The induction of Bacillus subtilis genes controlled by the extracytoplasmic function alternative sigma factor sigmaW is strongly impaired in a strain deleted for the ClpP peptidase gene and in a double knockout of the ClpX and ClpE ATPase genes. Truncated soluble forms of the sigmaW anti-sigma factor RsiW are stabilized in a clpP minus strain as revealed by the green fluorescent reporter protein fused to the N-terminus of RsiW and by pulse-chase experiments. Conserved alanine residues are present in the transmembrane region of RsiW, and mutations in these positions abolish induction of sigmaW-controlled genes. Following alkaline shock, a truncated cytoplasmic form of RsiW is detectable in a strain expressing a triple alanine mutant allele of rsiW. These data point to a mechanism where the trans-membrane segment of RsiW contains a cryptic proteolytic tag that is uncovered as a result of intramembrane proteolysis of RsiW by RasP (YluC). After RasP-clipped RsiW is detached from the membrane, this proteolytic tag becomes crucial for the complete degradation of RsiW by cytoplasmic proteases and the release of sigmaW. ClpXP plays a major role in this third proteolytic step of stress-induced degradation of RsiW. Overexpression of SsrA-tagged green fluorescent protein as a ClpXP substrate protein reduces alkali induction of a sigmaW-controlled gene by a factor of about three, indicating that a titration mechanism is able to tune the sigmaW-mediated stress response to the cellular state.
Adenosine Triphosphatases Alanine