Use of cell wall stress to characterize σ22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa

MucA sequesters extracytoplasmic function (ECF) σ²² ( algT/U encoded) from target promoters including P algD for alginate biosynthesis. We have shown that cell wall stress (e.g. d -cycloserine) is a potent inducer of the algD operon. Here we showed that MucB, encoded by the algT-mucABCD operon, interacts with MucA in the sigma–sequestration complex. We hypothesized that AlgW protease (a DegS homologue) is activated by cell wall stress to cleave MucA and release σ²². When strain PAO1 was exposed to d -cycloserine, MucA was degraded within just 10 min, and σ²² was activated. However, in an algW mutant, MucA was stable with no increased σ²² activity. Studies on a yaeL mutant, defective in an RseP/YaeL homologue, suggest that YaeL protease cleaves MucA only after cleavage by AlgW. A defect in mucD , encoding a periplasmic HtrA/DegP homologue, caused MucA instability, suggesting MucD degrades cell wall stress signals. Overall, these data indicate that cell wall stress signals release σ²² by regulated intramembrane proteolysis (RIP). Microarray analyses identified genes of the early and late cell wall stress stimulon, which included genes for alginate production. The subset of genes in the σ²² regulon was then determined, which included gene products predicted to contribute to recovery from cell wall stress.     

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.     

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.     

Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of σ32in vivo

The heat shock response of Escherichia coli is regulated by the cellular level and the activity of σ³², an alternative sigma factor for heat shock promoters. FtsH, a membrane-bound AAA-type metalloprotease, degrades σ³² and has a central role in the control of the σ³² level. The ftsH null mutant was isolated, and establishment of the Δ ftsH mutant allowed us to investigate control mechanisms of the stability and the activity of σ³² separately in vivo . Loss of the FtsH function caused marked stabilization and consequent accumulation of σ³² (≈20-fold of the wild type), leading to the impaired downregulation of the level of σ³². Surprisingly, however, Δ ftsH cells express heat shock proteins only two- to threefold higher than wild-type cells, and they also show almost normal heat shock response upon temperature upshift. These results indicate the presence of a control mechanism that downregulates the activity of σ³² when it is accumulated. Overproduction of DnaK/J reduces the activity of σ³² in Δ ftsH cells without any detectable changes in the level of σ³², indicating that the DnaK chaperone system is responsible for the activity control of σ³² in vivo . In addition, CbpA, an analogue of DnaJ, was demonstrated to have overlapping functions with DnaJ in both the activity and the stability control of σ³².     

Functional and Binding Properties of σ Receptors in Rat Cerebellum

Abstract: Autoradiographic studies have shown that σ receptors are enriched in the locus coeruleus, the origin of noradrenergic projections to the cerebellum, as well as in the Purkinje, molecular, and granular layers and the interpositus cerebellar nucleus of the cerebellum itself. In contrast, the cerebellum is relatively poor in phencyclidine (PCP) binding sites, which have been historically confused with σ sites. The high ratio of σ to PCP receptors in cerebellum is advantageous for discriminating σ-mediated physiological effects. σ agonists and antagonists have been shown to regulate N -methyl- d -aspartate (NMDA)-stimulated norepinephrine release in hippocampus, which is innervated by locus coeruleus projections. We now report that σ drugs also regulate norepinephrine release from cerebellum. In contrast to findings in the hippocampus, where regulation is via σ₁ and σ₂ receptors, σ-mediated regulation in cerebellum seems to be primarily via σ₁ receptors. In radioligand binding studies, we find that σ receptors primarily of the σ₁ type are present in the cerebellum. We further report that binding to σ receptors in cerebellum is not affected by the addition of NMDA or glycine or by the presence of NMDA antagonists, suggesting that σ receptors are not located within the NMDA-operated cation channel in this brain region.