Subtilosin A

Subtilosin A is a sactipeptide bacteriocin produced by Bacillus subtilis strain 168, containing intramolecular thioether bonds and a head-to-tail macrocyclic peptide bond. Macrocyclization is presumably catalyzed by AlbE and AlbF proteins encoded by the subtilosin A biosynthesis gene cluster. However, the underlying mechanism of macrocyclization remains uncertain as the tertiary structures of the proteins are undetermined. Here, we report the crystal structures of AlbE and AlbF homologs in Quasibacillus thermotolerans, wherein the subtilosin biosynthesis gene cluster is highly conserved. Structural analysis and pull-down assays revealed that AlbE and AlbF form heterodimeric complexes. Although the AlbEF complex shows structural similarity to M16B family metalloproteases, the substrate-binding chamber is shallower and more open than the other M16B family proteins. The chamber surface showed electrostatic complementarity to the precursor of subtilosin. Our findings provide insights into the role of AlbEF in metalloprotease catalysis and macrocyclic peptide bond formation.

It has been recently shown, that certain strains/isolates of Bacillus subtilis can be used as a probiotic for humans. The production of the macrocyclic sactibiotic subtilosin in B. subtilis ATCC 6633 is highly regulated. To improve the subtilosin productivity of B. subtilis, different growth conditions were compared for maximal expression of the sbo promoter that regulates the expression of the subtilosin biosynthetic gene cluster. Oxygen-limiting conditions led to a strong increase of sbo promoter activities compared to aerobic conditions, and accordingly, the subtilosin amount determined by reversed phase HPLC (7.8 mg/L) was 15-fold superior to the amount of aerobic grown cultures (0.5 mg/L). A further promising enhancement of the subtilosin yield was achieved using a deletion mutant that is avoiding the general transition state regulator protein AbrB. The subtilosin titer of 42 mg/L produced by ΔabrB cells grown under oxygen-limiting conditions corresponds to an 84-fold increase compared to the subtilosin titer obtained from B. subtilis wild type cells propagated in aerobic conditions. Furthermore, evidence is provided that oxygen-limiting conditions led to a strong decrease in the productivity of the lantipeptide subtilin suggesting contrary regulatory mechanisms for the B. subtilis antimicrobials subtilin and subtilosin.

In a designed study to screen for antimicrobial exhibiting bacteria using molecular aspects, Bacillus species were considered to investigate antibiotic biosynthesis genes. 28 bacterial strains and 3 induced mutants were screened for the presence of subtilosin gene (sbo) and subtilosin through PCR and Mass spectrometry respectively. Sbo gene was detected in 16 out of 28 Bacillus strains. The results from gene sequences deliberated by multiple sequence alignments revealed high-level homology to the sequences of the sbo-alb gene locus of B. subtilis 168 and the other limited reported strains. Hence, this report provided additional strains to support the idea of subtilosin gene predominance amongst Bacillus strains isolated from environment and to find different species containing homologous genes, furthermore the utilization of its conserved region as a means of identifying Bacillus spp. that produce subtilosin. This is the first report to confirm the detection of subtilosin production from B. amyloliquefaciens.