Anoxybacillus Pushchinensis Gen. Nov., Sp. Nov., A

International Journal of Systematic and Evolutionary Microbiology (2000), 50, 2109–2117 Printed in Great Britain

Anoxybacillus pushchinensis gen. nov., sp. nov., a novel anaerobic, alkaliphilic, moderately thermophilic bacterium from manure, and description of Anoxybacillus flavithermus comb. nov.

Elena Pikuta,2† Anatolyi Lysenko,1 Natalya Chuvilskaya,2 Ulrike Mendrock,3 Hans Hippe,3 Natalya Suzina,2 Dmitriy Nikitin,2 Georgiy Osipov4 and Kestas Laurinavichius2

Author for correspondence: Elena Pikuta. Tel: j1 256 544 7619. Fax: j1 256 544 5956. e-mail: elenapikuta!hotmail.com

1 Institute of Microbiology, A new strictly anaerobic, alkaliphilic, moderately thermophilic, fermentative, Russian Academy of spore-forming bacterium, strain K1T, was isolated from manure samples (pH Sciences, Pr. 60-letya Oktyabrya, Moscow 68). Cells were Gram-positive, straight, non-motile rods that grew at 117811, Russia temperatures of 37–66 SC (optimum at 62 SC) and in a pH range of 80–105 2 Institute of Biochemistry (optimum at 95–97). The bacterium fermented D-glucose, sucrose, D-fructose, and Physiology of D-trehalose and starch as carbon and energy sources. It required vitamins and Microorganisms, Russian its growth is stimulated by yeast extract. The major metabolic products were Academy of Science, pr. Nauki 5, Pushchino 142292, H2 and acetate. Cells were catalase-negative and could reduce nitrate to nitrite. Russia The GMC content of the DNA was 422 mol%. Based on the phenotypic 3 DSMZ, Mascheroder Weg properties and 16S rDNA sequencing and DNA–DNA hybridization data, strain 1b, D-38124 Braunschweig, K1T (l DSM 12423T l ATCC 700785T l VKM B-2193T) was assigned to the new Germany genus Anoxybacillus gen. nov., as a representative of a new species, 4 Academician Yu. Isakov Anoxybacillus pushchinensis sp. nov. ‘Bacillus flavothermus’ strain d.y., which Scientiﬁc Group, Russian was found to be closely related to strain K1T, is described as Anoxybacillus Academy of Medical T T Sciences, Moscow, Russia flavithermus comb. nov. (type strain l d.y. l DSM 2641 ).

Keywords: anaerobic alkaliphilic thermophilic bacterium, Anoxybacillus pushchinensis gen. nov., sp. nov., Anoxybacillus ﬂavithermus comb. nov.

INTRODUCTION philic alkaliphilic bacteria were found in soda lakes (Grant & Tindall, 1986; Krulwich & Guffanti, 1989; Alkaliphilic bacteria can be subdivided into two Zavarzin, 1993). More recently, thermophilic, alkali- groups which include obligately alkaliphilic species philic, anaerobic bacteria have been isolated from that are unable to grow at neutral pH, and alkali- sewage (Li et al., 1993, 1994) and thermal springs tolerant bacteria that are able to grow at high pH (Engle et al., 1995). These investigations expanded our values but have their growth optimum at or near a knowledge about the biodiversity and ecology of neutral pH (Grant & Tindall, 1986). Alkalitolerant alkaliphilic bacteria. For some of these bacteria, bacteria of the species Streptococcus faecalis were first however, the optimal growth conditions differ re- described in 1928 (Downie & Cruickshank, 1928). markably from the conditions in their habitats and the Obligately alkaliphilic bacteria of the genera Bacillus role of such micro-organisms in these ecosystems is not and Clostridium have been isolated from soils with well understood yet. In this work, Anoxybacillus neutral pH (Horikoshi & Akiba, 1982). Later, meso- pushchinensis gen. nov., sp. nov. is described, a new

