Gram-positive filamentous streptomycetes produce ?-butyrolactone (GBL) molecules in a growth rate- and cell density-dependent manner that regulate expression of genes involved in secondary metabolism and morphogenesis. GBL signaling involves the GBL autoregulator, required for GBL biosynthesis, and the GBL binding protein (GBLBP), a TetR-type transcriptional regulator with a GBL binding domain. Several streptomycetes produce secondary metabolite phytotoxins, thaxtomins, which are required for efficient host colonization and infection of plants. The elaboration of host-microbe interaction activities by many gram-negative prokaryotes relies on N-acylhomoserine lactone (AHSL) derivatives, which bear an appreciable structural similarity to GBLs. Since thaxtomins are potent secondary metabolite phytotoxins required for host-microbe interaction, a role for GBL compounds in the regulation of thaxtomin gene expression by gram-positive streptomycetes analogous to that of AHSLs in regulating gene expression in gram-negative organisms is envisioned. This project will employ genetic, physiological and biochemical methods to elucidate the role(s) played by GBL signaling in the biology of thaxtomin-producing organisms. GBL autoregulator and GBLBP mutants will be generated using disruption and replacement techniques and analyzed using physiological studies of growth and morphological development. Quantitative assays of production of thaxtomins and other metabolites will be carried out. Transcript mapping and growth-rate dependent transcriptional analysis of autoregulator and thaxtomin structural genes in wild type and mutant derivatives will be performed. Biochemical analysis of GBLBP will include electrophoretic mobility shift and DNase footprinting assays with DNA regulatory elements. With extensive undergraduate student involvement, the research is expected to advance the limited understanding of streptomycete GBL autoregulator-mediated quorum-sensing activities, particularly with regard to thaxtomin-dependent host-microbe interactions.
