A suppressor of cell death caused by the loss of sigmaE downregulates extracytoplasmic stress responses and outer membrane vesicle production in Escherichia coli

Abstract

When envelope biogenesis is compromised or damage to envelope components occurs, bacteria trigger signaling cascades, which lead to the production of proteins that combat such extracytoplasmic stresses. In Escherichia coli, there are three pathways known to deal with envelope stresses: the Bae, Cpx, and sigma(E) responses. Although the effectors of the Bae and Cpx responses are not essential in E. coli, the effector of the sigma(E) response, the sigma factor RpoE (sigma(E)), is essential for viability. However, mutations that suppress the lethality of an rpoE-null allele can be easily obtained, and here we describe how we have isolated at least four classes of these suppressors. We present the first description of one such suppressor class, loss-of-function mutations in ydcQ, a gene encoding a putative DNA-binding protein. In wild-type rpoE(+) strains, ydcQ mutants have two distinct phenotypes: extracytoplasmic stress responses are significantly downregulated, and the production of outer membrane vesicles is severely reduced. We present a model in which sigma(E) is not essential per se but, rather, we propose that rpoE mutant cells die, possibly because they overreact to the absence of this sigma factor by triggering a cell death signal.

FIG. 1.

Strategy used to map suppressors of rpoE::cam lethality. A detailed description of this strategy is given in Results and Materials and Methods. The rpoE::cam P1 donor strain used was CAG22216, which carries the unidentified suppressor sup^CAG.

FIG. 2.

Disruption of ydcQ suppresses lethality of rpoE::cam and downregulates degP transcription. (A) Genetic organization of the ydcQ locus and localization of various insertion elements and antibiotic resistance markers. Arrows indicate the direction of transcription. (B) Disruption of ydcQ but not of ydcR reduces expression of degP′-lacZ⁺ and suppresses lethality of rpoE::cam. Relative levels of LacZ expression from the degP′-lacZ⁺ fusion on lactose MacConkey agar are indicated with “+” signs. If a mutation is able to suppress the lethality of rpoE::cam, a “+” is shown on the right column, but if a mutation is not able to suppress lethality, a “−” is shown.

FIG. 3.

Loss of YdcQ downregulates envelope stress responses. (A) LacZ activity of various reporter fusions (shown in the x axis) controlled by envelope stress responses in a wild-type (▪) and a ydcQ::kan (░⃞) strain. The degP′-lacZ⁺ fusion is controlled by both σ^E and Cpx, rpoHP3′-lacZ⁺ by σ^E, cpxP′-lacZ⁺ by Cpx, and spy′-lacZ⁺ by Cpx and Bae. Relative LacZ activities are shown where 100% corresponds to the LacZ activity of the wild-type strain carrying each fusion. Shown is an experiment representative of at least three independent experiments. (B) Western blotting of whole-cell samples showing reduced levels of DegP and Spy in a ydcQ::kan mutant (NR905) with respect to the wild-type (wt) MC4100 and ydcR::kan mutant (NR906) strains.

FIG. 4.

Loss of YdcQ downregulates production of outer membrane vesicles. Western blot analysis of the outer membrane proteins OmpA and LamB, periplasmic protein MBP, and outer membrane lipid LPS shows that although the loss of YdcQ function in NR905 does not alter the levels of these envelope components in whole cells, it reduces the amount of outer membrane vesicles present in culture supernatants compared to the wild-type strain MC4100.

FIG. 5.

YdcQ downregulates Cpx in a CpxR-independent manner. LacZ activity of strains carrying the Cpx-regulated fusion cpxR′-lacZ⁺. Individually, the ydcQ::kan (ydcQ) and cpxR::spec (cpxR) alleles reduce the expression of cpxR with respect to wild type (wt). Together, both mutations reduce cpxR expression even further, demonstrating that they act in different pathways. Relative LacZ activities are shown where 100% corresponds to the LacZ activity of the wild-type strain carrying each fusion. Shown is an experiment representative of at least three independent experiments. From left to right, strains used were NR986, NR987, NR989, and NR990.