...... species of the strictly anaerobic, moderately thermo- † Present address: UAH Chemistry Department, Astrobiology Group, MSB philic, obligately alkaliphilic bacilli isolated from 203C, Huntsville, AL 35899, USA. manure with neutral pH. Since the new species was The GenBank accession number for the 16S rDNA sequence of Anoxy- found to be closely related genetically to ‘Bacillus bacillus pushchinensis strain K1T is AJ010478. ﬂavothermus’ (Heinen et al., 1982), this hitherto

01362 # 2000 IUMS 2109 E. Pikuta and others invalidly named species is described as Anoxybacillus with 1% OsO% in 0n05 M cacodylate buffer (pH 7n2) for 4 h flavithermus comb. nov. (name corrected). at 20 mC. After dehydration in a series of ethanol solutions, cells were embedded in Epon 812 and sectioned on a microtome, LKB 2128 Ultratome. Ultrathin sections were METHODS placed onto copper grids and negatively stained with 3% Strains, media and culture conditions. Strain K1T was uranyl acetate in ethanol with lead citrate (Reynolds, 1963). isolated from enrichment cultures that had been inoculated Thin sections were examined under a JEOL JEM-100B with mixed manure samples and grown under anaerobic electron microscope. conditions in Hungate tubes at 60 mC for 4 d. Samples of Analytical, physiological and biochemical methods. The equine and porcine manure (1:1, w\w) with a pH of 6n0–7n0 bacterial cell number was determined by direct counting were collected from farms in the Moscow region and stored under a light microscope. The optical density (OD) of cell at 4 mC before inoculation. suspensions was measured in 1 cm cuvettes at 600 nm using For the enrichment, isolation and cultivation of strain K1T, a model 11 Specol spectrophotometer. Catalase activity was a previously described anaerobic basal medium (Zhilina et assessed as described elsewhere (Gerhardt et al., 1984) with al., 1997) was used with certain modifications. The medium 3% H#O# added to the culture liquid. Volatile products " contained (g lV ): KH#PO%,0n2; MgCl#;6H#O, 0n1; KCl, of glucose fermentation were analysed with the use of a 0n2; NH%Cl, 1n0; Na#CO$,2n76; NaHCO$,10n0; NaCl, 5n0; Pye Unicam 304 GC with FID and a 0n9mi3n3mm Na#S;9H#O, 0n5. The pH of the medium after autoclaving Chromosorb 101 column kept at 160 mC; CO# served as the was 9 5–9 7. After sterilization at 120 C for 30 min, yeast carrier gas. NO# was determined by the colorimetric method n n " m " extract (0n01glV ) and glucose as carbon source (5n0glV ) (Tiedje, 1982). were added. Medium (1 l) was supplemented with 10 ml of Cellular fatty acids. Fatty acids and other lipid components a stock vitamin solution (Wolin et al., 1963) and 1 ml were extracted from cell biomass by acid methanolysis. Wet of a trace element solution containing (mg per 200 ml biomass (30 mg) was dried in a stream of nitrogen and water): MnCl#;4H#O, 720; Fe(NH%)(SO%)#;12H#O, 400; 400 ml of a 1 M solution anhydrous HCl in methanol was FeSO%;7H#O, 200; CoCl#;6H#O, 200; ZnSO%;7H#O, 200; added. The mixture was heated at 80 mC for 3 h. The methyl Na#MoO%;2H#O, 20; NiCl#, 100; CuSO%;5H#O, 20; esters of fatty acids and dimethylacetates (aldehyde deriva- AlK(SO%)#;12H#O, 20; H$BO$, 20; and 5 ml concentrated tives) were obtained as a result, and were extracted twice HCl. with 200 ml hexane. The extract was evaporated to dryness Unless otherwise indicated, enrichment and pure cultures and silylated in 20 ml N,O-bis(trimethylsilyl)trifluoro- were grown at 55–62 mC in 15 ml Hungate tubes containing acetamide for 15 min at 80 mC. A 1 ml portion of the reaction 10 ml medium and filled with N#. All sampling and mixture was analysed with a model QP-2000 GC-MS system