Adenylate Cyclase Mutations Rescue the degP Temperature-Sensitive Phenotype and Induce the Sigma E and Cpx Extracytoplasmic Stress Regulons in Escherichia coli

Abstract

Inactivation of the gene encoding the periplasmic protease DegP confers a high-temperature-sensitive phenotype in Escherichia coli. We have previously demonstrated that a degP mutant of E. coli strain CBM (W3110 pldA1) is not temperature sensitive and showed that this was most likely due to constitutive activation of the sigma E and Cpx extracytoplasmic stress regulons in the parent strain. In this study, further characterization of this strain revealed a previously unknown cryptic mutation that rescued the degP temperaturesensitive phenotype by inducing the extracytoplasmic stress regulons. We identified the cryptic mutation as an 11-bp deletion of nucleotides 1884 to 1894 of the adenylate cyclase-encoding cyaA gene (cyaA⌬11). The mechanism in which cyaA⌬11 induces the sigma E and Cpx regulons involves decreased activity of the mutant adenylate cyclase. Addition of exogenous cyclic AMP (cAMP) to the growth medium of a cyaA⌬11 mutant strain that contains a Cpx-and sigma E-inducible degP-lacZ reporter fusion decreased ␤-galactosidase expression to levels observed in a cyaA ؉ strain. We also found that a cyaA null mutant displayed even higher levels of extracytoplasmic stress regulon activation compared to a cyaA⌬11 mutant. Thus, we conclude that the lowered concentration of cAMP in cyaA mutants induces both sigma E and Cpx extracytoplasmic stress regulons and thereby rescues the degP temperature-sensitive phenotype.Gram-negative bacteria such as Escherichia coli have developed a set of response regulons designed to maintain cell viability under a variety of stressful environmental conditions. Under conditions that increase the amount of misfolded or aggregated proteins in the periplasm, such as those generated under conditions of heat shock or the overproduction of outer membrane porins, a pair of regulons known as sigma E and Cpx are induced. These regulons prevent cell death by inducing the expression of genes that encode chaperones and proteases that function to refold or degrade misfolded proteins in the periplasm (36).The sigma E extracytoplasmic stress response regulon initially described by Erickson and Gross (16) utilizes a regulated intramembrane proteolytic pathway (5) to transduce a stress signal from the periplasm to the cytoplasm, whereupon genes in the sigma E regulon are induced. A cascade of events initiating with detection of aberrant proteins in the periplasm results in the release of sigma E normally sequestered by the inner-membrane-spanning protein RseA to the cytoplasm. Release allows members of the sigma E regulon to be upregulated, including; rpoE, rseA, rseB, and rseC (14). Other sigma E-controlled genes include degP, which encodes the protease/ chaperone DegP (46); fkpA, which encodes the peptidyl/prolyl isomerase FkpA (31); rpoH, which encodes H of the cytoplasmic heat shock response regulon (16); and many others involved in performing basic cellular functions such as metabolism and phospholipid biosynthesis (37).The Cpx regulon is a three-component regulatory system composed of an inne...

Sigma E controls biogenesis of the antisense RNA MicA

“…The observation that a Δ cyaA strain displayed high levels of MicA even in exponential phase (Figure 2) was initially puzzling. Recently, however, it was found that the absence of cAMP results in strong induction of σ E activity (31); the mechanism by which this occurs is as yet unknown. Since the MicA-inducing stresses (in Figure 1) as well as stationary-phase growth have previously been associated with an increase in synthesis and/or activity of the ECF σ E (16,32), this factor is strongly suggested to be involved in micA regulation.…”

Activation of the σ E -Dependent Stress Pathway by Conjugative TraR May Anticipate Conjugational Stress

“…Thermotolerance was examined using a modified protocol from Strozen et al (24). Cultures were diluted 1:1,000 and grown at 30°C in LB for 1 h. The cultures then were aliquoted into a 96-well microtiter plate (Costar) and incubated at 42°C using an Optima microplate reader (BMG Scientific).…”

Influence of the Cpx Extracytoplasmic-Stress-Responsive Pathway on Yersinia sp.-Eukaryotic Cell Contact

“…When phosphorylated by CpxA, CpxR-P acts as a transcriptional factor to activate or repress Ͼ100 gene targets in Escherichia coli (17). Several inducing cues are known, including elevated pH (13,53); adenylate cyclase mutations (79); depletion of phosphatidylethanolamine (48) or outer membrane phospholipase A (PldA) (42); and the accumulation of enterobacterial common antigen lipid II biosynthetic intermediate (14), assembly intermediates of pili (41), type IV secretion systems (90), and the outer membrane lipoprotein NlpE (78). All of these characteristics were seen with the Cpx system of E. coli, and they probably cause inappropriate protein folding in the periplasm.…”