Date of Degree
PhD (Doctor of Philosophy)
John R. Kirby
Despite years of intensive research, many of the fundamental aspects of two-component signal transduction pathways are not yet understood. Interestingly these systems are found throughout all domains of life including archaea, bacteria and eukaryotes and are known to regulate diverse cellular processes such as motility, pathogenesis, development, biofilm formation, and toxin production. Despite many groups working on two-component systems it is not yet appreciated whether these systems have conserved features, amino acid requirements, structures and specificity. By understanding the mechanisms by which signals propagate through these systems we could perhaps develop novel therapeutics targeting these pathways.
In order to address these questions my thesis has focused on studying the signaling pathways which regulate multicellular development in the model soil bacterium Myxococcus xanthus. The developmental process is highlighted by large changes in gene expression patterns and motility resulting in the production of large macroscopic fruiting bodies composed of metabolically dormant myxospores. My initial work focused on characterizing the Che3 chemosensory pathway known to regulate time of aggregation required for fruiting body formation. I discovered an additional kinase CrdS which works with the Che3 system to regulate phosphorylation of the important developmental regulator CrdA.
Additionally I performed mutagenesis on the kinase CrdS to demonstrate that specific residues in CrdS are required for both kinase and phosphatase activities. A conserved Thr/Asn was required for phosphatase activity while a conserved acidic residue was required for kinase activity. Importantly, these residues are highly conserved and when we made mutations in multiple other kinases, we saw similar requirements, indicating the importance of these residues.
Further analysis focused on 26 other CrdA homologs found within the M. xanthus genome. Using phosphotransfer profiling and a newly created phosphatase profiling method we were able to demonstrate signaling specificity whereby each kinase was able to phosphorylate and dephosphorylate a single response regulator. Since phosphotransfer and phosphatase activities are predicated upon protein-protein interactions, we also determined that cognate pairs exhibited preferential binding. Cumulatively this research highlights some of the conserved mechanism governing the signal transduction pathways regulating multicellular development in M. xanthus.
Copyright 2012 Jonathan Willett