dispensing procedures were performed with sterile syringes (Shimadzu) equipped with a fused silica capillary column and needles. Prior to roll-tube cultivation on 3% Difco agar (25 mi0n25 mm) containing an Ultra-1 non-polar methyl- (w\v), aliquots of separately sterilized carbonate solutions silicone phase. The temperature profile included a 2 min isotherm at 120 mC and subsequent temperature program- were added, in addition to yeast extract and glucose, to V" Hungate tubes with 4 ml sterile basal medium. ming at a rate of 5 mC min to 280 mC. Data processing was carried out with standard programs of the GC-MS system. In the series of experiments designed to elucidate the optimal growth conditions, the pH of the medium was adjusted to Resistance to antibiotics. Resistance to antibiotics was required values with sterile stock solutions of HCl or NaOH evaluated by growing the bacterium in basal medium supplemented with filter-sterilized bacitracin, chloram- at 55 C under an N flow. pH measurements were done " m # phenicol (100 µgmlV ), penicillin, ampicillin, streptomycin using a model 121 pH meter (Russia) calibrated at 55 C. " m or vancomycin (all at 250 µgmlV ). After the inoculation of Cells were grown at 55 mC. To determine the range of growth temperatures, the isolate was cultivated in the basal medium antibiotic-containing media with exponentially growing cells, the cultures were incubated at 37 C for 12 h and then (pH 9n7). m at 55 mC for 14 d. The effect of NaCl on the bacterial growth was studied in GjC content determination and DNA hybridization. Cells basal medium containing 0n0, 0n5, 1n0, 2n0, 3n0, 5n0or7n0% were disrupted with a French pressure cell and DNA was (w\v) NaCl. The medium was modified to exclude other isolated by chromatography on hydroxyapatite by the sodium sources by replacing Na#CO$ and NaHCO$ with V" procedure of Cashion et al. (1977). The GjC content of the 5gl K#CO$, and Na#S with K#S. The NH%Cl-dependence DNA was determined by HPLC as described by Tamaoka & of the strain was estimated in medium containing 0, 0n5, 1n0 Komagata (1984) and Mesbah et al. (1989). Non-methylated or 3 0% (w\v) NH Cl. n % lambda phage DNA (Sigma) with a GjC content of Carbon sources other than glucose were added to the 49n858 mol% served as a standard. DNA–DNA hybrid- medium in the form of autoclaved or filter-sterilized " ization was performed as described by De Ley et al. (1970) solutions to a final concentration of 5 g lV . Sterile stock with modifications described by Huß et al. (1983) and solutions of electron acceptors were added to the medium " Escara & Hutton (1980), using a Gilford System 2600 with glucose (5 g lV ) to the following final concentrations: spectrophotometer equipped with a Gilford model 2527-R Na#SO%,20mM;Na#SO$,5mM;Na#S#O$.5H#O, 10 mM; thermoprogrammer and plotter. The renaturation rates were NaNO$, 10 mM; benzenesulfonate, 5 mM; fumarate, computed using the . program (Jahnke, 1992). ! V" 10 mM; S ,2gl . Phylogenetic analysis. Chromosomal DNA of strain K1T Light and electron microscopy. The morphology of the was isolated and purified as described elsewhere (Marmur, isolate was examined under an MBI-3 LOMO (Russia) 1961) and 16S rDNA was then amplified by Thermus microscope with a phase-contrast device. For electron aquaticus thermostable DNA polymerase (USB) via PCR in microscopy studies, harvested cells were pre-fixed with 1n5% a mixture containing 1i TaqPol buffer (USB), 0n2mg (v\v) glutaraldehyde in 0n05 M cacodylate buffer (pH 7n2) chromosomal DNA, 20 pM oligonucleotide primers pA and for 1 h at 5 mC, washed three times with the buffer, and fixed pHh (Edwards et al., 1989), 2n5 mM dNTP, 2n5 mM MgCl#

