RsbT and RsbV contribute to sigmaB-dependent survival under environmental, energy, and intracellular stress conditions in Listeria monocytogenes

Abstract

Sigma B (sigma(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 sigma(B) activity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway. RsbT contributes to regulation of sigma(B) activity in response to environmental stresses, while RsbV contributes to sigma(B) activation under both environmental and energy stresses in B. subtilis. To probe L. monocytogenes Rsb roles in sigma(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 DeltasigB 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 DeltarsbT and DeltarsbV showed survival reductions similar to that of the DeltasigB strain. These observations suggest that the pathways for Rsb-dependent regulation of sigma(B) activity differ between L. monocytogenes and B. subtilis. As sigma(B) also activates transcription of the L. monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of DeltaprfAP1rsbT and DeltaprfAP1rsbV double mutants in hemolysis and tissue culture assays. Both double mutants showed identical phenotypes to DeltaprfAP1P2 and DeltaprfAP1sigB 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 sigma(B) activation in L. monocytogenes during exposure to environmental and energy stresses as well as during tissue culture infection.

FIG. 1.

Viabilities of L. monocytogenes wild-type 10403S (black), ΔsigB (gray), ΔrsbT (hatched), and ΔrsbV (white) strains following exposure of mid-log (A and C) or stationary-phase (B, D, and E) cultures to synthetic gastric fluid at pH 2.5 (A and B), acid (pH 2.5) (C and D), and CHP (13 mM) (E). The results shown are mean values from three independent experiments; error bars indicate standard deviations.

FIG. 2.

Growth of L. monocytogenes 10403S (⧫), ΔsigB (□), ΔrsbT (▴), and ΔrsbV (○) in a defined medium with a limiting amount of glucose (0.04% [wt/vol]). OD₆₀₀ values were recorded. The results shown are mean values from three independent experiments; error bars indicate standard deviations.

FIG. 3.

Viabilities of L. monocytogenes 10403S (⧫), ΔsigB (□), ΔrsbT (▴), and ΔrsbV (○) cultures that had been grown statically in BHI at 37°C. Bacteria were harvested at indicated times, serially diluted, and plated for enumeration on BHI agar plates. The results shown are mean values from three independent experiments; error bars indicate standard deviations.

FIG. 4.

OD₆₀₀ values for L. monocytogenes 10403S (⧫), ΔsigB (□), ΔrsbT (▴), and ΔrsbV (○) strains after exposure of mid-log cells (OD₆₀₀ = 0.4) to 32-μg/ml CCCP. The results shown are mean values from three independent experiments; error bars indicate standard deviations.

FIG. 5.

Proposed model of Rsb-mediated σ^B activation in L. monocytogenes (B) in comparison to Rsb-mediated σ^B activation in B. subtilis (A).