INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1990, p. 297-301 Vol. 40, No. 3 0020-7713/9O/030297-05$02.OO/O Copyright 0 1990, International Union of Microbiological Societies

Amphibacillus xylanus gen. nov. , sp. nov. , a Facultatively Anaerobic Sporeforming Xylan-Digesting Bacterium Which Lacks Cytochrome, Quinone, and YOUICHI NIIMURA,l* ENKI KOH,l FUJITOSHI YANAGIDA,l KEN-ICHIRO SUZUKI,, KAZUO KOMAGATA,3 AND MICHIO KOZAKI' Tokyo University of Agriculture, Setagaya-ku, Tokyo 1.56,' Japan Collection of Microorganisms, RIKEN, Wako-shi, Saitama 351 -01 ,2 and Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku, Tokyo 113,3 Japan

Three strains of gram-positive, facultatively anaerobic, sporeforming, rod-shaped were isolated from composts of manure with grass and rice straw. These organisms grew well in an alkaline medium and digested xylan both in strictly anaerobic cultures when titanium(III) citrate was used as a reducing agent and in aerobic cultures with shaking. The cells contained meso-diaminopimelic acid, and their cellular fatty acids consisted of iso- and anteiso-branched acids and considerable amounts of straight-chain acids. The DNA base composition of these strains ranged from 36 to 38 mol% plus cytosine. Cytochromes, isoprenoid quinones, and catalase activity were not detected. DNA-DNA homology determinations did not show relatedness to strains of representative of the genera , , and . Considering the uniqueness of the characteristics, the sequence of the 5s rRNA, and the unique metabolic pathways, we propose xylanus gen. nov., sp. nov., for these strains. The type strain is strain EpOl (= JCM 7361).

Previously, Niimura et al. reported the isolation from an 10.0 with a 10% Na,CO, solution. Cultivation was carried alkaline compost of manure with grass and rice straw of a out at 393°C. For aerobic cultivation, a cotton-plugged facultatively anaerobic, sporeforming, xylan-digesting bac- culture flask containing medium (20% of the volume of the terium (strain EpOIT [T = type strain]) that lacks cy- flask) was shaken vigorously. For anaerobic cultivation, 0.13 tochromes, quinones, and catalase (10). This strain grew mM titanium(II1) citrate (19) was added to medium prepared well in xylan medium and digested xylan both under strictly anaerobically (10). The E, of the medium was decreased by anaerobic conditions and under aerobic conditions with adding titanium(II1) citrate, which produced values less than shaking. -300 mV (pH 10.0). Sporulation was determined by using In this work, we included two new isolates similar to strain cells cultivated under aerobic or anaerobic conditions by EpOIT in a taxonomic study. Our data revealed that the three phase-contrast microscopy and by heating culture broth at strains are members of an independent taxon that is different 80°C for 10 min. Utilization of sugars was determined by from members of the genera Bacillus, Clostridium, and measuring the turbidity and pH of the broth after 4 days of Sporolactobacillus with respect to phenotypic and chemo- incubation in glucose medium (see above) in which glucose taxonomic characteristics. Consequently, we propose a new was replaced with other carbohydrates and also in basal genus, Amphibacillus, and a new species, Amphibacillus medium (3). Turbidity was determined by using naked eye xylanus, for these organisms. observations, and pH was determined with a pH meter (Horiba Seisakusho, Tokyo, Japan), Nitrate reduction, H,S MATERIALS AND METHODS production, indole production, citrate utilization, and hy- drolysis of gelatin were determined as described by Holde- Strains and isolation. We examined three strains of a man et al. (3) and Komagata (6); the pH of the media for all facultatively anaerobic xylan-utilizing bacterium, strains of these tests was adjusted to 10.0 with a 10% Na,CO, EpOIT, SinIV, and 15abb. Strains SinIV and 15abb were solution. newly isolated from different composts of manure with grass and rice straw by using the same enrichment culture tech- nique that was used previously (10). TABLE 1. Effects of oxygen on the growth of A. xylanus strains Identification methods. Cell morphology, Gram reaction, in glucose medium motility, and flagella were determined by using cells grown CL-~ (h-9" Cell yield on glucose medium which contained (per liter) 10 g of Strain Anaerobic Aerobic Anaerobic Aerobic glucose, 1 g of K,HPO,, 2 g of NH,NO,, 200 mg of growth' growthd growth growth MgS04.7H,0, 5 mg of MnSO4.7H,O, 5 mg of ~ ~~ ~~ ~~ FeSO, - 7H,O, 100 mg of CaC1, . 2H20, 3 g of yeast extract EpOIT 0.82 0.70 0.65 0.72 (Difco Laboratories, Detroit, Mich.), 300 mg of Polypeptone SinIV 0.68 0.97 0.73 0.78 (Daigo Eiyo Chemical Co., Tokyo, Japan), and 1 mg of 15abb 0.56 0.26 0.58 0.43 resazurin. All of the media used for cultivation and biochem- " kmanwas measured at pH 9.5 (start). ical and physiological characterizations were adjusted to pH Cell yield was measured at pH 10.0 (start). Strictly anaerobic cultures in which titanium (111) citrate was used as a reducing agent. * Corresponding author. Aerobic cultures with shaking.

