Genetic Determinants of Growth Arrest Survival in the Bacterial Pathogen Pseudomonas aeruginosa and the Role of Proteases

Citation

Basta, David Wagdi (2019) Genetic Determinants of Growth Arrest Survival in the Bacterial Pathogen Pseudomonas aeruginosa and the Role of Proteases. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/K6X1-GS91. https://resolver.caltech.edu/CaltechTHESIS:05282019-090132145

Abstract

Growth arrest is the dominant mode of microbial existence on the planet, yet the molecular mechanisms that underpin survival during growth arrest remain far less studied than other growth states. A better understanding of these mechanisms would provide valuable insight into the activity of microbial communities in both biogeochemical and clinical contexts, including the treatment of chronic infections. This thesis investigates the genetic requirements for survival of the bacterium Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen that thrives in diverse environments in which growth arrest is often caused by energy limitation. After reviewing our current knowledge of the strategies used by growth-arrested bacteria to adjust metabolism, regulate transcription and translation, and maintain the chromosome, I perform a functional genomic screen to identify genes that promote fitness of P. aeruginosa during growth arrest caused by carbon or oxygen starvation. I find that P. aeruginosa can survive for days to weeks in these energy-starved conditions by maintaining a reduced steady-state level of ATP, and that many functional classes of genes are required for fitness. Intriguingly, a majority of genetic fitness determinants differ between carbon and oxygen starvation, despite the common endpoint of reduced ATP levels in these two conditions. Among the few genes generally required for fitness are the stress response sigma factor encoded by rpoS and the heat shock protease encoded by ftsH. Using independently-generated deletion strains, I show that mutants in distinct functional categories exhibit temporal fitness dynamics during oxygen starvation: regulatory genes generally manifest a phenotype early during growth arrest, whereas genes involved in cell wall metabolism are required later. Building on these findings, I investigate the functional role of FtsH during growth arrest more deeply and find a surprising negative genetic interaction between ftsH and rpoS, with mutations in rpoS alleviating the fitness defects of ΔftsH during growth arrest. I also find that FtsH functions coordinately with the other conserved heat shock proteases to maintain cellular integrity and delay aging of P. aeruginosa during growth arrest. Finally, I investigate the role of FtsH and the other heat shock proteases in a novel N-terminal protein degradation pathway and find that the molecular details of this pathway likely differ between E. coli and P. aeruginosa. Together, these findings uncover essential molecular processes that promote fitness of an important bacterial pathogen during growth and survival.

Thesis Files