2110 International Journal of Systematic and Evolutionary Microbiology 50 Anoxybacillus pushchinensis gen. nov., sp. nov. and 2 U Taqr. After denaturation at 94 mC for 5 min, the mV) in medium with glucose as the carbon and energy reaction mixture was subjected to 30 thermal cycles (52 mC, source; growth was stimulated by vitamins and yeast 1 min; 72 mC, 1n5 min; 94 mC, 1 min). PCR products were extract. The bacterium was catalase-negative and able sequenced using the FemtoMol kit (Promega). 16S rDNA to reduce nitrate to nitrite. It did not hydrolyse gelatin was sequenced in both strands by Sanger’s method using or casein. next primers: pA, pC, pE, pDh,pFh and pHh (Edwards et al., 1989). All procedures were done as directed in the manu- Growth was observed in a pH range of 8n0–10n5 (Fig. facturer’s protocol. The 16S rDNA sequence of strain K1T 2a), with an optimum at 9n5–9n7. At pH 9n7, cells began was aligned against those of closely related strains using the to multiply after a very short lag phase. Strain K1T  program (version 1.60). Pairwise evolutionary could not grow in media with pH values of 7n5or11n0. distances were computed by the correction of Jukes and The bacterium obligately required carbonate for Cantor (Felsenstein, 1989) using the  program from growth; it could not grow in carbonate-free glycine- or the  software package (version 3.5). A 16S rDNA V" sequence of 1340 nt was registered in GenBank under the serine-buffered media (pH 9n5) with glucose (5 g l ). Strain K1T grew well, however, in carbonate-con- accession number AJ010478. " taining medium with glycine and serine (5 g lV ); RESULTS therefore, these amino acids had no inhibitory effect. Hence, strain K1T is an alkaliphile that is obligately Enrichment and pure cultures dependent on carbonate. The concentrations of NaCl optimal for growth ranged from 0 5to1%(w\v). The A population of anaerobic, alkaliphilic, moderately n strain was tolerant to 3% (w\v) NaCl but could not thermophilic bacteria developed in glucose-containing grow at 5% (w\v) NaCl. Strain K1T was not obligately medium (pH 10 0) that had been inoculated with n Na+-dependent: it grew in medium with K+ salts mixed manure samples, which had a neutral pH (6 8), n substituted in equimolar amounts for Na+ salts (three and incubated under anaerobic conditions at 60 C for m successive inoculations). Since strain K1T was isolated 4d. from manure, it was important to study its requirement + From this enrichment culture (OD of 0n4), serial for NH%. The strain showed good growth at NH%Cl dilutions were prepared and used for inoculation of concentrations of 0n1–0n5%(w\v), with an optimum at the same medium supplemented with penicillin " " 0n1% (w\v) NH%Cl, but not at 1% (w\v) NH%Cl. The (500 µgmlV ) and streptomycin (500 µgmlV ). The temperature range for growth was 37–66 mC, with an T highest dilution exhibiting growth of anaerobic alkali- optimum at 62 mC (Fig. 2b). Therefore, strain K1 is a philic bacteria was pure as judged from the absence of moderate thermophile. growth at pH 7n0 in glucose-peptone medium with In addition to -glucose, strain K1T was capable of large amounts of yeast extract. This culture was fermenting sucrose, -fructose, -trehalose and starch " anaerobically grown in roll-tubes with agar medium, (5 g lV ). The major metabolic products were H and and one of the colonies formed (referred to as strain # T acetate. No growth was observed with formate, acet- K1 ) was selected for further studies. ate, propionate, pyruvate, lactate, butyrate, methanol, ethanol, glycerol, yeast extract, peptone, methylamine, Morphology casein, -cysteine, -valine, sorbitol, dulcitol, man- Strain K1T was a Gram-positive, non-motile, straight, nitol, inositol, -sorbose, -melezitose, -rhamnose, spore-forming rod with rounded ends, measuring -arabinose, -galactose, -melibiose, -raffinose, - 0n4–0n5i2n5–3n0 µm. It formed terminal and sub- ribose, -lactose or -xylose. terminal spherical endospores. Interestingly, spores Strain K1T was able to reduce NaNO to NaNO , but $ # ! were found only after storage at V40 mC and 12 h it could not use Na#SO%,Na#SO$,Na#S#O$;5H#O, S post-incubation at 22 mC. Cells occurred singly, in or benzenesulfonate as electron acceptors; no for- pairs or in short or, sometimes, long chains (Fig. 1a). mation of H#S was observed during growth in media Cells reproduced by binary fission; Y-shaped cells containing sulfur compounds. sometimes occurred (Fig. 1b, f). The cell wall, visible The bacterium studied was sensitive to bacitracin (at " as a narrow electron-dense layer, was of the Gram- 100 µglV ), but resistant to penicillin, vancomycin, " positive type. The outer part of the cell envelope ampicillin and streptomycin (all at 250 µgmlV ), and " consisted of two structurally ordered S-layers covered chloramphenicol (100 µgmlV ). with a thick peptidoglycan layer (Fig. 1d). Cellular fatty acids Cultural, physiological and biochemical properties The cellular fatty acid profile of strain K1T (Table 1) After 2–3 d cultivation on agar medium at 55 mC, the T differed (at a correlation level of k " 0n5) from that of colonies of strain K1 were white, matt, dentate, rough 435 strains of various taxonomic groups, including 80 and from 0n5to2n0 mm in diameter. Surface colonies species of Bacillus, available in the database (obtained were round and granular, with a dense centre. Sub- from one of the authors, G. Osipov, private collection). surface colonies were round, white, with a dense This implies that the studied bacterium cannot be yellowish centre and uneven edges. consigned to any of the known species of the genus T Strain K1 grew strictly anaerobically (Eh ! V100 Bacillus.

