Subtilosin Production by Two Bacillus subtilis Subspecies and Variance of the sbo-alb Cluster

Abstract

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...

Ecology and genomics of Bacillus subtilis

“…For many years most of the available evidence concerning genotypic variation among different B. subtilis isolates came from the assessment of phenotypic variation, principally strain-tostrain variation in the ability to make various antibiotics 26,29 . It wasn't until the 1990s that loci other than 16S rRNA genes were examined among multiple strains [30][31][32] .…”

Bacteria Exhibiting Antimicrobial Activities; Screening for Antibiotics and the Associated Genetic Studies

“…Detection and Sequencing of sbo Genes sbo and its flanking region were detected from the environmental Bacillus strain.Genomic DNA was extracted from overnight inoculated bacterial culture in L-broth at 37°C with 120 rpm. PCR amplification of ~1375-bp consisted of sbo and flanking region was performed successfully with TS13C (GAATTGACACTATCTAG AGAAATGC CG) and TS14 (ATCCGGTGGTGCGGAATT CGATGA) [16]. The optimum PCR conditions were determined as heating 94°C for 5min through 30 cycles as follows: denaturation for 30s at 94 °C primer annealation for 30s at 59°C and extension at 72°C for 1.2 min.…”

“…Subtilosin A is one of many antibiotics produced by Bacillus strains [15][16][17]. The spectrum of activity was investigated and proved antagonism against a wide range of bacteria including Gram-positive and Gram-negative bacteria and both aerobes and anaerobes [18].…”

Antibacterial peptides from thermophilic bacteria

“…2F) has a macrocyclic structure with three inter-residual linkages (Marx et al, 2001) and is produced by B. subtilis (Zheng et al, 1999;Stein et al, 2004). In the processing of subtilolisin AlbF (YwhN) protein and immunity proteins AlbB-D (YwhQPO) are included .…”