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.     

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.     

Virulence and vaccine potential of Salmonella typhlmurium mutants deficient in the expression of the RpoS (σs) regulon

The alternative sigma factor RpoS (σ^s) is required for Salmonella virulence in mice. We report the immunizing capacity of Salmonella typhimurlum rpoS and rpoS aroA mutants to protect susceptible BALB/c mice against subsequent oral challenge with virulent S. typhimurium. When administered orally or intraperitoneally, rpoS derivatives of the mouse-virulent S. typhimurium strains, C52 and SL1344, were highly attenuated and were efficient single-dose live vaccines. rpoS aroA mutants were more attenuated than corresponding single aroA or rpoS mutants, as assessed after oral or intraperitoneal administration, but retained significant ability to protect mice against salmonellosis. Salmonella rpoS and rpoS aroA mutants therefore deserve serious consideration for rational vaccine design. Consistent with this, Salmonella typhi Ty2, a ‘wild-type’ strain used widely for the development of human live-vaccine candidates against typhoid fever, was shown to be defective for rpoS. In addition, our results demonstrate that rpoS not only controls the growth and persistence of S. typhimurium in deep lymphoid organs, but also plays a role during the initial stages of oral infection.     

Non-specific, general and multiple stress resistance of growth-restricted Bacillus subtilis cells by the expression of the σB regulon

Bacillus subtilis cells respond almost immediately to different stress conditions by increasing the production of general stress proteins (GSPs). The genes encoding the majority of the GSPs that are induced by heat, ethanol, salt stress or by starvation for glucose, oxygen or phosphate belong to the σ^B-dependent general stress regulon. Despite a good understanding of the complex regulation of the activity of σ^B and knowledge of a very large number of general stress genes controlled by σ^B, first insights into the physiological role of this non-specific stress response have been obtained only very recently. To explore the physiological role of this regulon, we and others identified σ^B-dependent general stress genes and compared the stress tolerance of wild-type cells with mutants lacking σ^B or general stress proteins. The proteins encoded by σ^B-dependent general stress genes can be divided into at least five functional groups that most probably provide growth-restricted B. subtilis cells with a multiple stress resistance in anticipation of future stress. In particular, sigB mutants are impaired in non-specific resistance to oxidative stress, which requires the σ^B-dependent dps gene encoding a DNA-protecting protein. Protection against oxidative damage of membranes, proteins or DNA could be the most essential component of σ^B-mediated general stress resistance in growth-arrested aerobic Gram-positive bacteria. Other general stress genes have both a σ^B-dependent induction pathway and a second σ^B-independent mechanism of stress induction, thereby partially compensating for a σ^B deficiency in a sigB mutant. In contrast to sigB mutants, null mutations in genes encoding those proteins, such as clpP or clpC, cause extreme sensitivity to salt or heat.     

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.     

Developmental control of the heat-shock stress regulon in Streptomyces coelicolor

In the differentiating eubacterium Streptomyces coelicolor , nutritional imbalances activate a developmental programme which involves the heat-shock stress regulon. In liquid batch cultures, the growth curve could be separated into four components: rapid growth 1 (RG1), transition (T), rapid growth 2 (RG2) and stationary (S). Patterns of gene expression in cultures subjected to heat shock in various phases were recorded on two-dimensional gels and analysed using advanced statistical methods. The responses of all heat-shock proteins (HSPs) were highly dependent upon the growth phase, thus demonstrating that the four phases of growth were physiologically distinct. For many HSPs, the levels of thermal induction attained were closely related to growth stage-determined levels of synthesis before heat shock, thus supporting the idea that developmental and thermal induction of this stress regulon have common control elements. Cluster analysis identified five groups of HSPs displaying similar kinetics of heat and developmentally induced synthesis, probably reflecting the influence of major regulatory systems. Methods introduced here to analyse the response of groups of genes to multiple simultaneous stimuli should find broad applications to studies of other prokaryotic and eukaryotic regulons.     

Mutations in genes downstream of the rpoN gene (encoding σ54) of Klebsiella pneumoniae affect expression from σ54-dependent promoters

Two open reading frames (ORFs), designated ORF95 and ORF162, downstream of the Klebsiella pneumoniae sigma 54 structural gene (rpoN) have been sequenced and shown to encode polypeptides of 12 kD and 16 kD, respectively. ORFs homologous to ORF95 are present downstream of four out of five rpoN genes sequenced to date from a range of Gram-negative bacteria, and ORF162 is also conserved, at least in Pseudomonas putida. Chromosomal mutations have been created in each gene using a kan cassette and both have the same phenotype, i.e. they cause an increase in the level of expression from sigma 54-dependent promoters. We propose that the products of both genes function to modulate the activity of E sigma 54, although a physiological role for these proteins has not yet been identified.