The long-term objective of the proposed research is to understand how cell-cell interactions control developmental gene expression. The gram- negative bacterium, Myxococcus xanthus, provides a very attractive experimental system for these studies because it undergoes a simple multicellular development requiring cell-to-cell signaling, and it is amenable to both biochemical and genetic analyses. The proposed studies seek to elucidate the molecular mechanism(s) that connects a signaling interaction to gene activation. These studies are of broad significance because cell-to-cell communication controls both organismal development and many processes within mature multicellular organisms, as well as host/pathogen and host/symbiont interactions. In addition, it is becoming increasingly clear that interactions between microbes influence many important biological phenomena. At least four signaling interactions control Myxococcus development and the best-characterized of these is that mediated by csgA, which encodes a 17 Kda protein that serves as the C-signal molecule. A csgA mutant fails to normally express many developmentally regulated Myxococcus genes, which were originally identified by transposon-generated lacZ fusions. These C-dependent genes provide a starting point for the proposed investigation which is aimed at exploring the molecular mechanism(s) that connects C-signaling to gene activation. The regulatory regions of several C-dependent genes will be cloned and characterized by testing for promoter activity in vivo, then subjecting promoter regions to deletion analyses, transcriptional start site mapping, and DNA sequencing. Regulatory proteins that interact wit the regulatory regions of C-dependent genes will be identified using a mobility shift assay for sequence-specific DNA-binding proteins and by reconstructing C-dependent gene transcription in vitro. The regulatory proteins will be purified, the corresponding genes will be cloned and sequenced, and both gene and antibody probes will be developed for use in studies aimed at understanding how the regulatory proteins respond to C-signaling and control C-dependent gene expression.