International Journal of Systematic and Evolutionary Microbiology 50 2111 E. Pikuta and others

(a) (b)

(e) (f)

...... Fig. 1. Electron micrographs of cells of strain K1T. (a) Light micrograph (bar, 16n0 µm); (b) light micrograph of a branching form (bar, 3 µm); (c) ultrathin section (bar, 1n0 µm); (d) ultrathin section of the Gram-positive cell wall (Sout, outer S-layer, Sin, inner S-layer, PG, peptidoglycan layer; bar, 0n2 µm); (e) ultrathin section of a sporulating cell (Ps is a forespore; bar, 0n5 µm); (f) ultrathin section of a branching form (bar, 0n5 µm).

2112 International Journal of Systematic and Evolutionary Microbiology 50 Anoxybacillus pushchinensis gen. nov., sp. nov.

0·40 pasteurii VKM B 513 (GjC content of 38n5mol%) (a) (b) and Thermoanaerobacter thermohydrosulfuricus DSM T 0·35 567 (GjC content of 37 mol%) were 21, 20 and 4%, respectively. 0·30

0·25 Phylogenetic analysis )

–1 T 0·20 A length of 1340 bp of 16S rDNA of strain K1 was (h

l sequenced on both strands. The sequence was com- 0·15 pared with the 16S rDNA sequences of some representatives of the Bacillus group by using the Phylo- 0·10 genetic Inference Package (version 3.5) (Felsenstein, 0·05 1989). Phylogenetic analysis revealed a clusterization with ‘B. 0·00 20 30 40 50 60 70 8 9 10 11 flavothermus’ (98n89% sequence similarity). These Temperature (°C) pH bacteria differed by 7–16% from other species of the genus Bacillus and they can, therefore, be distinguished ...... T Fig. 2. Dependence of the growth of strain K1T on temperature as a separate genus. Strain K1 and ‘B. flavothermus’ (a) and pH (b). form an individual branch among the other bacillus rDNA groups. Table 2 shows the position of strain K1T in the phylogenetic cluster of related bacteria. Table 1. Fatty acids of Anoxybacillus pushchinensis K1T ...... DISCUSSION " The medium contained (g lV ): KH#PO%,0n2; MgCl#;6H#O, 0n1; KCl, 0n2; NH%Cl, 1n0; Na#CO$,2n76; NaHCO$,10n0; Table 3 summarizes data of bacteria of the genera Bacillus and Thermoanaerobacter, which are most NaCl, 5n0; Na#S;9H#O, 0n5; yeast extract, 0n01 (Difco); and T glucose, 5n0. The pH of the medium was of 9n5–9n7. The similar in their physiology to strain K1 , despite some culture was grown at 55–62 mC. remarkable differences existing between them. Thus, strain K1T differs from ‘B. flavothermus’ in its mor- Fatty acid Percentage of total fatty acids in phology, cell size, motility (no peritrichous flagel- Anoxybacillus pushchinensis K1T lation), and pH range and optimum. Unlike ‘B. T flavothermus’, strain K1 cannot grow at 30 or 70 mC; C 6 9 its growth temperature ranges from 37 to 65 mC. In "#:! n T C"%:! 7n3 addition, strain K1 is unable to utilize peptone and iso-C"&:! 38n7 yeast extract as substrates, does not hydrolyse casein, anteiso-C"&:! 2n0 cannot grow under aerobic conditions and is catalase- C"&:! 0n9 negative. Cells of Thermoanaerobacter thermohydro- iso-C 0 3 sulfuricus are motile, have a lower DNA GjC content "':! n T C"':" 2n6 than strain K1 cells, exhibit a remarkably different C"':! 