Reversal of the Δ<em>degP</em> Phenotypes by a Novel <em>rpoE</em> Allele of <em>Escherichia coli</em>

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.当包膜应激处于低水平时，RseA会将RpoE（σE）束缚在大肠杆菌（Escherichia coli）的内膜上。升高的包膜应激会触发两种膜蛋白酶DegS和RseP依次切割RseA，释放σE以激活降低包膜应激的通路。 一株因高包膜应激而无法在37℃生长的ΔdegP ΔbamB突变株的回复突变株，其rseA和rpoE基因中存在突变。rseA基因的无效突变与错义突变组成型过度激活了σE调节子（regulon），并显著降低了主要外膜蛋白（outer membrane protein, OMP）的水平。 与之相反，一种由rpoE基因部分重复产生的新型rpoE等位基因rpoE3，其σE水平的升高幅度大于rseA突变背景下的情况，但并未降低OMP水平。依赖σE的RybB::LacZ融合构建体仅显示出rpoE3对σE通路的微弱激活作用。尽管如此，rpoE3完全逆转了ΔdegP突变株的生长缺陷与包膜囊泡化表型。 有趣的是，rpoE3还将ΔdegP菌株中适度激活的Cpx包膜应激通路下调至野生型水平，这表明σE通路与Cpx通路具有互补性。 通过使用不稳定的突变周质蛋白AcrAL222Q，本研究证实rpoE3突变可部分通过激活σE依赖的蛋白水解通路来克服ΔdegP的表型。我们的数据表明，OMP水平的降低并非σE介导的降低包膜应激机制的固有特征。 研究结果还提示，在极端包膜应激条件下，RseA与σE之间的紧密稳态环路可能部分参与了细胞死亡，而这类环路可通过降低RseA活性或升高σE水平的突变被打破。