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1 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität Wien, A-1210 Wien, Austria
2 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany
Correspondence
Hans-Jürgen Busse
hans-juergen.busse{at}vu-wien.ac.at
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Published online ahead of print on 13 May 2005 as DOI 10.1099/ijs.0.63660-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of D-1,5aT is AJ781029.
Genomic fingerprints and polar lipid profiles of strains D-1,5aT and D-1,5b are available as supplementary figures in IJSEM Online.
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, 2002), Brachybacterium fresconis, Brachybacterium sacelli, Halomonas muralis, Bacillus decolorationis, Virgibacillus picturae and Brevibacterium picturae (Heyrman et al., 2002a, b, 2003a, b, 2004). During our studies of the microbiological diversity of the wall painting, we isolated, in addition to A. citreus and M. luteus, other isolates, which were preliminarily identified as members of the Bacillaceae (four isolates), the Microbacteriaceae (one isolate) and the Moraxellaceae (one isolate) (M. Wieser, unpublished results). Here we report the detailed characterization of two strains from this collection, which, in preliminary examinations, differed by only a single band in the protein patterns after SDS-PAGE (results not shown) performed as described by Altenburger et al. (1996).
Strains were isolated and investigated morphologically as described by Wieser et al. (1999). NaCl tolerance was tested on PYES agar (0·3 % peptone, 0·3 % yeast extract, 0·23 % disodium succinate, 1·5 % agar, pH 7·2) supplemented with 1, 3, 5, 7 and 10 % NaCl, respectively. Tolerance towards different pH values was examined on PYES agar adjusted with HCl (37 %) or 4 M NaOH to pH 6, 7, 8, 9, 10, 11 and 12, respectively, or on buffered medium as described by Nielsen et al. (1995). Hydrolysis of starch was examined on PYES medium as described by Sneath (1986). Other tests were done as described by Kämpfer et al. (1991). Optimal growth was obtained on PYES agar or broth, pH 8·0, supplemented with 1 % NaCl (w/v). The characteristics are listed in the species description below and in Table 1.
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) confirmed indications from protein patterns (results not shown) that these strains were members of a single species. The 16S rRNA gene sequence (1464 bases) of D-1,5aT was analysed and sequenced according to Wieser et al. (1999), using primers 27f, 342f, 519r and 1992r (Lane, 1991). It should be mentioned here that sequencing of the 16S rRNA-encoding gene of D-1,5aT caused unexpected problems. When primer 27f was used, the sequence could be determined only up to position 192. Ambiguous results were obtained for subsequent bases. A similar observation was made at the same position in the sequence when primer 519r was employed, and the base at position 193 (A or G) could not be determined in either of the two sequencing directions. This observation might be explained by the presence of two rRNA-encoding genes in D-1,5aT, one of which contains a gap or insertion at this position. Sequence comparisons (Pearson & Lipman, 1988) revealed the highest scores with the type strains of established species Bacillus flexus IFO 15715T (96·5 %), Bacillus fastidiosus DSM 91T (96·1 %), Bacillus indicus SD/3T (96·1 %), Bacillus cibi JG-30T (96·0 %), Bacillus megaterium IAM 13418T (96·0 %), Bacillus cohnii DSM 6308T (96·0 %), Bacillus bataviensis LMG 21833T (96·0 %), Bacillus soli LMG 21838T (96·0 %), Bacillus asahii MA001T (95·8 %), Bacillus muralis LMG 20238T (95·8 %), Bacillus novalis LMG 21837T (95·7 %), Bacillus niacini IFO 15566T (95·7 %) and Bacillus firmus IAM 12464T (95·6 %), indicating no relatedness between D-1,5aT and any of them at the species level. Interestingly, the 16S rRNA gene sequence of D-1,5aT shared the highest similarity (99·5 %) with the sequence of Bacillus sp. LMG 20243, which was recently isolated from a mural painting in St. Martins church in Greene–Kreiensen (Lower Saxony, Germany; Gorbushina