14n5 range and optimum of temperatures and pH for 10Me-C"':! 0n9 growth, and are able to utilize yeast extract. Cells of iso-C 0 8 Bacillus pasteurii (Sneath, 1986) are larger in size than "(:! n T anteiso-C"(:! 0n1 cells of strain K1 , exhibit different optimal and hydroxyiso-C"&:! 0n3 ultimate temperature and pH values, are tolerant to C"(:! 0n5 NaCl up to 10%, can hydrolyse gelatin and casein, C 2 2 and, finally, grow as facultatively anaerobic bacteria. "):# n T C"):"δ* 4n3 It should be emphasized that strain K1 can be C"):"δ" 1n0 distinguished from all other strains chosen for com- C"):! 10n4 parison by the obligate dependence of its growth on #V C#!:! 0n6 the CO$ ion and a tendency toward the formation of branched cells. The 16S rDNA of strain K1T was compared with GenBank database sequences using the Genotypic characteristics  program (Pearson & Lipman, 1988). The comparison revealed the highest degree of similarity of the T The GjC content of the DNA of strain K1 was strain with representatives of the genus Bacillus sensu 42n2p0n2 mol%, a value similar to that of ‘B. stricto and related taxa. A strictly anaerobic species of flavothermus’ DSM 2641 (41n6p0n2 mol%, as deter- the genus Bacillus, Bacillus infernus, has already been mined in this work). The level of DNA relatedness described (Boone et al., 1995) but this species clearly between these strains was 58n8%. The levels of DNA belongs to a different cluster within this phyletic group. relatedness of strain K1T with Clostridium stercorarium Comparisons of 16S rDNA sequences indicated the T NCIMB 11754 (GjC content of 40n2 mol%), Bacillus significant difference (" 80%) between strain K1 and

International Journal of Systematic and Evolutionary Microbiology 50 2113 E. Pikuta and others

Table 2. 16S rDNA difference values between strain K1T and related taxa ...... Values for Anoxybacillus pushchinensis sp. nov. and Anoxybacillus flavithermus comb. nov. DSM 2641T (Z26932) (formerly ‘Bacillus flavothermus’) are shown in bold. Species are as follows (accession numbers are given in parentheses): 1, Anoxybacillus pushchinensis sp. nov.; 2, Bacillus sp. strain 13 DSM 2349 (Z26929); 3, Bacillus pallidus DSM 3670T (Z26930); 4, ‘Bacillus thermoalkalophilus’ DSM 6866 (Z26931); 5, Alicyclobacillus acidocaldarius DSM 446T (X60742); 6, Bacillus pasteurii NCIB 8841T (X60631); 7, Anoxybacillus flavithermus comb. nov. DSM 2641T (Z26932) (formerly ‘Bacillus flavothermus’); 8, Bacillus pseudofirmus DSM 8715T (X76439); 9, Bacillus cereus NCTC 11143 (55063); 10, Bacillus alcalophilus DSM 485T (X76436); 11, Bacillus pseudalcaliphilus DSM 8725T (X76449); 12, Bacillus gibsonii DSM 8722T (X76446); 13, Bacillus clausii DSM 8716T (X76440); 14, Bacillus megaterium DSM 32T (X60629); 15, Bacillus horikoshii DSM 8719T (X76443); 16, Aneurinibacillus aneurinolyticus ATCC 12856T (D78455); 17, Bacillus brevis NCIB 9372T (X60612); and 18, Thermoanaerobacter thermohydrosulfuricus E100-69T (L09161).

