Sensing Extracellular Stress: Key Features of the Anti-Sigma Factor RsiV in Sensing Lysozyme

Bacillus subtilis is a soil dwelling organism which uses alternative extracytoplasmic function (ECF) sigma factors that respond to environmental stressors encountered by a cell. Upon encountering a signal, ECF sigma factors are activated and bind RNAP to initiate transcription of genes that confer resistance to stressors that threaten cellular integrity [5]. One such factor, sigmaV (s^V), is required by B. subtilis for resistance to lysozyme. In the absence of lysozyme, s^V is inhibited by the anti-sigma factor, RsiV, which is a transmembrane protein that has an extracellular lysozyme sensing domain. RsiV binds lysozyme, allowing for regulated intramembrane proteolysis (RIP) of the anti-sigma factor. The rate limiting step in degradation of RsiV is site-1 cleavage, which is performed by signal peptidase. Once RsiV is degraded it releases s^V,allowing it to bind RNAP and transcribe lysozyme resistance genes. We sought to understand how RsiV avoids signal peptidase cleavage in the absence of lysozyme. To investigate this, we fused various lengths of RsiV to GFP in order to identify portions of RsiV needed to shield it from signal peptidase cleavage in the absence of lysozyme. We also defined the transmembrane domain of RsiV using the substituted cysteine accessibility method (SCAM). These experiments contribute to our investigation of a putative amphipathic helix with protective properties from signal peptidase.