et al., 2004) and shared 97·1 % similarity with Bacillus sp. LMG 19415, which, like D-1,5aT, had been isolated from the wall painting in the chapel of Castle Herberstein (Gurtner et al., 2000). The phylogenetic position of strain D-1,5aT was calculated using the PHYLIP software package (Felsenstein, 1993) after alignment of the 16S rRNA gene sequences using CLUSTAL X (Thompson et al., 1997) and manual editing of sequences for ambiguous nucleotides and gaps using BioEdit (Hall, 1999). In all algorithms, the phylogenetically next most closely related species to strain D-1,5aT were (i) the type strain of the type species of the genus Bacillus, Bacillus subtilis DSM 10T (Fig. 1), although the two strains share only 95·3 % 16S rRNA gene sequence similarity, and (ii) Bacillus fastidiosus DSM 91T. This branching was statistically supported by a moderate bootstrap value (81 %). However, these results indicate that strain D-1,5aT may be considered as a species of the genus Bacillus sensu stricto. For G+C content determination, DNA was extracted and purified as described by Auling et al. (1985). The DNA G+C contents of strains D-1,5aT and D1,5b were determined by HPLC (Kaneko et al., 1986) as 36·2 and 36·9 mol%, respectively. These values differ only slightly from those reported for Bacillus fastidiosus (34·3–35·1 mol%; Claus & Berkeley, 1986), Bacillus flexus (37·0–37·9 mol%; Priest et al., 1988), Bacillus megaterium (37·3 mol%; Claus & Berkeley, 1986) and Bacillus niacini (37·0–39·0 mol%; Nagel & Andreesen, 1991).
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; Ventosa et al., 1993; Altenburger et al., 1996). Fatty acid methyl esters were extracted from biomass grown on nutrient agar (l–1; 5 g peptone from casein, 3 g meat extract, 3 g yeast extract, 15 g agar) and was analysed as described by Kämpfer (1994). The diagnostic cell-wall diamino acid was determined as described by Schleifer (1985).
The major respiratory menaquinone in the two strains was found to be MK-7 and the diagnostic diamino acid in their cell walls was meso-diaminopimelic acid. These characteristics are in agreement with those of numerous species of the genus Bacillus, including the type species, Bacillus subtilis (Claus & Berkeley, 1986). The polar lipid profiles of D-1,5aT (see Supplementary Fig. S2 in IJSEM Online) and D1,5b (results not shown) were identical. Major to moderate amounts of diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids, which might correspond to monoglucosyl diacylglycerol and diglucosyldiacyl glycerol (as reported to be present in a strain of Geobacillus stearothermophilus; Minnikin et al., 1974), and moderate to minor amounts of two unknown phospholipids and two unknown polar lipids were detected. The presence of two glycolipids and the absence of phosphatidylethanolamine clearly distinguish the two strains from Bacillus flexus DSM 1320T, Bacillus cohnii LMG 16678T and Bacillus megaterium DSM 32T (Täubel et al., 2003). On the other hand, the presence of the two glycolipids might confirm the closer phylogenetic relatedness to Bacillus subtilis (Fig. 1
), which has been reported to contain glycolipids (Brundish et al., 1965; Bishop et al., 1967). The fatty acid profiles of strains D-1,5aT and D1,5b contained the major compounds C15 : 0 iso, C15 : 0 anteiso, C14 : 0 iso, C16 : 0 iso, C16 : 0 and C17 : 0 iso, which are characteristic of numerous taxa within the bacilli (Kämpfer, 1994). The relative fatty acid concentrations are listed below in the species description.
All of the characteristics determined for strain D-1,5aT are in accordance with those of the genus Bacillus. On the basis of phylogenetic distance from established Bacillus species, also indicated by relatively low 16S rRNA gene sequence similarities (<97 %) and the combination of unique phenotypic characteristics, it is demonstrable that D-1,5aT is not affiliated with any species of this genus. In conclusion, we describe D-1,5aT as the type strain of a novel species, for which we propose the name Bacillus herbersteinensis sp. nov.
Description of Bacillus herbersteinensis sp. nov.
