Abstract
RseA sequesters RpoE (σE) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σE to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σE regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σE levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σE-dependent RybB::LacZ construct showed only a weak activation of the σE pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σE and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrAL222Q, it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σE-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σE-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σE may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σE levels.
Highlights
DegP is the major periplasmic protease in Escherichia coli [1,2]
Temperature resistant colonies, which arose at a frequency of around 1027, were purified at 30uC and suppressor mutants categorized based on growth robustness and outer membrane protein (OMP) levels
The DdegPmediated elevated CpxP::LacZ activity returned to the wild type level in the rpoE3 DdegP double mutant background. These results showed the complementary nature of the two envelope stress response pathways controlled by the sequesters RpoE (sE) and Cpx systems
Summary
DegP is the major periplasmic protease in Escherichia coli [1,2]. Its proteolytic activity towards outer membrane proteins (OMPs) was initially observed against a mutant LamB protein with a temperature sensitive folding defect [3]. A series of structural and biochemical studies on DegP provided important mechanistic clues [11,12,13,14] These studies showed that the binding of unfolded substrate proteins to DegP triggers its reversible oligomerization into a cage-like structure and membrane association greatly influences DegP’s assembly and activity. The expression of degP is under the control of the sE and Cpx regulons [15,16,17,18], which together constitute the two major envelope stress response pathways [19,20,21] Both pathways are required to elevate degP expression to overcome the potentially lethal envelope stress caused by aberrant OMP assembly [9]
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