Molecular regulation of bacterial stress responses and pyocin production

Abstract

Bacteria have a variety of systems to sense stress and respond ensuring the survival of cells. Bacillus subtilis uses stressosomes—large cytoplasmic multiprotein complexes—to sense environmental stressors and trigger the general stress response through activation of the alternative sigma factor σB. Stressosomes are comprised of 40 RsbR proteins made up of four paralogous (RsbRA, RsbRB, RsbRC, and RsbRD) putative stress sensors. Previous work uncovered differences in the timing and magnitude of the RsbR paralogs’ σB response profiles to the same stressor. In chapter 1, we use microfluidic-coupled microscopy to investigate the σB responses mediated by each paralog and how they differ in the presence of different environmental stressors. Wild-type and RsbRA-only cells activate σB with a characteristic transient response irrespective of the stressor, modulating the magnitude of the response. Other individual RsbR paralogs show distinct timing and magnitude of responses to stressors, implying RsbR proteins can distinguish among stressors. In Chapter 2 we explore how these differences in σB activation affect the fitness of cells by conducting competition experiments under stress conditions. Our data suggests that the dynamics of the σB responses, which are impacted by the single RsbR paralogs, is capable of affecting fitness of cells.

Pyocins are bacteriophage tail-like complexes released via cell lysis and kill other strains of P. aeruginosa, thought to aid in the elimination of competition for resources within a niche. The production of pyocins in xerC mutant strains occurs through a previously unknown non-canonical pathway. Normally the production of pyocins and resulting cell death is kept off by the transcriptional repressor PrtR, which undergoes autocleavage allowing pyocin production to occur only when RecA binds to damaged DNA. In chapter 3 we investigate the genetic regulation of pyocins via PrtR which appears to have previously unappreciated targets. In a recA and xerC double deletion, pyocins are still produced despite the trigger (RecA) for cleaving the repressor (PrtR) being absent, suggesting the xerC deletion is bypassing the PrtR repressor. Surprisingly, replacement of PrtR with a non-cleavable version still allows pyocin expression but somehow blocks production of functional pyocins, suggesting other previously unknown targets of PrtR.