Bacillus herbersteinensis (her.ber.stein'en.sis. N.L. masc. adj. herbersteinensis pertaining to Castle Herberstein in Styria, in which the chapel with the medieval wall painting is located from which the type strain was isolated).
Cells are motile, rod-shaped, Gram-positive in the KOH and aminopeptidase tests, rod-shaped and produce oval spores in terminal, unswollen sporangia. Colonies on PYES agar are 2–3 mm in diameter, slightly raised, irregular and cream-coloured to beige. Older colonies are more translucent. Catalase- and oxidase-positive. Growth occurs at 4 and 28 °C, in the presence of 0, 1, 3 and 5 % NaCl (w/v) but not at 7 or 10 % NaCl (w/v). On buffered medium, growth is observed at pH 7 (weakly), 8 and 9. On unbuffered medium, growth is observed at pH 7 (weakly), 8, 9, 10, 11 and 12 but not at pH 6. Negative for nitrate reduction and haemolysis. Starch, p-nitrophenyl (pNP) 
-D-galactopyranoside, pNP 
-D-glucopyranoside, pNP -D-glucopyranoside and bis-pNP phosphate are hydrolysed. pNP -D-xyloside, pNP phenylphosphonate, pNP phosphorylcholine, 2-deoxythymidine-5'-pNP phosphate, L-alanine pNA (pNA, p-nitroanilide), L-glutamate 
-3-carboxy-pNA, L-proline pna and pnp -D-glucoronide are not hydrolysed. Acid is not produced from adonitol, L-arabinose, D-arabitol, cellobiose, dulcitol, erythritol, glucose, inositol, lactose, maltose, D-mannitol, D-mannose, melibiose, methyl D-glucoside, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose or D-xylose. N-Acetyl-D-glucosamine, L-arabinose (weakly), p-arbutin (weakly), D-cellobiose, D-fructose, D-galactose, gluconic acid, D-glucose, D-maltose, D-mannitol, D-mannose, -D-melibiose, L-rhamnose, D-ribose, salicin, D-sorbitol, D-sucrose, D-trehalose, D-xylose, acetate (weakly), cis-aconitate (weakly), L-aspartate, citrate, fumarate, DL-3-hydroxybutyrate, DL-lactate, L-malate, L-ornithine (weakly), 2-oxoglutarate (weakly), pyruvate and L-proline are assimilated. Adonitol, i-inositol, maltitol, putrescine, azelate, glutarate, itaconate, trans-aconitate, adipate, propionate, 4-aminobutyric acid, 4-hydroxybenzoic acid, mesaconate, suberate, L-alanine, -alanine, L-serine, L-histidine, L-leucine, L-phenylalanine, L-tryptophan, 3-hydroxybenzoic acid and phenylacetate are not assimilated. The fatty acid profile consists of C14 : 0 iso (8·7–14·2 %), C14 : 0 (0–0·5 %), C15 : 0 iso (17·7–27·4 %), C15 : 0 anteiso (17·0–23·4 %), C15 : 0 (1·9–3·2 %), C16 : 1
7c (2·9–5·1 %), C16 : 111c (1·9–2·4 %), C16 : 0 iso (8·4–15·8 %), C16 : 0 (4·8–5·9 %), C17 : 110c iso (2·4–2·5 %), C17 : 15c iso (1·0 %), C17 : 0 iso (3·7–5·1 %), C17 : 0 anteiso (1·1–1·2 %) and C17 : 0 (1·1–1·6 %). The diamino acid in the cell wall is meso-diaminopimelic acid. The predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. Additionally, moderate to minor amounts of a second unknown glycolipid, two phospholipids and four polar lipids are present. The major respiratory quinone is MK-7. The DNA G+C content is 36·2–36·9 mol% (HPLC).
The type strain is strain D-1,5aT (=DSM 16534T=CCM 7228T). Strains D-1,5aT and D-1,5b were both isolated from a damaged wall painting in the chapel of Castle Herberstein, Styria (Austria).
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ACKNOWLEDGEMENTS |
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