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.     

Dynamic control of Dps protein levels by ClpXP and ClpAP proteases in Escherichia coli

The Escherichia coli starvation-induced DNA protection protein Dps was observed to be degraded rapidly during exponential growth. This turnover is dependent on the clpP and clpX genes. The clpA gene is not required for Dps proteolysis, suggesting that Dps is a substrate for ClpXP protease but not for ClpAP protease. Dps proteolysis was found to be highly regulated. Upon carbon starvation, Dps is stabilized, which together with increased Dps synthesis allows strong accumulation of Dps in the stationary phase. The addition of glucose to starving cells results in rapid resumption of Dps proteolysis by ClpXP. Oxidative stress also leads to efficient stabilization of Dps. After hyperosmotic shift, however, proteolysis remains unaffected. Thus, regulated proteolysis of Dps strongly contributes to controlling Dps levels under very specific stress conditions. In contrast to the regulated degradation of RpoS by ClpXP, Dps proteolysis is independent of the recognition factor RssB. In addition, during starvation, clpP and, to a somewhat lesser extent, clpA are involved in maintaining ongoing Dps synthesis (acting at the level of Dps translation), which is required for strong Dps accumulation in long-term stationary phase cells. In summary, both ClpXP and ClpAP exert significant control of Dps levels by affecting log phase stability and stationary phase synthesis of Dps respectively.     

Regulation of sigma S degradation in Salmonella enterica var typhimurium: in vivo interactions between sigma S, the response regulator MviA(RssB) and ClpX

The alternate sigma factor sigmaS plays an important role in the survival of Salmonella typhimurium following sudden encounters with a variety of stress conditions. The level of sigmaS is very low in rapidly growing cells but dramatically increases as those cells encounter environmental stress or enter into stationary phase. This increase is due in large measure to the stabilization of sigmaS protein against degradation by the ClpXP protease. The MviA protein, also known as RssB or SprE in Escherichia coli, is a putative member of a two component signal transduction system that plays a central role in facilitating sigmaS degradation by ClpXP. In contrast to most two-component systems, MviA does not appear to regulate gene expression but is believed to interact directly with sigmaS and somehow facilitate degradation. We now provide evidence that MviA(RssB) directly interacts both with sigmaS and ClpX in vivo, presumably enabling presentation of sigmaS to the ClpP protease. Interactions were demonstrated using a bacterial two-hybrid system in which sigmaS, MviA, and ClpX were fused to separate moieties of Bordetella pertussis CyaA (adenylate cyclase). Paired hybrid plasmids containing Cya'-MviA/RpoS-'Cya or Cya'-MviA/ClpX-'Cya successfully reconstituted adenylate cyclase activity in both S. typhimurium and E. coli. However, no direct interactions were detected between ClpX and RpoS. A second series of experiments has indicated that the interaction between MviA and sigmaS requires the N-terminus but not the C-terminus of MviA. Cellular levels of MviA appear to be very low in the cell based on lacZ fusion, Western blot and Northern blot analyses suggesting a catalytic role for MviA in sigmaS degradation. Mutagenesis of MviA residue D58, a canonical residue subject to phosphorylation in many two-component systems, decreased the ability of MviA to facilitate sigmaS turnover in vivo confirming that phosphorylation of MviA increases MviA activity.     

Multiple pathways for regulation of sigmaS (RpoS) stability in Escherichia coli via the action of multiple anti-adaptors

σ ^S, the stationary phase sigma factor of Escherichia coli and Salmonella , is regulated at multiple levels. The σ^S protein is unstable during exponential growth and is stabilized during stationary phase and after various stress treatments. Degradation requires both the ClpXP protease and the adaptor RssB. The small antiadaptor protein IraP is made in response to phosphate starvation and interacts with RssB, causing σ^S stabilization under this stress condition. IraP is essential for σ^S stabilization in some but not all starvation conditions, suggesting the existence of other anti-adaptor proteins. We report here the identification of new regulators of σ^S stability, important under other stress conditions. IraM (inhibitor of RssB activity during Magnesium starvation) and IraD (inhibitor of RssB activity after DNA damage) inhibit σ^S proteolysis both in vivo and in vitro . Our results reveal that multiple anti-adaptor proteins allow the regulation of σ^S stability through the regulation of RssB activity under a variety of stress conditions.     

