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.     

Identification and classification of two-component systems that affect rpoS expression in Escherichia coli

The rpoS-encoded sigmaS subunit of RNA polymerase regulates the expression of stationary phase and stress response genes in Escherichia coli. Recent study of our DNA microarray analysis suggested that the rpoS expression is affected by multiple two-component systems. In this study, we identified two-component-system mutants in which the rpoS expression increased. The regulatory manner of the systems on rpoS expression is suggested.     

The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein.

The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage. Among the OxyS targets is the rpoS-encoded sigma(s) subunit of RNA polymerase. Sigma(s) is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase. We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message. This repression is dependent on the hfq-encoded RNA-binding protein (also denoted host factor I, HF-I). Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity.     

The response regulator RssB, a recognition factor for sigmaS proteolysis in Escherichia coli, can act like an anti-sigmaS factor

sigmaS (RpoS) is the master regulator of the general stress response in Escherichia coli. Several stresses increase cellular sigmaS levels by inhibiting proteolysis of sigmaS, which under non-stress conditions is a highly unstable protein. For this ClpXP-dependent degradation, the response regulator RssB acts as a recognition factor, with RssB affinity for sigmaS being modulated by phosphorylation. Here, we demonstrate that RssB can also act like an anti-sigma factor for sigmaS in vivo, i.e. RssB can inhibit the expression of sigmaS-dependent genes in the presence of high sigmaS levels. This becomes apparent when (i) the cellular RssB/sigmaS ratio is at least somewhat elevated and (ii) proteolysis is reduced (for example in stationary phase) or eliminated (for example in a clpP mutant). Two modes of inhibition of sigmaS by RssB can be distinguished. The 'catalytic' mode is observed in stationary phase cells with a substoichiometric RssB/sigmaS ratio, requires ClpP and therefore probably corresponds to sequestering of sigmaS to Clp protease (even though sigmaS is not degraded). The 'stoichiometric' mode occurs in clpP mutant cells upon overproduction of RssB to levels that are equal to those of sigmaS, and therefore probably involves binary complex formation between RssB and sigmaS. We also show that, under standard laboratory conditions, the cellular level of RssB is more than 20-fold lower than that of sigmaS and is not significantly controlled by stresses that upregulate sigmaS. We therefore propose that antisigma factor activity of RssB may play a role under not yet identified growth conditions (which may result in RssB induction), or that RssB is a former antisigma factor that during evolution was recruited to serve as a recognition factor for 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.     

Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5'-leader

The intricate regulation of the Escherichia coli rpoS gene, which encodes the stationary phase sigma-factor sigmaS, includes translational activation by the noncoding RNA DsrA. We observed that the stability of rpoS mRNA, and concomitantly the concentration of sigmaS, were significantly higher in an RNase III-deficient mutant. As no decay intermediates corresponding to the in vitro mapped RNase III cleavage site in the rpoS leader could be detected in vivo, the initial RNase III cleavage appears to be decisive for the observed rapid inactivation of rpoS mRNA. In contrast, we show that base-pairing of DsrA with the rpoS leader creates an alternative RNase III cleavage site within the rpoS/DsrA duplex. This study provides new insights into regulation by small regulatory RNAs in that the molecular function of DsrA not only facilitates ribosome loading on rpoS mRNA, but additionally involves an alternative processing of the target.