Our major research project is based upon results we obtained from global transcriptional analysis of alkaline stressed B. subtilis cells. Using DNA-macroarrays, we defined the B. subtilis alkali stress stimulon and could show that a major part of genes induced at least four-fold after alkaline shock is member of the Sigma-W regulon. The alternative sigma factor Sigma-W belongs to the group of ECF sigma factors which control genes of extracytoplasmic function, and alkaline stress was the first stimulus described to induce the Sigma-W regulon, allowing us to identify additional Sigma-W-controlled genes and to define the promoter consensus sequence in more detail (Wiegert et al., 2001). Later it was proposed that Sigma-W constitutes an antibiosis regulon, reacting on defects in cell wall biosynthesis (Helmann, 2002), suggesting that an elevated extracellular pH imposes envelope stress to B. subtilis cells. We decided to further analyse the mechanism of Sigma-W induction, because we also came across Sigma-W when we tried to identify substrates of the B. subtilis FtsH protease (Zellmeier et al., 2003). We could show that Sigma-W is sequestered by the cytoplasmic part of a transmembrane protein which acts as an anti-sigma factor (RsiW), abolishing interaction of Sigma-W with the RNA polymerase core enzyme and transcription of Sigma-W–controlled genes (Schöbel et al., 2004). RsiW belongs to a new family of zinc-binding anti-sigma factors. Sigma-W is released in a process of regulated proteolysis of RsiW. Alkaline shock or addition of antibiotics like vancomycin activate a protease by an unknown mechanism, removing most of the extracytoplasmic part of RsiW. The truncated RsiW created in this site-1 proteolyic step is subjected to site-2 proteolysis. The site-2 protease was identified as RasP (regulating alternative sigma factor protease, formerly YluC), which belongs to the group of iClips (intramembrane cleaving proteases). Therefore, the Sigma-W / RsiW system represents, besides Sigma-E / RseA from Escherichia coli (Alba and Gross, 2004), the second bacterial sigma factor / anti-sigma factor system modulated through regulated intramembrane proteolysis (RIP). Cleavage of the transmembrane part of RsiW by RasP uncovers a cryptic proteolytic tag that is recognized mainly by the cytoplasmic ClpXP protease, which is important to completely degrade RsiW (Zellmeier et al., 2006). Most recently, Ellermeier and Losick (2006) and our group (Heinrich and Wiegert, 2006) independently identified the ypdC gene being involved in site-1 proteolysis of RsiW. YpdC was renamed PrsW (protease responsible for activating Sigma-W).

Fig.1 Factors involved in proteolytic degradation of the B. subtilis Sigma-W anti-sigma factor RsiW. RsiW sequesters and inactivates Sigma-W. PrsW: probable Site-1 protease. RasP: Site-2 protease.

Fig.2 Model for the stress induced proteolytic cleavage of the B. subtilis Sigma-W anti-sigma factor RsiW. Alkaline shock imposes a kind of cell wall stress that activates PrsW to remove most of the extracytoplasmic part of RsiW (Site-1). The truncated RsiW created in this site-1 proteolytic step is substrate for RasP in site-2 proteolysis, where the transmembrane part of RsiW is clipped (Site-2). The ClpXP protease completely degrades truncated RsiW and as a consequence Sigma-W is released to interact with the RNA polymerase core enzyme and Sigma-W-controlled promoters are transcribed.

ECF sigma factors seem to be of crucial importance for prokaryotes in the adaptation to changing environmental conditions. For Streptomyces coelicolor and a Bacteroides strain living in the human gut more than 50 ECF sigma factors have been discovered. The function and regulation of ECF sigma factors is mostly unknown, some publications denote a role in cell wall stress response, antibiosis and pathogenesis. Our data on the Sigma-W / RsiW system of sigma factor /anti-sigma factor point to a general mechanism to modulate the activity of ECF anti-sigma factors in bacteria through regulated intramembrane proteolysis. The process of regulated intramembrane proteolysis has been discovered first in eukaryotes and is of major interest, because it is for example involved in the formation of amyloid plaques in Alzheimer's disease. The Sigma-W / RsiW system is an excellent model to investigate regulated intramembrane proteolysis and to analyse its relevance in prokaryotic regulation of gene expression. We therefore want to decipher all steps and components which are involved in degrading RsiW. Furthermore we will examine a possible role of the RasP intramembrane cleaving protease in other regulatory processes, for example in the activation of the remaining six ECF sigma factors of B. subtilis.

Relevant publications Alba, B.M., and[...].A. (2004) Regulation of the Escherichia coli Sigma-W -dependent envelope stress response. Mol Microbiol 52, 613-619. Ellermeier, C.D[...] R. (2006) Evidence for a novel protease governing regulated intramembrane proteolysis and resistance to antimicrobial peptides in Bacillus subtilis. Genes Dev 20. 1911-22. Heinrich, J., a[...] T. (2006). YpdC determines site-1 degradation in regulated intramembrane proteolysis of the RsiW anti-sigma factor of Bacillus subtilis. Mol Microbiol 62, 566-579. Heinrich, J., L[...] T. (2008) The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP. Microbiology 154, 1989-1997 Helmann, J. D. (2002). The extracytoplasmic function (ECF) sigma factors. Adv Microb Phys 46, 47-110. Schöbel, S[...] T. (2004). The Bacillus subtilis SigmaW anti-sigma factor RsiW is degraded by intramembrane proteolysis through YluC. Mol Microbiol 52, 1091-1105. Wiegert, T., Ho[...] W. (2001). Alkaline shock induces the Bacillus subtilis SigmaW regulon. Mol Microbiol 41, 59-71. Zellmeier, S., [...] T. (2006). Involvement of Clp protease activity in modulating the Bacillus subtilis SigmaW stress response. Mol Microbiol 61, 1569-1582.