SUMMARY The σS (RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and related bacteria. While rapidly growing cells contain very little σS, exposure to many different stress conditions results in rapid and strong σS induction. Consequently, transcription of numerous σS-dependent genes is activated, many of which encode gene products with stress-protective functions. Multiple signal integration in the control of the cellular σS level is achieved by rpoS transcriptional and translational control as well as by regulated σS proteolysis, with various stress conditions differentially affecting these levels of σS control. Thus, a reduced growth rate results in increased rpoS transcription whereas high osmolarity, low temperature, acidic pH, and some late-log-phase signals stimulate the translation of already present rpoS mRNA. In addition, carbon starvation, high osmolarity, acidic pH, and high temperature result in stabilization of σS, which, under nonstress conditions, is degraded with a half-life of one to several minutes. Important cis-regulatory determinants as well as trans-acting regulatory factors involved at all levels of σS regulation have been identified. rpoS translation is controlled by several proteins (Hfq and HU) and small regulatory RNAs that probably affect the secondary structure of rpoS mRNA. For σS proteolysis, the response regulator RssB is essential. RssB is a specific direct σS recognition factor, whose affinity for σS is modulated by phosphorylation of its receiver domain. RssB delivers σS to the ClpXP protease, where σS is unfolded and completely degraded. This review summarizes our current knowledge about the molecular functions and interactions of these components and tries to establish a framework for further research on the mode of multiple signal input into this complex regulatory system.

SummaryDuring carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30-50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation-induced csi::/acZ fusions using the x.p/acMu syste-;". One operon fusion (csi2::/acZ) has been studied in detail. csi2::/acZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi2::/acZ did not synthesize at least 16 proteins present in an isogenic csi2+ strain. Cells containing csi2::/acZ or csi2::Tn 10 did not produce glycogen, did not develop thermotolerance and H20 2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::/acZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi2::/acZ defines a regulatory gene of central importance for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi2 demonstrated that the csi2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi21 katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose 'rpoS' (' us,) as appropriate designations.

The second vegetative sigma factor crs (encoded by the rpoS gene) is the master regulator in a complex regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coil Using a combination of gene-fusion technology and quantitative immunoblot, pulse-labeling, and immunoprecipitation analyses, we demonstrate here that rpoS/~r s expression is not only transcriptionally controlled, but is also extensively regulated at the levels of translation and protein stability. rpoS transcription is inversely correlated with growth rate and and is negatively controlled by cAMP-CRP. In complex medium rpoS transcription is stimulated during entry into stationary phase, whereas in minimal media, it is not significantly induced, rpoS translation is stimulated during transition into stationary phase as well as by an increase in medium osmolarity. A model involving mRNA secondary structure is suggested for this novel type of post-transcriptional growth phase-dependent and osmotic regulation. Furthermore, ~r s is a highly unstable protein in exponentially growing cells (with a half-life of 1.4 min), that is stabilized at the onset of starvation. When cells are grown in minimal glucose medium, translational induction and ~r s stabilization occur in a temporal order with the former being stimulated already in late exponential phase and the latter taking place at the onset of starvation. Although crs does not control its own transcription, it is apparently indirectly involved in a negative feedback control that operates on the post-transcriptional level. Our analysis also indicates that at least five different signals [cAMP, a growth rate-related signal (ppGpp?I, a cell density signal, an osmotic signal, and a starvation signal] are involved in the control of all these processes that regulate rpoS/~r s expression.

The protein encoded by katF (also known as nur, appR, csi-2, abrD, and rpoS in various alleles) has been biochemically confirmed to be an alternate sigma transcription factor and renamed sigma S. Its synthesis is controlled transcriptionally and posttranscriptionally by as yet undefined mechanisms that are active well into stationary phase. sigma S controls a regulon of 30 or more genes expressed in response to starvation and during the transition to stationary phase. Proteins in the regulon, many of which have not been characterized, enhance long-term survival in nutrient-deficient medium and have a diverse group of functions including protection against DNA damage, the determination of morphological changes, the mediation of virulence, osmoprotection, and thermotolerance. Differential expression of subfamilies of genes within the regulon is effected by supplementary regulatory factors, working both individually and in combination to modulate activity of different sigma S-dependent promoters.

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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 σ s subunit of RNA polymerase. σ 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 hfqencoded 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 rpoS (katF) gene of Escherichia coli encodes a putative sigma factor (ajS) required for the expression of a variety of stationary phase-induced genes, for the development of stationary-phase stress resistance, and for long-term starvation survival (R. Lange and R. Hengge-Aronis, Mol. Microbiol. 5:49-59, 1991). Here we show that the genes otsA, otsB, treA, and osmB, previously known to be osmotically regulated, are also induced during transition into stationary phase in a rs-dependent manner. otsA and otsB, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively, are involved in os-dependent stationary-phase thermotolerance. Neither os nor trehalose, however, is required for the development of adaptive thermotolerance in growing cells, which might be controlled by cJE.

The novel sigma factor (as) encoded by rpoS (katF) is required for induction of many growth phase-regulated genes and expression of a variety of stationary-phase phenotypes in Escherichia coli. Here we demonstrate that wild-type cells exhibit spherical morphology in stationary phase, whereas rpoS mutant cells remain rod shaped and are generally larger. Size reduction of E. coli cells along the growth curve is a continuous and at least biphasic process, the second phase of which is absent in rpoS-deficient cells and correlates with induction of the morphogene bol in wild-type cells. Stationary-phase induction of boUl is dependent on as. The "gearbox," a characteristic sequence motif present in the cS-dependent growth phase-and growth rate-regulated bolAp1 promoter, is not recognized by cs, since stationary-phase induction of the mcbA promoter, which also contains a gearbox, does not require rs, and other crs-controlled promoters do not contain gearboxes. However, good homology to the potential -35 and -10 consensus sequences for crs regulation is found in the bolAp, promoter.