Signal Peptidase Is Necessary and Sufficient for Site 1 Cleavage of RsiV in Bacillus subtilis in Response to Lysozyme

Abstract

Extracytoplasmic function (ECF) σ factors are a diverse family of alternative σ factors that allow bacteria to sense and respond to changes in the environment. σ^V is an ECF σ factor found primarily in low-GC Gram-positive bacteria and is required for lysozyme resistance in several opportunistic pathogens. In the absence of lysozyme, σ^V is inhibited by the anti-σ factor RsiV. In response to lysozyme, RsiV is degraded via the process of regulated intramembrane proteolysis (RIP). RIP is initiated by cleavage of RsiV at site 1, which allows the intramembrane protease RasP to cleave RsiV within the transmembrane domain at site 2 and leads to activation of σ^V Previous work suggested that RsiV is cleaved by signal peptidase at site 1. Here we demonstrate in vitro that signal peptidase is sufficient for cleavage of RsiV only in the presence of lysozyme and provide evidence that multiple Bacillus subtilis signal peptidases can cleave RsiV in vitro This cleavage is dependent upon the concentration of lysozyme, consistent with previous work that showed that binding to RsiV was required for σ^V activation. We also show that signal peptidase activity is required for site 1 cleavage of RsiV in vivo Thus, we demonstrate that signal peptidase is the site 1 protease for RsiV.IMPORTANCE Extracytoplasmic function (ECF) σ factors are a diverse family of alternative σ factors that respond to extracellular signals. The ECF σ factor σ^V is present in many low-GC Gram-positive bacteria and induces resistance to lysozyme, a component of the innate immune system. The anti-σ factor RsiV inhibits σ^V activity in the absence of lysozyme. Lysozyme binds RsiV, which initiates a proteolytic cascade leading to destruction of RsiV and activation of σ^V This proteolytic cascade is initiated by signal peptidase, a component of the general secretory system. We show that signal peptidase is necessary and sufficient for cleavage of RsiV at site 1 in the presence of lysozyme. This report describes a role for signal peptidase in controlling gene expression.

Keywords: cell envelope; regulation of gene expression; sigma factors; signal transduction; stress response; σ factors.