Regulation of the Escherichia coli sigma-dependent envelope stress response

The Escherichia colisigma(E)-dependent stress response pathway controls the expression of genes encoding periplasmic folding catalysts, proteases, biosynthesis enzymes for lipid A (a component of lipopolysaccharide or LPS) and other proteins known or predicted to function in or produce components of the envelope. When E. coli is subjected to heat or other stresses that generate unfolded envelope proteins, sigma(E) activity is induced. Four key players in this signal transduction pathway have been identified: RseA, an inner membrane sigma(E) antisigma factor; RseB, a periplasmic protein that binds to the periplasmic face of RseA; and the DegS and YaeL proteases. The major point of regulation, the interaction between sigma(E) and RseA, is primarily controlled by the stability of RseA. Envelope stress promotes RseA degradation, which occurs by a proteolytic cascade initiated by DegS. There is evidence that one sigma(E)-inducing stress (OmpC overexpression) directly activates DegS to cleave RseA. Secondarily, envelope stress may relieve RseB-mediated enhancement of RseA activity. Additional levels of control upon sigma(E) activity may become evident upon further study of this stress response pathway.     

Acid stress activation of the σE stress response in Salmonella enterica serovar Typhimurium

The alternative sigma factor σ^E is activated by unfolded outer membrane proteins (OMPs) and plays an essential role in Salmonella pathogenesis. The canonical pathway of σ^E activation in response to envelope stress involves sequential proteolysis of the anti-sigma factor RseA by the PDZ proteases DegS and RseP. Here we show that σ^E in Salmonella enterica sv. Typhimurium can also be activated by acid stress. A σ^E-deficient mutant exhibits increased susceptibility to acid pH and reduced survival in an acidified phagosomal vacuole. Acid activation of σ^E-dependent gene expression is independent of the unfolded OMP signal or the DegS protease but requires processing of RseA by RseP. The RseP PDZ domain is indispensable for acid induction, suggesting that acid stress may disrupt an inhibitory interaction between RseA and the RseP PDZ domain to allow RseA proteolysis in the absence of antecedent action of DegS. These observations demonstrate a novel environmental stimulus and activation pathway for the σ^E regulon that appear to be critically important during Salmonella –host cell interactions.     

Regulation of the alternative sigma factor sigma(E) during initiation,adaptation, and shutoff of the extracytoplasmic heat shock response in Escherichia coli

The alternative sigma factor sigma(E) is activated in response to stress in the extracytoplasmic compartment of Escherichia coli. Here we show that sigma(E) activity increases upon initiation of the stress response by a shift to an elevated temperature (43 degrees C) and remains at that level for the duration of the stress. When the stress is removed by a temperature downshift, sigma(E) activity is strongly repressed and then slowly returns to levels seen in unstressed cells. We provide evidence that information about the state of the cell envelope is communicated to sigma(E) primarily through the regulated proteolysis of the inner membrane anti-sigma factor RseA, as the degradation rate of RseA is correlated with the changes in sigma(E) activity throughout the stress response. However, the relationship between sigma(E) activity and the rate of degradation of RseA is complex, indicating that other factors may cooperate with RseA and serve to fine-tune the response.     

Escherichia coli rpoS gene has an internal secondary translation initiation region

Sigma S (sigma(s)) encoded by rpoS in Escherichia coli is a stationary phase specific sigma subunit of the RNA polymerase holoenzyme. Widespread among the E. coli K12 strains is an amber mutation that prematurely terminates sigma(s). These rpoSAm mutants would be expected to show no sigma(s) activity. However, suppressor free rpoSAm mutants retain an intermediate catalase activity, a sigma S controlled function. By analyzing the sequence of the rpoS gene we hypothesize that a 277 amino acids long delta1-53 sigma(s) of about 30 kDa can be translated from an internal secondary translation initiation region (STIR, AGGGAGN11GUG) that is located downstream of the amber codon. By cloning this rpoSAm gene, following the expression, function, and N-terminal sequence of this mutant protein, we report the presence of a functional internal STIR in E. coli rpoS, from where a truncated but nevertheless functional form of sigma(s) can be synthesized.     

