International Journal of Systematic and Evolutionary Microbiology (2001), 51, 447–455 Printed in Great Britain Ureibacillus gen. nov., a new genus to accommodate Bacillus thermosphaericus (Andersson et al. 1995), emendation of Ureibacillus thermosphaericus and description of Ureibacillus terrenus sp. nov. M. Grazia Fortina,1 Ru$ diger Pukall,2 Peter Schumann,2 Diego Mora,1 Carlo Parini,1 P. Luigi Manachini1 and Erko Stackebrandt2 Author for correspondence: M. Grazia Fortina. Tel: j39 2 23955830. Fax: j39 2 70630829. e-mail: grazia.fortina!unimi.it 1 Department of Food A polyphasic taxonomic study was performed on the type strain of Bacillus Science and Microbiology– thermosphaericus DSM 10633T and three related soil isolates. On the basis of Industrial Microbiology Section, University of phenotypic characteristics, chemotaxonomic profiles and phylogenetic data a Milan, 20133 Milan, Italy new genus, Ureibacillus gen. nov., is proposed for the strains in the Bacillus 2 Deutsche Sammlung von thermosphaericus cluster. Strains of this cluster fall into two DNA–DNA Mikroorganismen und similarity groups: while one group contains the type strain of Ureibacillus Zellkulturen GmbH, 38124 thermosphaericus comb. nov. and a single soil isolate, the other contains two Braunschweig, Germany soil isolates. The two groups differed in the composition of isoprenoid quinones and some phenotypic properties. These data support the description of a novel species of Ureibacillus for which the name Ureibacillus terrenus is proposed. The type strain of this new species is TH9AT (l DSM 12654T l LMG 19470T). Keywords: Ureibacillus gen. nov., Ureibacillus terrenus sp. nov., Ureibacillus thermosphaericus INTRODUCTION have been isolated. Among the thermophilic bacilli Bacillus thermosphaericus is rather unique as it stands Recent taxonomic studies on thermophilic bacilli have phylogenetically isolated and possesses unique physio- shown that this biotechnologically important group of logical properties. These features have already been bacteria is still in need of further study to improve its stressed in the original description of the species classification and identification. Indeed, in recent years (Andersson et al., 1995) and the new taxon was there has been a proliferation of new species described tentatively assigned to the genus Bacillus. (Manachini et al., 1985; Demharter & Hensel, 1989; As part of a study investigating genotypic diversity Combet-Blanc et al., 1995; Meier-Stauffer et al., 1996; among thermophilic bacilli (Mora et al., 1998), three Pettersson et al., 1996) and, through more detailed strains were isolated from uncultivated soil in three phylogenetic and chemosystematic studies, the pro- different geographical areas. These strains showed posal of new genera (Wisotzkey et al., 1992; Shida et morphological and phenotypic features typical of al., 1996; Heyndrickx et al., 1998, Wainø et al., 1999). Bacillus thermosphaericus. Phylogenetic and chemo- In this framework Bacillus thermosphaericus represents taxonomic data supported the affiliation but indicated a thermophilic Bacillus species which is not closely that two of the soil isolates formed a novel species, the related to members of any of the newly described description of which is presented in this communi- genera that accommodate former Bacillus species. This cation. species was isolated in 1995 from urban air in Southern Finland (Andersson et al., 1995), but its habitat is as yet unknown as no additional strains of this species METHODS ................................................................................................................................................. Bacterial strains, growth conditions and phenotypic tests. T The GenBank accession number for the 16S rDNA sequence of Ureibacillus The strains used in this study, designated TH9A , TH29A terrenus is AJ276403. and TU1A, had been previously isolated from soil samples 01590 # 2001 IUMS 447 M. G. Fortina and others from Italy, Egypt and Turkey, respectively (Mora et al., Plasmid detection. Detection of plasmid DNA followed the 1998). Bacillus thermosphaericus DSM 10633T was used as alkaline extraction procedure described by Sambrook et al. reference strain. Strains were routinely maintained at 4 mC (1989). after growth at 55 mC on CESP agar (casitone, 15 g; yeast 16S rDNA sequence determination and phylogenetic analy- extract, 5 g; soytone, 3 g; peptone, 2 g; MgSO%,0n015 g; sis. Genomic DNA extraction, PCR-mediated amplification FeCl$,0n007 g; MnCl#,0n002 g; made up to 1 l with distilled of the 16S rDNA and sequencing of the PCR products was water, pH 7n2). For long-term maintenance, cell suspensions carried out as described previously (Rainey et al., 1994). The were stored at k80 mC in broth cultures