Species 1234567891011121314151617

27n1 37n2 0n1 47n1 0n60n6 518n4 17n517n517n9 610n9 10n410n510n718n0 71n16n16n36n617n19n5 811n7 9n49n39n618n3 10n3 10n4 910n6 9n49n49n420n0 10n7 9n77n2 10 11n5 9n08n99n519n1 11n0 10n24n28n8 11 11n3 8n68n79n119n8 11n4 10n44n88n42n1 12 13n0 9n79n910n019n6 11n8 11n66n610n46n46n8 13 12n2 8n08n28n519n1 11n3 10n96n39n55n86n16n0 14 10n1 9n49n59n619n4 9n7 9n17n15n79n28n810n19n5 15 13n0 9n79n910n019n6 11n8 11n66n610n36n46n80n06n010n1 16 15n4 12n512n513n118n5 13n5 13n912n013n113n814n514n613n213n614n6 17 14n3 11n611n612n118n7 12n0 13n011n912n611n711n712n111n712n012n110n3 18 33n0 35n935n835n835n7 38n4 34n334n135n234n034n335n833n536n735n835n134n8

B. infernus. Due to this large difference, the latter However, based on a comparison of physiological and species was not included in Table 2. ‘B. flavothermus’ chemotaxonomic data and genetic characterizations, T was the closest relative of strain K1 , their 16S rDNA including determination of GjC content of DNA, sequences exhibiting 98n8% similarity to each other. DNA–DNA hybridization and 16S rDNA analysis However, the 16S rDNA relatedness of strain K1T and (our data; Heinen et al., 1982; Claus & Berkeley, 1986; ‘B. flavothermus’ is inconsistent with the phenotypic Ash et al., 1991; Sharp et al., 1992; Rainey et al., features which are clearly different for these two 1994), this new genus should also include the species organisms. It is known that the analysis of 16S rDNA ‘B. flavothermus’(strain d.y l DSM 2641), the name sequences may be insufficient to distinguish between having not been validly published. It is, therefore, species (Vandamme et al., 1996). Therefore, DNA– proposed that ‘B. flavothermus’ be placed also in the DNA hybridization was performed, which showed genus Anoxybacillus as Anoxybacillus flavithermus 58n8% similarity between the two strains. This indi- comb. nov. cates that strain K1T and ‘B. flavothermus’ represent different species. Because 16S rDNA sequence similarity of these bacteria differed by 7–16% from those Description of Anoxybacillus gen. nov. of other representatives of the genus Bacillus and its Anoxybacillus (An.o.xy.ba.cilhlus. Gr. pref. an without; relatives, they can be distinguished as a separate taxon M.L. oxy shortened from oxygenium, oxygen; L. masc. at the generic level (Tables 3 and 4). Based on the n. bacillus small rod; N.L. masc. n. Anoxybacillus morphological and physiological features determined, small rod living without oxygen). as well as the data of genetic analyses, it is proposed that strain K1T be placed in a new genus, Anoxy- Cells are rod-shaped and straight, 0n4–0n85i2n5– bacillus, as a new species, Anoxybacillus pushchinensis 3n0 µm, often arranged in pairs or chains, with rounded gen. nov., sp. nov. ends. Gram-positive. Endospores are round and resistant to heating and freezing. Spores are located at The aim of the present investigation was mainly the the end of the cell. There is not more than one spore elucidation of the taxonomic structure of strain K1T. per cell. Obligately anaerobic or facultatively anaer-

2114 International Journal of Systematic and Evolutionary Microbiology 50 Anoxybacillus pushchinensis gen. nov., sp. nov.

