Sigma B (B ) 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 B activity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway. RsbT contributes to regulation of B activity in response to environmental stresses, while RsbV contributes to B activation under both environmental and energy stresses in B. subtilis. To probe L. monocytogenes Rsb roles in B -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 ⌬sigB 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 ⌬rsbT and ⌬rsbV showed survival reductions similar to that of the ⌬sigB strain. These observations suggest that the pathways for Rsb-dependent regulation of B activity differ between L. monocytogenes and B. subtilis. As B also activates transcription of the L. monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of ⌬prfAP1rsbT and ⌬prfAP1rsbV double mutants in hemolysis and tissue culture assays. Both double mutants showed identical phenotypes to ⌬prfAP1P2 and ⌬prfAP1sigB 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 B activation in L. monocytogenes during exposure to environmental and energy stresses as well as during tissue culture infection.Listeria monocytogenes, a gram-positive, non-spore-forming rod-shaped bacterium, is recognized as a foodborne pathogen. This organism is capable of surviving in a broad range of ecological niches (e.g., in farm environments and food processing plants) and in a wide range of hosts, including humans and many species of animals. In L. monocytogenes, the alternative sigma factor B ( B ) contributes to survival under stressful environmental conditions, such as exposure to low pH, oxidizing conditions, and starvation (17, 18). Loss of B also reduces L. monocytogenes virulence in a murine model (32, 44).The sigB gene, which encodes B , lies seventh in the sigB operon. This operon also includes seven additional genes, which encode the following regulator of sigma B proteins: RsbR, RsbS, RsbT, RsbU, RsbV, RsbW, and RsbX (4,19,25,44,45). In B. subtilis, activation of B by the Rsb proteins is achieved through a complex phosphorylation/dephosphorylation cascade in response to various cellular stimuli, which have been catego...