Eight different Bacillus subtilis strains and Bacillus atrophaeus were found to produce the bacteriocin subtilosin A. On the basis of the subtilosin gene (sbo) sequences two distinct classes of B. subtilis strains were distinguished, and they fell into the two B. subtilis subspecies (B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii). The entire sequence of the subtilosin gene cluster of a B. subtilis subsp. spizizenii strain, B. subtilis ATCC 6633, was determined. This sequence exhibited a high level of homology to the sequence of the sbo-alb gene locus of B. subtilis 168. By using primer extension analysis the transcriptional start sites of sbo in B. subtilis strains ATCC 6633 and 168 were found to be 47 and 45 bp upstream of the sbo start codon, respectively. Our results provide insight into the incipient evolutionary divergence of the two B. subtilis subspecies.Almost 4% of the 4.2-Mbp Bacillus subtilis 168 genome codes for proteins similar to the proteins involved in the biosynthesis of antimicrobial metabolites (17). However, B. subtilis 168 produces only a few antibiotics because several of the biosynthetic pathways are not functional, most likely because of the X-ray mutation of the original Marburg strain (6). In contrast, various other B. subtilis wild-type strains produce characteristic cocktails of numerous peptide antibiotics (1, 18). For example, a well-established bioindicator strain for sterilization control, ATCC 6633 (11), was investigated with respect to biosynthesis of the lantibiotic subtilin (4,8,16,27) and its regulation (26,28). In a series of B. subtilis strains production of the nonribosomally synthesized cyclic lipopeptides surfactin, fengycin, and the iturins, including mycosubtilin, with different compositions has been observed (9,18,20,31).Subtilosin is a macrocyclic bacteriocin with three intramolecular bridges (14,19). An acidic isoelectric point differentiates subtilosin from the basic lantibiotics (13, 24). Subtilosin transcription is increased under oxygen-limited and anaerobic conditions (22; T. Stein, S. Düsterhus, A. Stroh, and K.-D. Entian, 10th Int. Conf. Bacilli, abstr. P103, p. 65, 1999). The production of mature subtilosin is based on the expression of the sbo-alb gene cluster encompassing the subtilosin structural gene sbo and genes involved in posttranslational modification and processing of presubtilosin and in immunity (34, 35).Here we describe subtilosin production by eight different B. subtilis wild-type strains and Bacillus atrophaeus. The sbo genes of these organisms, as well as the entire subtilosin gene cluster of B. subtilis ATCC 6633, were sequenced in order to analyze the genetic variation between B. subtilis wild-type strains. MATERIALS AND METHODSStrains and plasmids. Strains used in this work are listed in Table 1. Recombinant plasmids were amplified in Escherichia coli DH5␣ or TG1 grown in Luria-Bertani medium (GIBCO, Neu-Isenburg, Germany). B. subtilis was grown either on TY (0.8% tryptone, 0.5% yeast extract [Difco, Detroit, Mich.], 0.5% N...