Suppressor mutations in degS overcome the acute temperature-sensitive phenotype of ΔdegP and ΔdegP Δtol-pal mutants of Escherichia coli

In Escherichia coli , the periplasmic protease DegP plays a critical role in degrading misfolded outer membrane proteins (OMPs). Consequently, mutants lacking DegP display a temperature-sensitive growth defect, presumably due to the toxic accumulation of misfolded OMPs. The Tol-Pal complex plays a poorly defined but an important role in envelope biogenesis, since mutants defective in this complex display a classical periplasmic leakage phenotype. Double mutants lacking DegP and an intact Tol-Pal complex display exaggerated temperature-sensitive growth defects and the leaky phenotype. Two revertants that overcome the temperature-sensitive growth phenotype carry missense mutations in the degS gene, resulting in D102V and D320A substitutions. D320 and E317 of the PDZ domain of DegS make salt bridges with R178 of the DegS9 protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS9 basal protease activity. Although the D102V substitution is as effective as D320A in suppressing the temperature-sensitive growth phenotype, the molecular mechanism behind its effect on DegS9 protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, σ E -mediated envelope stress response pathway and elevate periplasmic protease activity to restore envelope homeostasis. Based on the release of a cytoplasmic enzyme in the culture supernatant, we conclude that the conditional lethal phenotype of Δ tolB Δ degP stems from a grossly destabilized envelope structure that causes excessive cell lysis. Together, the data point to a critical role for periplasmic proteases when the Tol-Pal complex-mediated envelope structure and/or functions are compromised. IMPORTANCE The Tol-Pal complex plays a poorly defined role in envelope biogenesis. The data presented in this study show that DegP9s periplasmic protease activity becomes crucial in mutants lacking the intact Tol-Pal complex, but this requirement can be circumvented by suppressor mutations that activate the basal protease activity of a regulatory protease, DegS. These observations point to a critical role for periplasmic proteases when Tol-Pal-mediated envelope structure and/or functions are perturbed.