297 298 NIIMURA ET AL. INT.J. SYST.BACTERIOL.

TABLE 2. Products of xylan by A. xylanus strainsa Concn of the following products Strain Culture (mmol/liter): Formic acid EpOIT Anaerobicb 22 22 46 Aerobic" 35 SinIV Anaerobic 26 25 57 Aerobic 22 15abb Anaerobic 28 32 67 Aerobic 35

a The medium contained 1%xylan as a carbon source. StrictIy anaerobic culture in which titanium (111) citrate was used as a reducing agent. Aerobic culture with shaking.

Quinone systems were determined as described by Koma- gata and Suzuki (7). Amino acids in the cell wall peptidoglycan were analyzed with a model 835 automatic amino acid analyzer (Hitachi, Ltd., Tokyo, Japan) (7). The isomers of diaminopimelic acid were determined by thin-layer chromatography, using the modified method (7) of Staneck and Roberts (14). Cellular fatty acid composition was determined by gas FIG. 1. Phase-contrast photomicrograph of A. xylanus EpOIT chromatography as described by Suzuki and Komagata (15). cells grown on glucose agar at 39.5"C. Bar * = 5 pm. The arrow indicates an . DNA base composition and DNA-DNA hybridization. DNA base composition was determined by the thermal melting point method as described previously (10). The levels of The fermentation products from xylan were analyzed by DNA-DNA relatedness with representatives of the genera gas chromatography and high-performance liquid chroma- Bacillus, Clostridium, and Sporolactobacillus were deter- tography (10). The xylan was prepared from oat spelt (Ald- mined by using the sodium dodecyl sulfate membrane filter rich Chemical Co., Milwaukee, Wis.). method (4, 5). Strains of the following species were used in Cytochrome systems were determined by spectropho- the homology experiments: Bacillus alcalophilus, Bacillus tometry as described previously (10). cereus, Bacillus lentus, Bacillus megaterium, Bacillus sub-

FIG. 2. Electron micrograph of A. xylanus 15abb. The cell was negatively stained with phosphotungstic acid. Bar = 0.5 pm. VOL. 40, 1990 GEN. NOV., SP. NOV. 299

TABLE 3. Cellular fatty acid compositions of A. xylanus strains

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

c14:0 c16:0 c14:o c16:0 c15:o c17:0 c15:o c17:0 EpOIT Anaerobic" 11 27 8 17 10 1 19 5 Aerobicb 11 20 11 19 10 1 21 4 SinIV Anaerobic 12 24 7 11 10 1 28 5 Aerobic 8 14 6 7 12 1 38 4 15abb Anaerobic 10 36 6 15 8 1 15 5 Aerobic 5 21 9 22 10 1 22 6

