C Signal-Dependent Gene Expression in Myxococcus xanthus

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In nature, bacteria exist as members of complex communities. Cells interact in these communities, sending signals to each other and changing their behavior in response. These groups of bacteria play a vital role in recycling carbon, nitrogen, and other elements in the global ecosystem. Often, the mixed population of bacteria is on or in another living organism, and interacts with its host. Understanding how bacteria interact with each other and with their hosts is a fundamental challenge in biology. Myxococcus xanthus provides a very attractive experimental system to elucidate the molecular mechanisms of interactions between bacterial cells. Swarms of these bacteria glide over solid surfaces, secreting substances that kill and digest prey bacteria. When starved, M. xanthus cells move to aggregation centers and construct a nascent fruiting body. Within the fruiting body, rod-shaped cells differentiate into spherical, dormant spores. This spectacular developmental process is regulated by cell-cell interactions. M. xanthus is very amenable to genetic and biochemical analyses at the molecular level. In contrast, molecular analysis of cell-cell interactions in mixed populations of bacteria is extremely difficult. As methods to study complex communities improve, research to understand their mechanisms of signaling and response will be facilitated by the paradigms learned from model organisms like M. xanthus. Studies of C signaling during M. xanthus development are establishing a new paradigm for how bacterial cells can interact. C signaling involves CsgA, a protein produced in the developing cells that becomes associated with the cell surface. CsgA appears to have enzymatic activity, but the substrate is unknown. It has been proposed that either CsgA itself or the product of its enzymatic activity is exchanged upon end-to-end contact between cells. C signaling induces several responses in recipient cells. It appears to maintain the stringent response, preventing cells from resuming growth on amino acids that are produced early in development, so these amino acids are instead used for macromolecular synthesis during development. C signaling also regulates cell movements, allowing macroscopic patterns called ripples and aggregates to form, and it regulates developmental gene expression and sporulation. Intriguingly, the different responses to C signaling require different levels of CsgA, and the level of CsgA rises during development because C signaling stimulates CsgA production. Extensive end-to-end contacts between cells in the nascent fruiting body have been proposed to allow efficient C signaling. The high level of CsgA that results within the fruiting body may trigger sporulation. This research aims to determine the molecular mechanisms of developmental gene expression in response to C signaling. Preliminary studies have characterized the DNA regulatory regions of two loci that depend partially on C signaling for expression and one that depends absolutely on C signaling. All three regions have one or more sequences matching the consensus CAYYCCY (called the C box; Y means pyrimidine) near the transcriptional start site. The one that depends absolutely on C signaling also has an upstream repeat sequence that is needed for expression. Mutational analyses will be used to define the sequence features of the C box and the upstream repeat that are important for expression. To identify proteins directly involved in the regulation of C signal-dependent genes, several approaches will be used. Preliminary results showing that the FruA protein binds to promoter regions of C signal-dependent genes will be extended in collaboration with L. Sogaard-Andersen, using in vitro footprinting and electrophoretic mobility shift assays. The technique of in vivo cross-linking and immunoprecipitation will be adapted for use in bacteria to examine FruA binding to promoter regions. Yeast one-hybrid screens will be performed to identify proteins that bind specifically to DNA sequences shown by mutational analyses to be important for expression. Transcription of a C signal-dependent gene will be reconstituted in vitro to identify the form of RNA polymerase involved and its associated sigma factor. Insights gained from research in this area should find application in environmental cleanup, industrial and agricultural practices.