The activation and response of Bacillus subtilis ECF sigma factor sigma V to lysozyme

Abstract

Extra-cytoplasmic function (ECF) σ factors are a subset of σ factors bacteria use to transcribe specific genes in response to environmental cues. In the absence of an inducing signal, ECF σ factors are inhibited by an anti-σ factor that prevents the ECF σ factor from interacting with RNA polymerase. The ECF σ factor σ V from Bacillus subtilis is activated in response to lysozyme stress. Lysozyme damages bacterial peptidoglycan by cleaving at the 1,4-ß-linkage between N-acetylmuramic acid and Nacetylglucosamine. In the absence of lysozyme, the activity of σ V is inhibited by the antiσ factor RsiV, a single pass transmembrane protein. The two main components of this project have been to elucidate the mechanism of σ V activation and examine how this system senses lysozyme stress. In Chapter 2 we show the activation of σ V is specific to lysozyme and the anti-σ factor RsiV is degraded by a step-wise proteolytic cascade known as Regulated Intramembrane Proteolysis (RIP). In the presence of lysozyme, the extracellular domain of RsiV is removed by cleavage at site-1. Upon removal of the extracellular domain, the site-2 protease, RasP, cleaves RsiV within the membrane. The remainder of RsiV is degraded by cytosolic proteases allowing σ V to interact with RNA polymerase. In response to lysozyme stress σ V activates an O-actyltransferase, OatA, which modifies the peptidoglycan to prevent further lysozyme damage. Our studies in Chapter 3 identifed the protease(s) responsible for site-1 cleavage of RsiV and revealed RsiV directly binds to lysozyme. We determined the cleavage site of the site-1 protease using N-terminal sequencing and demonstrate that disruption of site-1 cleavage blocks σ V activation. Site-1 cleavage occurs at what appears to be a signal peptide cleavage site. We demonstrate that four out of the five signal peptidases from B. subtilis are able to cleave RsiV at site-1 in vitro only in the presence of lysozyme. Additionally, we show that the extracellular domain of RsiV directly binds the inducing substrate lysozyme. v In Chapter 4 we focus on determining if the interaction between RsiV and lysozyme is necessary for σ V activation. Based on the co-crystal structure of RsiV and lysozyme we mutated sveral residues predicted to be involved in binding. One combination of RsiV mutations (S169W, P259A, Y261A) was unable to activate σ V and subsequently was unable to bind lysozyme. We propose a RIP dependent mechanism of σ V activation that is contingent upon the anti-σ factor (RsiV) directly binding the inducing signal (lysozyme) to present the site-1 cleavage site to signal peptidase. The co-crystal structure of RsiV and lysozyme revealed that RsiV interacts with the active site of lysozyme. We demonstrate that purified RsiV inhibits lysozyme activity suggesting RsiV provides an additional lysozyme response mechanism. Thus, the anti-σ factor RsiV senses the presence of lysozyme, activates σ V , and protects against further lysozyme damage. vi

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“…The Eep gene is not directly involved in quorum sensing but plays a crucial role in the production and maturation of specific peptide pheromones in E. faecalis [180,181]. Additionally, the extracytoplasmic function (ECF) sigma factor RsiV, the anti-sigma factor for sigV, is processed more easily by the protease encoded by Eep [182], which contributes to stress resistance. The protease is also essential for the activation of the extracytoplasmic function sigma factor SigV, which contributes to lysozyme resistance in E. faecalis [183].…”