a Strictly anaerobic culture in which titanium(II1) citrate was used as a reducing agent. Aerobic culture with shaking. tilis, Clostridiurn arninovalericum, Clostridiurn butyricum, These results suggest that our isolates differ from mem- Clostridium mangenotii, Clostridium thermocellurn, and bers of the genera Bacillus, Clostridium, and Sporolactoba- Sporolactobacillus inulinus (see Table 4 for strain designa- cillus (Table 5). Furthermore, these three isolates were not tions). identified as any previously described species by using the data in references 2 and 13. Thus, we propose a new genus RESULTS AND DISCUSSION and new species for these isolates, Amphibacillus xylanus. Description of Amphibacillus gen. nov. Amphibacillus Strains EpOIT, SinIV, and 15abb grew well under anaer- (Am. phi. ba. cil' lus. Gr. pref. amphi, both sides or double; obic and aerobic conditions (Table 1) and formed heat- L. dim. n. bacillus, a small rod; M. L. masc. n. Amphiba- resistant spores under both aerobic and anaerobic conditions cillus, rod capable of both aerobic and anaerobic growth). (Fig. 1). Spore formation under both conditions differenti- Cells are rods that are 0.3 to 0.5 km in diameter and 0.9 to ated these organisms from species of Bacillus, Clostridium, 1.9 pm long. Gram positive in the very early stages of growth and Sporolactobacillus (13). Strains SinIV and 15abb were and loosely gram positive in stationary growth phase. Heat- motile by means of peritrichous flagella (Fig. 2), and strain resistant, oval are formed, but the sporangia are EpOIT was nonmotile. These three strains produced ethanol, rapidly lysed and the spores are liberated. Spores are formed acetic acid, and formic acid from xylan under anaerobic under both aerobic and anaerobic conditions. Grows well conditions and acetic acid under aerobic conditions (Table both in well-aerated cultures and in aerobic cultures (Eh, 2). -370 mV; pH 10) when titanium(II1) citrate is used as a Catalase and oxidase were not produced, and the isolates reducing agent. Does not grow in nutrient broth. Chemoor- had neither cytochromes a, 6, c, or d nor any respiratory ganotrophic. Produces ethanol, acetic acid, and formic acid isoprenoid quinones, as reported previously (10). The oxy- under anaerobic conditions and acetic acid under aerobic gen consumption of the isolates did not occur through the conditions from glucose (9). Grows at pH values between 8.0 cytochrome system (lo), but NADH oxidase played a role in and 10.0 but not at pH 7.0. Contains mesa-diaminopimelic oxygen consumption (9), as it does in several lactic acid acid in the cell wall, and the peptidoglycan structure is the bacteria (12). This system explains the mechanism direct-linkage type which corresponds to type Aly of Schle- of growth of the isolates under both aerobic and anaerobic conditions (9). The major cellular fatty acids of our isolates were anteiso- and iso-branched and straight-chain acids (Table 3). This TABLE 4. Levels of DNA-DNA homology between A. xylanus composition is similar to that of members of the genera strains and some representative strains of sporeforming bacteria Bacillus and Sporolactobacillus (13), but our isolates also % Homology with contained considerable amounts of saturated straight-chain G+C labeled DNA from: acids. The cellular fatty acids of the genus Clostridium Strain content (mol%) A. &anus A. xylunus mainly consist of both saturated and unsaturated straight- EpOIT SinIV chain acids (1, 13). Therefore, the fatty acid pattern of our isolates differed from the patterns of species in the previ- A. xylanus strains ously described sporeforming genera. EpOIT 36 100 100 92 The DNA base compositions of our isolates ranged from SinIV 36 108 84 100 36 to 38 mol% guanine plus cytosine (G+C) (Table 4). These l5abb 38 78 90 isolates exhibited high levels of DNA-DNA relatedness to B. megaterium JCM 2506T 37.6" 9 B. cereus JCM 2152= 35.7" 12 one another, but not to the other bacteria studied (Table 4). B. lentus JCM 2511T 36.3" 12 When from B. alcalophilus JCM 5262T, B. megate- B. subtilis JCM 1465T 42.9" 11 rium JCM 2506T, and C. thermocellum ATCC 27405T were B. alcalophilus JCM 5262T 37.0" 9 12 used as probes, lower levels of relatedness were observed S. inulinus JCM 6014T 47b 4 4 (data not shown). C. aminovalericum ATCC 13725= 33" 4 4 As previously reported (17), the strain EpOIT 5s rRNA C. butyricum JCM 1391T 27-28' 18 was 116 nucleotides long, and there were 37 base differences C. mangenotii ATCC 25761T 31 13 compared with C. carnis 5s rRNA, 13 base differences C. thermocellum ATCC 27405T 38-39" 8 compared with B. subtilis 5s rRNA (8), and 25 base differ- a Data from reference 13. ences compared with S. inulinus 5s rRNA (16). Data from reference 18. 300 NIIMURA ET AL. INT.J. SYST.BACTERIOL.

TABLE 5. Differentiation of the genus Amphibacillus from the genera Bacillus, Clostridium, and Sporolactobacillus" Spore formation under: Genus Major fatty acids

Amphibacillus +b + + + - - - Straight-chain (saturated), iso- and anteiso-branched Bacillus f + - + + Iso- and anteiso-branched + -* - Clostridium + T + - + + T Straight-chain (saturated and unsaturated) Sporolactobacillus +c +c NT~ NT - - + Iso- and anteiso-branched Data from this study and from reference 13. +, Positive; -, negative; +, generally positive; +, generally negative. Microaerophilic . NT, Not tested.

