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.     

degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide σE activity

DegS (HhoB), a putative serine protease related to DegP/HtrA, regulates the basal and induced activity of the essential Escherichia coli sigma factor sigma (E), which is involved in the cellular response to extracytoplasmic stress. DegS promotes the destabilization of the sigma (E)-specific anti-sigma factor RseA, thereby releasing sigma (E) to direct gene expression. We demonstrate that degS is an essential E. coli gene and show that the essential function of DegS is to provide the cell with sigma (E) activity. We also show that the putative active site of DegS is periplasmic and that DegS requires its N-terminal transmembrane domain for its sigma (E)-related function.     

Role of the response regulator RssB in σS recognition and initiation of σS proteolysis in Escherichia coli

In growing Escherichia coli cells, the master regulator of the general stress response, sigmaS (RpoS), is subject to rapid proteolysis. In response to stresses such as sudden carbon starvation, osmotic upshift or shift to acidic pH, sigmaS degradation is inhibited, sigmaS accumulates and numerous sigmaS-dependent genes with stress-protective functions are activated. sigmaS proteolysis is dependent on ClpXP protease and the response regulator RssB, whose phosphorylated form binds directly to sigmaS in vitro. Here, we show that substitutions of aspartate 58 (D58) in RssB, which result in higher sigmaS levels in vivo, produce RssB variants unable to bind sigmaS in vitro. Thus, RssB is the direct substrate recognition factor in sigmaS proteolysis, whose affinity for sigmaS depends on phosphorylation of its D58 residue. RssB does not dimerize or oligomerize upon this phosphorylation and sigmaS binding, and RssB and sigmaS exhibit a 1:1 stoichiometry in the complex. The receiver as well as the output domain of RssB are required for sigmaS binding (as shown in vivo and in vitro) and for complementation of an rssB null mutation. Thus, the N-terminal receiver domain plays an active and positive role in RssB function. Finally, we demonstrate that RssB is not co-degraded with sigmaS, i.e. RssB has a catalytic role in the initiation of sigmaS turnover. A model is presented that integrates the details of RssB-sigmaS interaction, the RssB catalytic cycle and potential stress signal input in the control of sigmaS proteolysis.     

Positive and negative feedback regulatory loops of thiol-oxidative stress response mediated by an unstable isoform of σR in actinomycetes

Alternate sigma factors provide an effective way of diversifying bacterial gene expression in response to environmental changes. In Streptomyces coelicolor where more than 65 sigma factors are predicted, σ^R is the major regulator for response to thiol-oxidative stresses. σ^R becomes available when its bound anti-sigma factor RsrA is oxidized at sensitive cysteine thiols to form disulphide bonds. σ^R regulon includes genes for itself and multiple thiol-reducing systems, which constitute positive and negative feedback loops respectively. We found that the positive amplification loop involves an isoform of σ^R (σ^R') with an N-terminal extension of 55 amino acids, produced from an upstream start codon. A major difference between constitutive σ^R and inducible σ^R' is that the latter is markedly unstable ( t _1/2 ∼ 10 min) compared with the former (> 70 min). The rapid turnover of σ^R' is partly due to induced ClpP1/P2 proteases from the σ^R regulon. This represents a novel way of elaborating positive and negative feedback loops in a control circuit. Similar phenomenon may occur in other actinomycetes that harbour multiple start codons in the sigR homologous gene. We observed that sigH gene, the sigR orthologue in Mycobacterium smegmatis , produces an unstable larger isoform of σ^H upon induction by thiol-oxidative stress.     

The PhyR-σEcfG signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum

PhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens . Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF σ factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF σ factor σ^EcfG are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and σ^EcfG control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein–protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/σ^EcfG regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium –legume interaction via yet undisclosed mechanisms.