Dataset Information

Suppressor Mutations in degS Overcome the Acute Temperature-Sensitive Phenotype of ?degP and ?degP ?tol-pal Mutants of Escherichia coli.

ABSTRACT: 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 DegS's protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS's 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 DegS's protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, ?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 ?tolB ?degP mutants 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 here show that DegP's 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.

SUBMITTER: Kern B

PROVIDER: S-EPMC6509649 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Suppressor Mutations in degS Overcome the Acute Temperature-Sensitive Phenotype of ΔdegP and ΔdegP Δtol-pal Mutants of Escherichia coli.

Kern Brea B Leiser Owen P OP Misra Rajeev R

Journal of bacteriology 20190508 11

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 T ...[more]

PMID: 30858298

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Project description:Salmonellae regulate membrane lipids during infection, but the exact proteins and mechanisms that promote their survival during bacteremia remain largely unknown. Mutations in genes encoding the conserved Salmonella enterica serovar Typhimurium (S Typhimurium) Tol-Pal apparatus caused the outer membrane (OM) sensor lipoprotein, RcsF, to become activated. The capsule activation phenotype for the mutants suggested that Tol-Pal might influence envelope lipid homeostasis. The mechanism involves reducing OM glycerophospholipid (GPL) levels, since the mutant salmonellae similarly accumulated phosphatidylglycerols (PGl) and phosphatidylethanolamines (PE) within the OM in comparison to the wild type. The data support the Escherichia coli model, whereby Tol-Pal directs retrograde GPL translocation across the periplasm. The S Typhimurium mechanism involves contributions from YbgC, a cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase, and CpoB, a periplasmic TolA-binding protein. The functional relationship between Tol-Pal and YbgC and CpoB was previously unresolved. The S Typhimurium Tol-Pal proteins contribute similarly toward promoting OM-GPL homeostasis and Rcs signaling inactivity but differently toward promoting bacterial morphology, rifampin resistance, survival in macrophages, and survival in mice. For example, tolQ, tolR, tolA, and cpoB mutants were significantly more attenuated than ybgC, tolB, and pal mutants in a systemic mouse model of disease. Therefore, key roles exist for TolQ, TolR, TolA, and CpoB during murine bacteremia, which are independent of maintaining GPL homeostasis. The ability of TolQR to channel protons across the inner membrane (IM) is necessary for S Typhimurium TolQRA function, since mutating conserved channel-facing residues rendered TolQ ineffective at rescuing deletion mutant phenotypes. Therefore, Tol-Pal promotes S Typhimurium survival during bacteremia, in part, by reducing OM GPL concentrations, while TolQRA and CpoB enhance systemic virulence by additional mechanisms.

Project description:Chemoreceptors are localized at the cell poles of Escherichia coli and other rod-shaped bacteria. Over the years, different mechanisms have been put forward to explain this polar localization, including stochastic clustering, membrane curvature-driven localization, interactions with the Tol-Pal complex, and nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at the cell poles. Interestingly, Tar still accumulated at midcell in tol and in pal deletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterized by strong membrane curvature. Chemoreceptors, like Tar, form trimers of dimers that bend the cell membrane due to a rigid tripod structure. The curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favorable, as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or the cell poles. These findings favor a model where chemoreceptor localization in E. coli is driven by strong membrane curvature and association with the Tol-Pal complex.IMPORTANCE Bacteria have exquisite mechanisms to sense and adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar in E. coli and found that membrane curvature at cell division sites and the Tol-Pal protein complex localize Tar at cell division sites, the future cell poles. This study shows how membrane curvature can guide localization of proteins in a cell.

Dataset Information

Suppressor Mutations in degS Overcome the Acute Temperature-Sensitive Phenotype of ?degP and ?degP ?tol-pal Mutants of Escherichia coli.

Publications

Suppressor Mutations in <i>degS</i> Overcome the Acute Temperature-Sensitive Phenotype of Δ<i>degP</i> and Δ<i>degP</i> Δ<i>tol-pal</i> Mutants of Escherichia coli.

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