supplemented with sequence reactions were electrophoresed using a model 373A 15% (w\v) glycerol. automatic DNA sequencer (Applied Biosystems). The 16S rDNA sequences were manually aligned with those of The morphological life-cycle was photographed in a phase- members of thermophilic bacilli and related organisms, contrast microscope by using cells grown on slides coated deposited in the 16S rDNA database of the DSMZ. with a thin layer of CESP agar. Gram staining was done as Evolutionary distances were calculated by the method of described by Salle (1961). Colony characteristics were Jukes & Cantor (1969). Phylogenetic dendrograms were determined from 24–36-h-old cultures grown at 55 mC. reconstructed using the treeing algorithm of DeSoete (1983) Anaerobic growth in glucose broth, anaerobic production of and Felsenstein (1993). gas from nitrate, citrate utilization, starch and casein hydrolysis, indole, acetoin and catalase production were Nucleotide sequence accession numbers. The accession determined by the method described by Smith et al. (1952). numbers of sequences used in the construction of the Reduction of nitrate was examined using the method of phylogenetic tree are: Bacillus thermosphaericus DSM 10633T, X90640; Bacillus thermocloaceae DSM 5250T, Lanyi (1987); the urease test followed the method described T by Atlas (1993). Utilization of different carbon sources was Z26939; Bacillus pasteurii NCIMB 884 , X60631; Bacillus " globisporus DSM 4T, X68415; Bacillus insolitus DSM 5T, tested on agar plates containing (g lV ) (NH%)#HPO% (1), KCl (0 2), MgSO (0 2), yeast extract (0 2), bromocresol purple X60642; Bacillus subtilis NCDO 1769, X60646; Bacillus n % n n smithii T Bacillus coagulans T (0 004) (pH 7 0) supplemented with 0 5% (w\v) of each DSM 4216 , Z26935; IAM 12463 , n n n Bucillus fastidiosus T Brevi carbon source sterilized separately. The temperature range DSM 91 , X60615; D16267; - bacillus brevis T Paenibacillus poly for growth was determined on CESP agar plates incubated NCIMB 9372 , X60612; - myxa DSM 36T, X57308; Exiguobacterium aurantiacum at different temperatures over the range 35–70 C. For pH m NCDO 2321T, X70316; Planococcus citreus NCIMB 1493T, studies, the pH of the medium was adjusted from 5 0to110. n n X62172; Kurthia zopfii NCIMB 9878T, X70321; and Tolerance to salinity was determined in CESP broth supple- Caryophanon latum NCIMB 9533, X70314. mented with 2–7% (w\v) NaCl. Riboprinting. Ribotyping was performed as described by Chemotaxonomy. Peptidoglycan structure was elucidated by Allerberger & Fritschel (1999). Analysis was done with the analyses of cell-wall hydrolysates employing the following RiboPrinter Microbial Characterization System (Qualicon) methods: qualitative analysis of amino acids and peptides by (Bruce, 1996). Each sample lane was normalized to a two-dimensional TLC on cellulose plates using described standard marker set, characterized and identified using solvent systems (Schleifer & Kandler, 1972), quantitative similarity measurements to previously run strains and amino acid analysis by GC and GC\MS (MacKenzie, 1987; reference patterns. Binary similarity values of riboprint Groth et al., 1996), dinitrophenylation of N-terminal amino patterns were used to generate a mean linkage clustering acids of the interpeptide bridge (Schleifer, 1985) and enantio- dendrogram (Sneath & Sokal, 1973). meric amino acid analysis using a chiral gas chromato- graphic column (Groth et al., 1997). Menaquinones were RESULTS analysed by HPLC as described previously (Groth et al., 1996). GC determination of cellular fatty acid profiles was Phenotypic properties performed as described by Schumann et al. (1997). Polar The three isolates could not be distinguished from one lipids extracted by the method of Minnikin et al. (1979) were another nor from Bacillus thermosphaericus DSM identified by two-dimensional TLC on silica gel and spraying T with specific reagents (Collins & Jones, 1980). 10633 by any of the physiological, morphological or metabolic characteristics tested. Cells were rod- Determination of GjC content of DNA. DNA base com- shaped, varying in length from 1 to 6 µm and approxi- position was determined by the thermal denaturation mately 0n5–0n7 µm in diameter, motile and occurred method described by Marmur & Doty (1962), using the singly or in chains. Round spores were located termin- equation of Owen & Hill (1979). DNA from Escherichia coli ally or subterminally within a swollen sporangium strain B (Sigma) was used as internal standard. (Fig. 1). The Gram reaction was negative. Circular, DNA–DNA hybridization method. DNA was isolated and entire, flat, transparent and swarming colonies were purified as described previously (Manachini et al., 1985). produced. The three isolates grew optimally