Bernhardt, ThomasHill, Ian2019-12-122019-052019-05-132019Hill, Ian. 2019. Loss of RNA Chaperone Hfq Unveils a Toxic Pathway in P. Aeruginosa. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029518Hfq is an RNA chaperone that operates as a global regulator of gene expression in many bacteria. In this work, we show that the loss of Hfq in the opportunistic pathogen Pseudomonas aeruginosa can result in a dramatic reduction in growth in LB media. We provide evidence that the growth defect observed in PAO1 Δhfq mutant cells is dependent upon MexT, a transcription regulator that governs antibiotic resistance in this organism. Using chromatin immunoprecipitation and high-throughput sequencing, we identified direct regulatory targets of MexT, including novel MexT-activated genes. We utilized a combination of whole genome sequencing of spontaneous suppressor mutants and transposon insertion sequencing to identify genes responsible for mediating the growth defect of hfq mutant cells. These include the MexT-activated genes mexEF, encoding components of a multidrug efflux pump, and PA1942. These data suggest that MexT-dependent activation of the mexEF and PA1942 genes contributes to the growth defect observed in PAO1 Δhfq mutant cells. We sought to characterize the novel MexT-controlled gene PA1942 and to identify factors that contribute to MexT-dependent gene activation. We demonstrate that a small ORF within the annotated PA1942 gene, which we refer to as hilR, encodes a small toxic protein. We show that hilR expression is negatively regulated by Hfq, which suggests Hfq influences the growth of P. aeruginosa by limiting the toxic effects of specific MexT-regulated genes. We demonstrate that in wild-type cells the ectopic expression of mexT or the loss of MexS, a MexT inhibitor, results in a growth defect on LB. We present evidence that enzymes involved in the biosynthesis of glutathione influence the expression of MexT-activated genes, suggesting that this metabolic pathway is important for regulating the in vivo activity of MexT. Taken together, this work demonstrates that the activation of MexT-regulated genes is an important determinant of the in vitro growth of P. aeruginosa.application/pdfenRNA binding protein MexT Transcription Factors Chromatin Immunoprecipitation Transposon mutagenesis GlutathioneThesis or Dissertation2019-12-120000-0002-9200-5268