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The Bacillus subtilis s W regulon is induced by different stresses that most probably affect integrity of the cell envelope. The activity of the extracytoplasmic function (ECF) sigma factor s W is modulated by the transmembrane anti-sigma factor RsiW, which undergoes stress-induced degradation in a process known as regulated intramembrane proteolysis, finally resulting in the release of s W and the transcription of s W -controlled genes. Mutations in the ecsA gene, which encodes an ATP binding cassette (ABC) of an ABC transporter of unknown function, block site-2 proteolysis of RsiW by the intramembrane cleaving protease RasP (YluC). In addition, degradation of the cell division protein FtsL, which represents a second RasP substrate, is blocked in an ecsA-negative strain. The defect in s W induction of an ecsA-knockout strain could be partly suppressed by overproducing RasP. A B. subtilis rasP-knockout strain displayed the same pleiotropic phenotype as an ecsA knockout, namely defects in processing a-amylase, in competence development, and in formation of multicellular structures known as biofilms. INTRODUCTIONGenes of Bacillus subtilis controlled by the alternative extracytoplasmic function (ECF) sigma factor s W constitute an antibiosis regulon (Helmann, 2002(Helmann, , 2006 that responds to a variety of envelope stresses such as certain antibiotics (Cao et al., 2002) and antimicrobial peptides (Pietiäinen et al., 2005;Butcher & Helmann, 2006), and also alkaline shock and phage infection . Its activity is modulated by RsiW, a transmembrane anti-sigma factor that sequesters and inactivates s W . Upon a stress signal, RsiW is degraded by the mechanism of regulated intramembrane proteolysis (RIP). In a concerted action, at least three proteases in three different compartments of the cell degrade the RsiW anti-sigma factor in a sequential manner, finally resulting in the release of s W and the transcription of s Wcontrolled genes. The first protease that has been identified to be involved in that process is RasP (regulating anti-sigma factor protease; formerly YluC). RasP belongs to the group of zinc-dependent intramembrane cleaving proteases (iClips) and cleaves RsiW in its transmembrane domain after the extracytoplasmic part of the anti-sigma factor has been removed by a site-1 protease (Schöbel et al., 2004). The site-2 degradation step catalysed by RasP uncovers a cryptic proteolytic tag that ensures further degradation of the RsiW remnant by cytoplasmic proteases, mainly ClpXP (Zellmeier et al., 2006). These two steps are related to RIP of the s E anti-sigma factor RseA of Escherichia coli (Ades, 2004;Alba & Gross, 2004). RasP is an orthologue of E. coli RseP, and the concept of a cryptic proteolytic tag recognized by ClpXP was first described for that system (Akiyama et al., 2004;Flynn et al., 2004). However, there is a marked difference in the site-1 proteolytic step, because no dependency on B. subtilis Deg (Htr) proteases was found and the inducing stress is apparently different. The probable site-1 prot...

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The Dlt system modulates the density of negative charge in the cell wall of Gram-positive bacteria by substituting anionic polymers (wall and lipoteichoic acids) with D-alanine. The htrA and htrB genes, regulated by the CssRS two-component system (TCS) and encoding membrane-associated protein quality control proteases, were expressed at strongly decreased levels in a mutant with defective Dlt (dltD : : miniTn10) as compared to the dlt + wild-type strain under a secretion stress condition (hypersecretion of AmyQ a-amylase). The level of HtrA protein in the extracellular proteome of the dltD mutant was decreased consistently. Expression from the promoter of the liaIHGFSR (yvqIHGFEC) operon (P liaI ) is dependent on the LiaRS TCS. The Dlt defect increased the expression from P liaI under two stress conditions, AmyQ hypersecretion and treatment with a cationic antimicrobial peptide (LL-37), but decreased the expression in vancomycin-treated cells. Furthermore, Dlt inactivation enhanced the expression of the YxdJK-regulated yxdL gene in LL-37-treated cells. The increased net negative charge of the cell wall seems to cause varied and opposite effects on the expression of CssRS-, LiaRS-and YxdJK-regulated genes under different stress conditions. The results suggest that TCSs which sense misfolded proteins or peptides are modulated by the density of negative charge in the cell wall. The density of negative charge on the outer surface of the cell membrane did not have a similar effect on TCSs.