Extra Cytoplasmic Function (ECF) σ factors are a diverse group of alternate σ factors bacteria use to respond to changes in the environment. The Bacillus subtilis ECF σ factor σ V responds to lysozyme. In the absence of lysozyme, σ V is held inactive by the anti-σ factor, RsiV. In the presence of lysozyme RsiV is degraded via regulated intramembrane proteolysis, which results in the release of σ V and thus activation of lysozyme resistance genes. Signal peptidase is required to initiate degradation of RsiV. Previous work indicated that RsiV only becomes sensitive to signal peptidase upon direct binding to lysozyme. We have identified a unique domain of RsiV that is responsible for protecting RsiV from cleavage by signal peptidase in the absence of lysozyme. We provide evidence that this domain contains putative amphipathic helices. Disruption of the hydrophobic surface of these helices by introducing positively charged residues results in constitutive cleavage of RsiV by signal peptidase and thus constitutive σ V activation. We provide further evidence that this domain contains amphipathic helices using a membrane-impermeable reagent. Finally, we show that upon lysozyme binding to RsiV, the hydrophobic face of the amphipathic helix becomes accessible to a membrane-impermeable reagent. Thus, we propose the amphipathic helices protect RsiV from cleavage in the absence of lysozyme. Additionally, we propose the amphipathic helices rearrange to form a suitable signal peptidase substrate upon binding of RsiV to lysozyme leading to the activation of σ V . Author summary Signal transduction involves (i) sensing a signal, (ii) a molecular switch triggering a response, and (iii) altering gene expression. For Bacillus subtilis’ response to lysozyme, we have a detailed understanding of (i) and (iii). Here we provide insights for a molecular switch that triggers the lysozyme response via σ V activation. RsiV, an inhibitor of σ V activity, is cleaved by signal peptidase only in the presence of lysozyme. Signal peptidase constitutively cleaves substrates that are translocated across the membrane. A domain-of-unknown-function (DUF4179) in RsiV contains the signal peptidase cleavage site, and protects RsiV from cleavage in the absence of lysozyme via amphipathic helices. In addition to RsiV, DUF4179 is found in an unrelated and uncharacterized anti-σ factor present in Firmicutes including within some clinically-relevant species.
【초록키워드】 Gene Expression, Bacillus subtilis, membrane, cleavage, Bacteria, molecular, Degradation, inhibitor, function, binding, Evidence, Trigger, Disruption, triggers, Activation, Factor, signal, domain, peptidase, cleavage site, hydrophobic, substrate, substrates, resistance genes, firmicutes, lysozyme, Bacillus, triggering, PROTECT, responsible, indicated, addition, required, changes in, absence, unique, regulated, respond, cleaved, inactive, charged residue, cleave, Firmicute, translocated, 【제목키워드】 activating,