Sequential recognition of two distinct sites in sigma(S) by the proteolytic targeting factor RssB and ClpX

sigma(S) (RpoS), the master regulator of the general stress response in Escherichia coli, is a model system for regulated proteolysis in bacteria. sigma(S) turnover requires ClpXP and the response regulator RssB, whose phosphorylated form exhibits high affinity for sigma(S). Here, we demonstrate that recognition by the RssB/ClpXP system involves two distinct regions in sigma(S). Region 2.5 of sigma(S) (a long alpha-helix) is sufficient for binding of phosphorylated RssB. However, this interaction alone is not sufficient to trigger proteolysis. A second region located in the N-terminal part of sigma(S), which is exposed only upon RssB-sigma(S) interaction, serves as a binding site for the ClpX chaperone. Binding of the ClpX hexameric ring to sigma(S)-derived reporter proteins carrying the ClpX-binding site (but not the RssB-binding site) is also not sufficient to commit the protein to degradation. Our data indicate that RssB plays a second role in the initiation of sigma(S) proteolysis that goes beyond targeting of sigma(S) to ClpX, and suggest a model for the sequence of events in the initiation of sigma(S) proteolysis.     

Regulation of RssB-dependent proteolysis in Escherichia coli: a role for acetyl phosphate in a response regulator-controlled process

σ^S (RpoS) is a highly unstable global regulatory protein in Escherichia coli , whose degradation is inhibited by various stress signals, such as carbon starvation, high osmolarity and heat shock. As a consequence, these stresses result in the induction of σ^S-regulated stress-protective proteins. The two-component-type response regulator, RssB, is essential for the rapid proteolysis of σ^S and is probably involved in the transduction of some of these stress signals. Acetyl phosphate can be used as a phosphodonor for the phosphorylation of various response regulators in vitro and, in the absence of the cognate sensor kinases, acetyl phosphate can also modulate the activities of several response regulators in vivo . Here, we demonstrate increased in vivo half-lives of σS and the RpoS742::LacZ hybrid protein (also a substrate for RssB-dependent proteolysis) in acetyl phosphate-free ( pta – ackA ) deletion mutants, even though no sensor kinase was eliminated. The in vivo data indicate that acetyl phosphate acts through the response regulator, RssB. In vitro , efficient phosphotransfer from radiolabelled acetyl phosphate to the Asp-58 residue of RssB (the expected site of phosphorylation in the RssB receiver domain) was observed. Via such phosphorylation, acetyl phosphate may thus modulate RssB activity even in an otherwise wild-type background. While acetyl phosphate is not essential for the transduction of specific environmental stress signals, it could play the role of a modulator of RssB-dependent proteolysis that responds to the metabolic status of the cells reflected in the highly variable cellular acetyl phosphate concentration.     

Genome-wide analysis of the general stress response network in Escherichia coli: sigmaS-dependent genes, promoters, and sigma factor selectivity

The sigmaS (or RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli. While nearly absent in rapidly growing cells, sigmaS is strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of the E. coli genes are under direct or indirect control of sigmaS and that sigmaS should be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a sigmaS consensus promoter in silico. Moreover, our results suggest that sigmaS-dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect sigmaS and/or sigma70 selectivity of many promoters. These observations indicate that certain modules of the sigmaS-dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with sigma70 RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation.     

The alternative sigma factor sigmaE controls antioxidant defences required for Salmonella virulence and stationary-phase survival

Bacteria must contend with conditions of nutrient limitation in all natural environments. Complex programmes of gene expression, controlled in part by the alternative sigma factors sigmaS (sigma38, RpoS) and sigmaH (sigma32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation. We show here that the alternative sigma factor sigmaE (sigma24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation. Expression of the sigmaE regulon is strongly induced upon entry of Salmonella into stationary phase. A Salmonella mutant lacking sigmaE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress. A Salmonella strain lacking both sigmaE and sigmaS is non-viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary-phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation. Moreover, the attenuated virulence of sigmaE-deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the sigmaE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts.     

The RNA-binding protein HF-I plays a global regulatory role which is largely, but not exclusively, due to its role in expression of the sigmaS subunit of RNA polymerase in Escherichia coli.

The hfq-encoded RNA-binding protein HF-I has long been known as a host factor for phage Qbeta RNA replication and has recently been shown to be essential for translation of rpoS, which encodes the sigmaS subunit of RNA polymerase. Here we demonstrate that an hfq null mutant does not synthesize glycogen, is starvation and multiple stress sensitive, and exhibits strongly reduced expression of representative sigmaS-regulated genes. These phenotypes are consistent with strongly reduced sigmaS levels in the hfq mutant. However, the analysis of global protein synthesis patterns on two-dimensional O'Farrell gels indicates that approximately 40% of the more than 30 proteins whose syntheses are altered in the hfq null mutant are not affected by an rpoS mutation. We conclude that HF-I is a global regulator involved in the regulation of expression of sigmaS and sigmaS-independent genes.