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RsbT and RsbV Contribute to σB-Dependent Survival under Environmental, Energy, and Intracellular Stress Conditions in Listeria monocytogenes

ABSTRACT Sigma B (σB) is a stress-responsive alternative sigma factor that has been identified in various gram-positive bacteria. Seven different regulators of sigma B (Rsbs) are located in the sigB operons of both Bacillus subtilis and Listeria monocytogenes. In B. subtilis, these proteins contribute to regulation of σB activity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway. RsbT contributes to regulation of σB activity in response to environmental stresses, while RsbV contributes to σB activation under both environmental and energy stresses in B. subtilis. To probe L. monocytogenes Rsb roles in σB-mediated responses to various stresses, in-frame deletions were created in rsbT and rsbV. Phenotypic characterization of the L. monocytogenes rsbT and rsbV null mutants revealed that both mutants were similar to the ΔsigB strain in their abilities to survive under environmental stress conditions (exposure to synthetic gastric fluid, pH 2.5, acidified brain heart infusion broth [BHI], or oxidative stress [13 mM cumene hydroperoxide]). Under energy stress conditions (carbon starvation in defined media, entry into stationary phase, or reduced intracellular ATP), both ΔrsbT and ΔrsbV showed survival reductions similar to that of the ΔsigB strain. These observations suggest that the pathways for Rsb-dependent regulation of σB activity differ between L. monocytogenes and B. subtilis. As σB also activates transcription of the L. monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of ΔprfAP1rsbT and ΔprfAP1rsbV double mutants in hemolysis and tissue culture assays. Both double mutants showed identical phenotypes to ΔprfAP1P2 and ΔprfAP1sigB double mutants, i.e., reduced hemolysis activity and reduced plaque size in mouse fibroblast cells. These findings indicate that RsbT and RsbV both contribute to σB activation in L. monocytogenes during exposure to environmental and energy stresses as well as during tissue culture infection.