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. σV 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, σV 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 σV. 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 σV 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.