ifer and Kandler (11). The major cellular fatty acids are ACKNOWLEDGMENTS anteiso- and iso-branched and straight-chain acids. Cy- We thank A. Hirata, Institute of Applied Microbiology, The tochromes, isoprenoid quinones, catalase, and oxidase are University of Tokyo, Tokyo, Japan, for taking electron micro- not detected. The DNA base composition ranges from 36 to graphs, Y. Benno, RIKEN, for the analysis of metabolic products, 38 mol% G+C. Probably widely distributed in composts of and K. Ohnishi for technical assistance. We also thank the late Y. manure with grass. Minoda, Y. Ikeda, and H. Oyaizu for their helpful suggestions. The type species is Amphibacillus xylanus. Description of Amphibacillus xylanus sp. nov. Amphibacil- LITERATURE CITED lus xylanus (xy. la' nus. M. L. adj. xylanus, xylan). The species has all of the characteristics that define the genus. In 1. Elsden, S. R., M. G. Hilton, K. R. Parsley, and R. Self. 1980. The lipid fatty acids of proteolytic clostridia. J. Gen. Microbiol. addition, it has the characteristics described below. Cells are 118:115-123. motile by means of flagella or nonmotile. In aerobic and 2. Gottshalk, G., J. R. Andressen, and H. Hippe. 1981. The genus anaerobic cultures, colonies on glucose agar are small, Clostridium (nonmedical aspects), p. 1767-1803. In M. P. Stan- circular, smooth, convex, entire, and white after 1 day of et al. (ed.), The prokaryotes, a handbook on habitats, isolation, incubation. Nitrate reduction, H,S production, indole pro- and identification of bacteria. Springer-Verlag, Berlin. duction, citrate utilization, and hydrolysis of gelatin are 3. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977. negative. Utilizes D-xylose, L-arabinose, D-ribose, D-glu- Anaerobe laboratory manual, 4th ed. Virginia Polytechnic In- cose, D-fI-UCtOSe, esculin, salicin, maltose, sucrose, cellobi- stitute and State University, Blacksburg. ose, trehalose, soluble starch, pectin, and xylan (oat spelt). 4. Ikemoto, S., K. Suzuki, T. Kaneko, and K. Komagata. 1980. Characterization of strains of Pseudomonas maltophilia which Grows at temperatures between 25 and 45°C but not at 50°C do not require methionine. Int. J. Syst. Bacteriol. 30:437447. and grows in the presence of 3% NaCl but not in the 5. Kaneko, T., R. Nozaki, and K. Aizawa. 1978. Deoxyribonucleic presence of 6% NaCl. The fermentation product from xylan acid relatedness between Bacillus anthracis, Bacillus cereus, in aerobic cultures is acetic acid, and the fermentation and Bacillus thuringiensis. Microbiol. Immunol. 22:639-641. products in anaerobic cultures are formic acid, ethanol, and 6. Komagata, K. 1984. Bacteria, part 1, p. 99-161. In T. Hasegawa acetic acid. Variable characteristics for the species are (ed.), Microbial classification and identification. Gakkai Shup- shown in Table 6. Cells of strain SinIV are weakly gram pan Center, Tokyo. (In Japanese.) positive. Strains have been isolated from composts of ma- 7. Komagata, K., and K. Suzuki. 1987. Lipid and cell-wall analysis nure with grass and rice straw. The type strain is strain in bacterial systematics, p. 161-207. In R. R. Colwell and R. EpOl. This strain is nonmotile, and its DNA base composi- Grigorova (ed.), Methods in microbiology, vol. 19. Academic Press, Inc. (London), Ltd., London. tion is 36 mol% G+C. It was isolated from an alkaline 8. Marotta, C. A., F. Varricchio, I. Smith, and S. M. Weissmann. compost of manure with grass and rice straw (10). 1976. The primary structure of Bacillus subtilis and Bacillus The type strain has been deposited in the Japan Collection stearothermophilus 5s ribosomal nucleic acids. J. Biol. Chem. of Microorganisms, RIKEN, Wako-shi, Saitama, Japan, as 251~3122-3127. strain JCM 7361. 9. Niimura, Y., E. Koh, T. Uchimura, N. Ohara, and M. Kozaki. 1989. Aerobic and anaerobic metabolism in a facultative anaer- obe EpOl lacking cytochrome, quinone and catalase. FEMS Microbiol. Lett. 61:79-84. 10. Niimura, Y., F. Yanagida, T. Uchimura, N. Ohara, K. Suzuki, TABLE 6. Characteristics for A. xylanus strains and M. Kozaki. 1987. A new facultative anaerobic xylan-using alkalophile lacking cytochrome, quinone, and catalase. Agric. Utilization of the following Biol. Chem. 51:2271-2275. compounds as carbon sources: 11. Schleifer, K. H., and 0. Kandler. 1972. Peptidoglycan types of Strain Gram Fla- G+C Xylan Xylan bacterial cell walls and their taxonomic implications. Bacteriol. reaction gella D-Man- E;!li Inulin from from Rev. 36:407-477. nose oat larch 12. Smart, J. B., and T. D. Thomas. 1987. Effect of oxygen on spelt wood lactose metabolism in lactic streptococci. Appl. Environ. Mi- EpOIT + -36++-++ crobiol. 53533-541. 13. Sneath, P. H. A. 1986. Endospore-forming gram-positive rods SinIV + (weak) + 36 - -++- and cocci, p. 1104-1207. In P. H. A. Sneath, N. S. Mair, M. E. 15abb + + 38 - -- +- Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic VOL. 40, 1990 AMPHIBACILLUS XYLANUS GEN. NOV., SP. NOV. 301

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