Fig. 1), and served in A. xylanus. taxonomic characteristics with the three species of 162 ARAI et al. Vol. 55

Amphibacillus and differentiated from other the spe- Niimura, Y. and Massey, V. (1996) Reaction mechanism of Am- cies in the HA group (An et al., 2007, Table 3). Accord- phibacillus xylanus NADH oxidase/alkyl hydroperoxide re- ingly, the genus Amphibacillus, including A. sediminis, ductase flavoprotein. J. Biol. Chem., 271, 30459‒30464. Niimura, Y., Nishiyama, Y., Saito, D., Tsuji, H., Hidaka, M., Miyaji, consists of 4 species now. T., Watanabe, T., and Massey, V. (2000) A hydrogen perox- In conclusion, not only the taxonomic characteris- ide-forming NADH oxidase that functions as an alkyl hy- tics of the genus Amphibacillus but also the growth droperoxide reductase in Amphibacillus xylanus. J. Bacte- characteristics based on the two metabolic pathways riol., 182, 5046‒5051. and unique oxygen metabolism are distinctive in those Niimura, Y., Poole, L. B., and Massey, V. 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