Table 3. Comparison of Anoxybacillus pushchinensis K1T with bacteria that have similar physiology and GjC content ...... Species are as follows: 1, Anoxybacillus pushchinensis strain K1T (this work); 2, ‘Bacillus ﬂavothermus’ strain d.y. DSM 2641 (Heinen et al., 1982); 3, Thermoanaerobacter thermohydrosulfuricus DSM 567T (Klaushofer & Parkkinen, 1965); 4, Bacillus pasteurii VKM B-513 (Sneath, 1986).

Characteristic 1 2 3 4

Morphology Shape of cells Rod Rod Rod Rod Motility kjjj Spore formation jjjj Size (µm) 0n5–0n6i3n0–5n00n85i2n3–7n10n4–0n6i1n8–13n00n5–1n2i1n3–4n0 DNA GjC (mol%) 42n242n3\61n0* 37 38n5–42n0 Temperature (mC): Range 37–66 30–72 42–78 15–40 Optimum 62 60–65 67–69 30 pH: Range 8n0–10n55n5–9n05n5–9n26n5–9n5 Optimum 9n5–9n77n06n9–7n59n0 Tolerance to NaCl (%) 3n02n5  10 Substrate: Glucose jkjj Sucrose jkjj Starch jk j  Peptone kjkj Yeast extract kj j  Lactate kk k  Relation to O# Anaerobe Facultative aerobe Anaerobe Facultative aerobe Catalase activity kj k  − NO$ reduction jjj\kj Hydrolysis of gelatin kkkj Hydrolysis of casein kjj\kj

*GjC values of 42n3 and 61n0 mol% from H. Hippe (DSMZ) and Heinen et al. (1982), respectively.

Table 4. Comparison of Anoxybacillus gen. nov. with genera that have similar endospore formation ...... Genera are as follows: 1, Anoxybacillus;2,Amphibacillus;3,Bacillus;4,Clostridium;5,Desulfotomaculum; and 6, Sporolactobacillus. All genera are rod-shaped and produce endospores.

Characteristic 1 2 3 4 5 6

Motility k\jj j j j j Gram reaction jjjj\k (rare) j (11–89% strains) j Relation to O# Anaerobe\ Facultative Anaerobe\ Anaerobe Obligate Anaerobe\ facultative aerobe facultative (aerotolerant) anaerobe facultative aerobe aerobe aerobe #− SO% reduction kkk k j k Catalase activity j\kk j k k k Oxidase activity j\kkj\kk k k NO$V reduction to NO#V jkj\kj\kk k NaCl requirement (3–12%) kkj\kk k k Lactate as sole product of kkj\kk k j carbohydrate fermentation

International Journal of Systematic and Evolutionary Microbiology 50 2115 E. Pikuta and others

T obic; catalase variable. Alkaliphilic or alkalitolerant, of 41n6 mol% (by HPLC method). Type strain: d.y. T thermophilic. Chemo-organotrophic, with a fermenta- (l DSM 2641 ). tive metabolism. DNA GjC content is around 42 mol%. Includes two species: Anoxybacillus push- ACKNOWLEDGEMENTS chinensis and Anoxybacillus flavithermus. The type species of the genus is Anoxybacillus pushchinensis. We are grateful to Dr A. V. Lebedinskyi and Dr A. L. Mulukin for editing the English version of this article. This work was in part supported by the RFFI grant 99-04-49144. Description of Anoxybacillus pushchinensis sp. nov. Anoxybacillus pushchinensis (push.chi.nenhsis. N.L. REFERENCES masc. adj. pushchinensis pertaining to Pushchino, a Ash, C., Farrow, A. E., Wallbanks, S. & Collins, M. D. 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