Studies of SpaK and SpaR, two components of a signal transduction system that regulate the expression of subtilin in Bacillus subtilis.

摘要

Ribosomally synthesized antimicrobial peptides have become an important field for investigation, resulting from a growing need for alternative antibiotics. Subtilin is a gene encoded, antimicrobial peptide with unusual post-translational modifications, including the thioether amino acid, lanthionine. Subtilin is a member of a growing class of antimicrobial peptides, termed “lantibiotics” because they contain lanthionine. The biosynthesis of subtilin is under complex regulation, dependent on environmental conditions. The regulatory features of subtilin have been investigated in this work. Subtilin expression is thought to be regulated by a two-component regulatory system. A system was developed to study the putative two-component signal transduction pathway and its role in transcriptional activation. The two components believed to regulate subtilin biosynthesis, SpaK and SpaR, have been characterized using biochemical analysis; The spaR and spaK genes were detected and cloned from the natural subtilin producing Bacillus subtilis 6633 strain and from Bacillus subtilis LH45, a 168 strain that had been converted into a subtilin producing strain. Expression systems were designed and constructed to produce N-terminal histidine-tagged SpaR and SpaK proteins. SpaK was found to have autophosphorylation, kinase and phosphatase activity and was found to phosphorylate SpaR. SpaR was found to bind to the promoter region of the spa operon (spa BTCS), a gene cluster partially responsible for subtilin production and containing the subtilin structural gene. SpaR was also found to bind to an intergeneic region upstream of the immunity factor gene cluster, spaIFG, a gene cluster that had been sequenced previously. The promoter region was identified and characterized in this work and found to be strikingly similar to the spaBTCS promoter.; A model of regulation is proposed that is in keeping with the purpose of antimicrobial peptides: antibiosis. The elucidation of the subtilin regulatory system suggests that we will find improved ways to regulate these antimicrobial peptides for our own combat against microbial takeovers. Principles learned from in vitro analysis of the SpaK/SpaR signal transduction pathway may further our global understanding of the complex mechanism of two-component signal transduction systems. This system offers an opportunity to further investigate mechanisms of transcriptional activation in Gram positive bacteria.