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

Sigma B (sigma(B)) is an alternative sigma factor that regulates the general stress response in Bacillus subtilis and in many other Gram-positive organisms. sigma(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 sigma(B) activity in Listeria monocytogenes, to inhibit sigma(B) activity in B. subtilis through perturbation of signal transduction cascades under various stress conditions. FPSS inhibited the activation of sigma(B) in response to multiple categories of stress known to induce sigma(B) activity in B. subtilis. Specifically, FPSS prevented the induction of sigma(B) activity in response to energy stress, including entry into stationary phase, phosphate limitation, and azide stress. FPSS also inhibited chill induction of sigma(B) activity in a Delta 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 sigma(B) activity. Genetic and biochemical experiments, including artificial induction of sigma(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 sigma(B) to inhibit activity, it appears to prevent the release of B. subtilis sigma(B) from its anti-sigma factor RsbW.