9631365 Gill Myxococcus xanthus is a Gram negative bacterium that undergoes a simple cycle of multicellular development. These studies will focus on one mutant group, bsgA, which is defective in producing one of the required cell-cell interactions occurring early in development. The primary defect in these mutants is loss of an ATP-dependent protease. As a result of the protease defect, these strains fail to transcribe any of the known developmentally induced genes, and fail to form fruiting bodies or spores. One of the components of the regulatory pathway through which the BsgA protease regulates transcription, and a putative substrate of the protease, has been identified. It bears significant homology to the NtrC family of response regulators of two component regulatory systems. These regulators are phosphorylated by their cognate histidine protein kinase in response to a specific input signal, and in their phosphorylated form bind to DNA in a sequence specific manner at promoters which are transcribed by a particular minor form of RNA polymerase. When bound to these promoters, these proteins stimulate transcription in an ATP-dependent fashion. The objective of this project is identify the components of the regulatory pathway through which bsgA-dependent cell-cell signaling regulates transcription and to determine the specific role of the BsgA protease in the pathway. The substrate of the BsgA protease which is critical for regulation will be identified among a collection of suppressor mutants which bypass the requirement for the protease. Candidates will be cloned, and expressed as fusion proteins in E. coli. The fusion proteins will be purified by affinity chromatography and used to produce antibodies. Immuno-blotting will then be used to assess whether the protein is degraded by the BsgA protease in vivo. A genetic approach may be attempted to identify additional components of this regulatory pathway. Mutants will be isolated which interfere with the BsgA dependent gene expression. The genes identified in this way will identify additional component involved in sensing and responding to BsgA signaling. The ultimate goal of this project is to understand the means by which this organism coordinates the behavior of individual cells during multicellular activities. The problem of multicellular coordination is a fundamental problem in developmental biology. The information provided by this work may help to provide a framework for thinking about cell interactions within tissues in more complex organisms. ***
