Insight on the roles of stringent response, stringent-like response, and general stress response in hyperosmotic shock tolerance in <i>Escherichia coli</i>

Bacteria frequently encounter osmotic stress in a variety of natural environments, and the primary protective systems they use to adapt to such stresses have been well-characterized. The work examining these mechanisms has largely focused on their role in adapting to moderate shock or continuous osmotic stress. However, survival of rapid, severe osmotic stress remains poorly understood. Here, we demonstrate that a pleiotropic mutation, <i>rpoB58</i>, in the beta subunit-coding gene of RNA polymerase in <i>Escherichia coli</i> confers tolerance to extreme salt stress. We found that the osmotic shock tolerance phenotype of the mutant is independent of the regulators of two major stress responses, the RelA/SpoT-mediated stringent response (SR) and RpoS-mediated general stress response (GSR). Rather, genetic analysis suggests that <i>rpoB58</i> partially mimics the effects of these pathways on gene expression. The data show that pre-activation of these stress responses by a constitutively active <i>relA</i> allele, overexpression of <i>relA</i> or <i>rpoS</i>, or environmental adaptation confers extreme osmotic shock tolerance similar to <i>rpoB58</i>. These findings suggest that several conditions, including SR pre-activation, GSR pre-activation, stationary phase, and environmental adaptation, cause cells to pre-emptively adopt a stress-tolerant physiological state and may confer tolerance to extreme osmotic shock by a shared mechanism. Furthermore, pre-activation of SR was found to confer osmotic shock tolerance independent of RpoS, indicating that SR has its own unique role in osmotic shock tolerance.IMPORTANCEThe overlapping regulation and effects of various stress response pathways in bacteria have been a major subject of study for several decades. This work examines the mechanisms by which a laboratory-acquired mutation in the <i>rpoB</i> gene conferring antibiotic tolerance also improves salt tolerance in <i>Escherichia coli</i>, an important pathogen of the human gut. We demonstrate that the <i>rpoB</i> mutation mimics the effects of multiple stress response pathways on gene expression and that pre-activation of these responses is critical for conferring hyperosmotic shock tolerance. These findings significantly advance our understanding of the genetic mechanisms controlling salt tolerance in bacteria and implicate the stringent response as one factor capable of conferring salt tolerance independent of the general stress response. Furthermore, these findings highlight the intricate connections between salt tolerance and other stress response pathways.