Autoregulation of subtilin biosynthesis in Bacillus subtilis:the role of the spa-box in subtilin-responsive promoters

The production of the type I antimicrobial peptide (AMP) subtilin by Bacillus subtilis is regulated in a cell-density-dependent manner. Three subtilin-responsive promoter elements within the spaBTCSIFEGRK are controlled by the specific cis-acting sequence element called the spa-box, which represents the binding site of the subtilin regulator SpaR. Here, we describe the functional characterization of the spaB, spaS and spaI promoters by transcriptional fusion with a promoterless β-glucuronidase encoding gusA gene. Within these gusA fusion constructs, transcription initiation start sites of the spaS and spaI promoters were mapped to be located downstream of the spa-box, which is in contrast to previous reports. Nevertheless, all spa-promoters displayed typical cell-density-dependent activity in a subtilin-producing strain B. subtilis ATCC6633. Moreover, analysis of β-glucuronidase activities in a spaB mutant of B. subtilis ATCC6633 and a derivative of strain 168 that harbors the spaRK genes integrated in the chromosomal amyE locus, confirmed that these promoters are activated by subtilin-triggered, SpaRK-mediated, quorum-sensing control. Quantitative analysis showed that the spaS promoter strength at a given subtilin concentration appeared to be approximately five-fold higher than the spaB promoter, which in turn is approximately two-fold higher than the spaI promoter. Finally, it is shown that the elementary components involved in subtilin-mediated regulation are the two-component system, SpaRK, and a spa-box containing promoter