Distinct and overlapping binding sites of Pseudomonas aeruginosa Hfq and RsmA proteins on the non-coding RNA RsmY

In Pseudomonas aeruginosa the Rsm system is involved in regulation of quorum-sensing and virulence gene expression. Our recent studies revealed that the stability and abundance of the non-coding RNA RsmY, which antagonizes the translational regulator RsmA, is dependent on Hfq. Here, we show that Hfq and RsmA bind concurrently to RsmY. Enzymatic probing of RsmY RNA in the presence of RsmA and Hfq verified the proposed -GGA- motifs as RsmA binding sites and located Hfq binding sites in single-stranded regions adjacent to stem-loop structures, respectively. We conclude that distinct binding of Hfq and RsmA on RsmY RNA permits RsmY-mediated RsmA titration upon binding to and stabilization of RsmY RNA by Hfq. In addition, we provide evidence that Hfq sequesters RNase E cleavage sites on RsmY, which explains the previously observed dependence of RsmY RNA stability on Hfq.     

Hfq modulates the sigmaE-mediated envelope stress response and the sigma32-mediated cytoplasmic stress response in Escherichia coli

Hfq, a chaperone for small noncoding RNAs, regulates many processes in Escherichia coli, including the sigma(S)-mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters. Many of these genes are involved in amino acid uptake and biosynthesis, sugar uptake and metabolism, and cell energetics. In addition, we find altered regulation of the sigma(E)- and sigma(32)-mediated stress responses, which we analyze further. We show that cells lacking Hfq induce the sigma(E)-mediated envelope stress response and are defective in sigma(E)-mediated repression of outer membrane proteins. We also show that the sigma(32)-mediated cytoplasmic stress response is repressed in cells lacking Hfq due to increased expression of DnaK. Furthermore, we show that cells lacking Hfq are defective in the "long-term adaptation" of sigma(32) to chronic chaperone overexpression. Together, our results indicate that Hfq may play a general role in stress response regulation in E. coli.     

Dispensable PDZ domain of Escherichia coli YaeL essential protease

In Escherichia coli, adaptation to extra-cytoplasmic stress relies on sigma(E) activation to induce a rescue pathway. Under non-stressed conditions, sigma(E) is sequestered by the inner membrane protein RseA. Extra-cytoplasmic stress activates DegS-dependent cleavage of RseA, rendering RseA sensitive to further degradation by the YaeL protease. YaeL contains two motifs characteristic of a family of metallo-proteases, as well as a periplasmic PDZ domain. We report results of mutational analyses of the YaeL domains. Surprisingly, expression in a strain depleted for wild-type YaeL or YaeL variants having a 40 amino acid deletion of the PDZ domain or amino acid substitutions of conserved amino acids of the YaeL PDZ domain did not affect cell viability. The proteolytic activity against RseA of these YaeL variants became independent of DegS. These observations suggest that the YaeL PDZ domain exerts a negative control on YaeL activity. Rather than being involved in substrate recognition, the PDZ domain of YaeL is likely to act as an inhibitor of proteolytic activity.     

RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences

Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP.     

A pair of circularly permutated PDZ domains control RseP, the S2P family intramembrane protease of Escherichia coli

The sigma(E) pathway of extracytoplasmic stress responses in Escherichia coli is activated through sequential cleavages of the anti-sigma(E) protein, RseA, by membrane proteases DegS and RseP. Without the first cleavage by DegS, RseP is unable to cleave full-length RseA. We previously showed that a PDZ-like domain in the RseP periplasmic region is essential for this negative regulation of RseP. We now isolated additional deregulated RseP mutants. Many of the mutations affected a periplasmic region that is N-terminal to the previously defined PDZ domain. We expressed these regions and determined their crystal structures. Consistent with a recent prediction, our results indicate that RseP has tandem, circularly permutated PDZ domains (PDZ-N and PDZ-C). Strikingly, almost all the strong mutations have been mapped around the ligand binding cleft region in PDZ-N. These results together with those of an in vitro reaction reproducing the two-step RseA cleavage suggest that the proteolytic function of RseP is controlled by ligand binding to PDZ-N.     

YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA

sigmaE is an alternative sigma factor involved in a pathway of extracytoplasmic stress responses in Escherichia coli. Under normal growth conditions, sigmaE activity is down-regulated by the membrane-bound anti-sigmaE protein, RseA. Extracytoplasmic stress signals induce degradation of RseA by two successive proteolytic events: DegS-catalyzed first cleavage at a periplasmic site followed by YaeL-mediated second proteolysis at an intramembrane region. Normally, the second reaction (site-2 proteolysis) only occurs after the first cleavage (site-1 cleavage). Here, we show that YaeL variants with the periplasmic PDZ domain deleted or mutated allows unregulated cleavage of RseA and consequent sigmaE activation. It was also found that a glutamine-rich region in the periplasmic domain of RseA was required for the avoidance of the YaeL-mediated proteolysis in the absence of site-1 cleavage. These results indicate that multiple negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine-rich region) determine the strict dependence of the site-2 proteolysis on the site-1 cleavage.