Proteolysis is an essential part of maintaining protein homeostasis in all living cells. Lon is a highly conserved AAA+ protease in bacteria, which performs protein quality control as well as regulatory roles in response to the requirements of the cell. In spite of growing evidence for its importance in bacterial survival and adaptation, there are major gaps in our understanding of how several of these pathways are regulated by Lon and how Lon-mediated proteolysis itself is controlled. These questions persist even in well-studied model organisms such as Pseudomonas aeruginosa and Caulobacter crescentus. In P. aeruginosa, Lon is known to affect many pathways but very few substrates were known. In addition, a Lon-like protein named AsrA remained largely uncharacterized with no known substrates. In C. crescentus, although several substrates have been discovered, no Lon-specific regulator was known. This thesis, aiming to fill some of these gaps, is a collection of three research studies.

In study I, we used quantitative proteomics to search for Lon substrates in P. aeruginosa, resulting in a list of putative substrates. We confirmed nine previously unknown substrates through in vitro assays, a majority of which were motility-related proteins. Through investigations of a lon loss-of-function mutant and its phenotypes, we observed defects in cell division and motility, and an accumulation of the substrate protein SulA, a cell division inhibitor under the control of the SOS response. Through suppressor mutational analysis, we found that Lon regulates motility indirectly through SulA under optimal conditions, and directly by degrading flagellar proteins, presumably under conditions of motility suppression. In study II, we characterized the AsrA protein biochemically through in silico and in vitro studies concluding that it is an active protease that performs functions distinct from Lon. We conducted a global search for its substrates, resulting in a list of putative substrates. Through this, we discovered that AsrA regulates the PQS pathway of quorum sensing through its substrate QslA, an anti-activator of LasR. This degradation is inhibited in vitro by a potential regulator of AsrA, a protein named Icp encoded by a gene adjacent to asrA. We also showed that AsrA is crucial for the survival of P. aeruginosa under heat-shock conditions. Together, this study reveals that AsrA is an important protease evolved to occupy distinct niches of proteolytic regulation in P. aeruginosa. In study III, we reported the discovery and biochemical investigation of a novel Lon regulator named LarA in C. crescentus, which enhances Lon-mediated degradation under proteotoxic stress. We analyzed the transferability of its degron through in vitro assays and concluded that its hydrophobic C-terminal degron is important but not sufficient for its regulation of Lon.

To summarize, we report new substrates and pathways linking Lon to P. aeruginosa motility, characterization of AsrA which regulates quorum sensing and heat-shock response, and LarA as a novel regulator of Lon activity in C. crescentus. Taken together, the research reported in this thesis expands our understanding of the substrates, cellular roles and regulatory mechanisms of three homologs of Lon proteases in two model organisms. This collection of studies can be a valuable resource in examining the impact and potential of bacterial proteolysis in environmental and healthcare research.