Fluoro-Phenyl-Styrene-Sulfonamide, a Novel Inhibitor of σ B Activity, Prevents the Activation of σ B by Environmental and Energy Stresses in Bacillus subtilis

Abstract

ABSTRACT Sigma B (σ B ) is an alternative sigma factor that regulates the general stress response in Bacillus subtilis and in many other Gram-positive organisms. σ B activity in B. subtilis is tightly regulated via at least three distinct pathways within a complex signal transduction cascade in response to a variety of stresses, including environmental stress, energy stress, and growth at high or low temperatures. We probed the ability of fluoro-phenyl-styrene-sulfonamide (FPSS), a small-molecule inhibitor of σ B activity in Listeria monocytogenes , to inhibit σ B activity in B. subtilis through perturbation of signal transduction cascades under various stress conditions. FPSS inhibited the activation of σ B in response to multiple categories of stress known to induce σ B activity in B. subtilis . Specifically, FPSS prevented the induction of σ B activity in response to energy stress, including entry into stationary phase, phosphate limitation, and azide stress. FPSS also inhibited chill induction of σ B activity in a Δ rsbV strain, suggesting that FPSS does not exclusively target the RsbU and RsbP phosphatases or the anti–anti-sigma factor RsbV, all of which contribute to posttranslational regulation of σ B activity. Genetic and biochemical experiments, including artificial induction of σ B , analysis of the phosphorylation state of the anti–anti-sigma factor RsbV, and in vitro transcription assays, indicate that while FPSS does not bind directly to σ B to inhibit activity, it appears to prevent the release of B. subtilis σ B from its anti-sigma factor RsbW.