Bacterial sensing: A putative amphipathic helix in RsiV is the switch for activating σV in response to lysozyme

Abstract

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 σV responds to lysozyme. In the absence of lysozyme, σV 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 σV 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 σV 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 σV.

Activation of the extracytoplasmic function σ factor σV by lysozyme

“…Lewerke et al determined that the region surrounding the signal peptidase cleavage site contains two predicted amphipathic helices separated by a turn (Lewerke et al , ). Disruption of these helices either by deletion or introduction of a charged residue on the hydrophobic face of the amphipathic helix leads to constitutive RsiV degradation and σ V activation (Lewerke et al , ). Thus, in the absence of lysozyme, the amphipathic helices protect RsiV from cleavage by signal peptidase.…”

“…To date, the amphipathic helices in RsiV have not been observed by X‐ray crystallography. However, the presence of the amphipathic helices is supported by substituted cysteine scanning mutagenesis and labeling experiments (Lewerke et al , ). In these experiments, cysteines were placed on either the hydrophobic or hydrophilic surface of the amphipathic helix and then labeled with a membrane impermeable reagent Na‐(3‐maleimidylpropionyl) biocytin.…”

“…However, in this structure most of the residues of the amphipathic helices were unstructured (Hastie et al , ). Interestingly although the cysteine labeling experiments suggest that Ile80 is part of the amphipathic helix, since it can only be labeled in the presence of lysozyme, in the RsiV–hen egg white lysozyme structure it forms part of a β‐sheet (Hastie et al , ; Lewerke et al , ). This suggests a possible mechanism by which RsiV becomes sensitive to signal peptidase in the presence of lysozyme.…”

Defining the regulon of genes controlled by σE, a key regulator of the cell envelope stress response in Streptomyces coelicolor

“…In Bacillus subtilis , four two‐component systems (LiaRS, BceRS, YvcPQ, YxdJK) and at least four of its seven ECF σ factors, σ M , σ X , σ V , σ W , have roles in the response to cell envelope stress (Jordan et al , ; Hastie et al , ; ; Lewerke et al , ). For example, BceRS is strongly induced by bacitricin and is involved in bacitracin detoxification (Mascher et al , ).…”

Co-evolutionary analysis reveals a conserved dual binding interface between extracytoplasmic function (ECF) σ factors and class I anti-σ factors

“…If the Zn-binding motif does not play an active sensory role, the general notion is that the ASDI domains have associated with additional protein domains that allow stimulus perception and ultimately trigger anti-σ factor release (24, 25). To assess the conservation of additional protein domains, usually located C-terminal of the ASDI-domain, we scanned full-length class I anti-σ factors with Pfam 31.0 models (26) as well as the extended model of the ASDI domain.…”