Involvement of Clp protease activity in modulating the Bacillus subtilisσ^W stress response

The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP

“…3a, compare lanes 3 and 9). GFP-RsiWD1, which is absent in the wild-type due to proteolysis by RasP and further cytoplasmic proteases (Zellmeier et al, 2006), was stabilized in the ecsA : : spec background (Fig. 3a, lanes 6 and 12).…”

“…To test for stability of FtsL, a fusion of GFP to the amino terminus of FtsL was constructed, essentially as described previously (Zellmeier et al, 2006). The ftsL coding region was PCR-amplified using primers (12) 59-GGCCATAGATCTATGAGCAATTTAGCTTACCAACCAGAG-39 and (13) 59-GGCCATAGATCTAGCGCTTCATTCCTGTATGTTT-TTCACTTT-39 and chromosomal DNA of B. subtilis 1012 as a template, and the product was restricted with BglII and ligated into plasmid pTW700 cut with the same enzyme.…”

“…Therefore, the ecsA : : spec mutation was introduced into strains expressing GFPRsiW and a GFP-RsiWD1 reporter, respectively. GFPRsiWD1 is a constitutive substrate for RasP and not dependent on site-1 proteolysis for degradation (Zellmeier et al, 2006). Colonies of both strains were highly fluorescent, whereas in the ecsA wild-type background Fig.…”

“…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).…”

“…b-Galactosidase activities and a-amylase activities of culture supernatants were measured as described previously Pummi et al, 2002). Northern blot analysis was performed according to a published procedure (Homuth et al, 1997) with antisense RNAs against yuaG, pbpE and yhaU (Zellmeier et al, 2006). Western blots were performed as described previously (Homuth et al, 1996) using a semi-dry blotting procedure (Bio-Rad, Trans Blot SD).…”