The rpoS‐encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase‐induced and osmotically regulated genes in Escherichia coli. sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover. We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content. rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post‐transcriptional growth phase‐related and osmotic regulation of sigma(S). Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover. RssB contains a unique C‐terminal output domain and is the first known response regulator involved in the control of protein turnover.

The rpoS-encoded cr s subunit of RNA polymerase in Escherichia coil is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of cr s, which in nonstressed cells is a highly unstable protein, the cellular ~r s content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Q~ RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced ~r s levels under all conditions tested and is deficient for growth phase-related and osmotic induction of ~r s. Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock ~r factor cr 32 (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli.

TheS subunit of RNA polymerase (encoded by the rpoS gene) is the master regulator in a complex regulatory network that controls stationary-phase induction and osmotic regulation of many genes in Escherichia coli. Here we demonstrate that the histone-like protein H-NS is also a component of this network, in which it functions as a global inhibitor of gene expression during the exponential phase of growth. On two-dimensional gels, at least 22 S -controlled proteins show increased expression in an hns mutant. H-NS also inhibits the expression of S itself by a mechanism that acts at the posttranscriptional level. Our results indicate that relief of repression by H-NS plays a role in stationary-phase induction as well as in hyperosmotic induction of rpoS translation. Whereas certain S -dependent genes (e.g., osmY) are only indirectly regulated by H-NS via its role in the control of S expression, others are also H-NS-regulated in a S -independent manner. (For this latter class of genes, rpoS hns double mutants show higher levels of expression than mutants deficient in rpoS alone.) In addition, we demonstrate that the slow-growth phenotype of hns mutants is suppressed in hns rpoS double mutants and that many second-site suppressor mutants that spontaneously arise from hns strains carry lesions that affect the expression of S . TheS subunit of RNA polymerase acts as a master regulator in a regulatory network that controls the expression of numerous stationary-phase-induced and osmotically regulated genes in Escherichia coli (11,12,15). rpoS, the structural gene for S , is itself induced during entry into the stationary phase (22,23,25,30,34,39) and in response to an increase in medium osmolarity (23). Under both conditions, control of the cellular S level is largely posttranscriptional, involving stimulation of translation (23, 30) as well as changes in S stability (23,43). So far, more than 30 genes or operons that are under direct or indirect control of S have been identified. Within this large S regulon, differential regulation has been observed for subsets of genes, for instance in response to anaerobiosis (3, 5, 6) or oxidative stress (1, 26). In addition, during entry into the stationary phase, the induction of various S -dependent genes follows different kinetics. These observations indicate that additional factors besides S participate in the fine regulation of these genes. The cyclic AMP (cAMP)-cAMP receptor protein complex (13,20,21,29,47), integration host factor (1, 20), and Lrp (20) have been identified as modulating factors in the control of various S -dependent genes. The data presently available indicate that various combinations of regulatory factors that can act either positively or negatively are used for the control of various stationary-phaseinducible genes. This regulatory strategy results in a high degree of specific fine modulation of S -controlled genes with respect to the time of induction during entry into the stationary phase and in response to additional environmental parameters.In the prese...

rpoS is the structural gene for the s subunit of RNA polymerase which controls the expression of a large number of genes in Escherichia coli that are induced during entry into stationary phase or in response to increased medium osmolarity. Using a combination of primer extension experiments and a 5 deletion analysis of the region upstream of rpoS, we show that rpoS transcription is mainly driven by a single promoter (rpoSp 1 ) located within the nlpD gene upstream of rpoS (the two relatively weak nlpD promoters contribute to the low level of rpoS expression during early exponential phase). In addition, we demonstrate that the expression of both transcriptional and translational rpoS::lacZ fusions as well as the level of rpoS mRNA originating at rpoSp 1 is strongly reduced in ppGpp-deficient relA spoT mutants. However, experiments with the 5 deletion constructs indicate that a lack of ppGpp does affect transcriptional elongation rather than initiation.The s subunit of RNA polymerase controls the expression of more than 30 genes or operons that are involved in starvation survival, multiple stress resistance during stationary phase, and coping with a high osmolarity environment. s is coded for by the rpoS gene (formerly also designated katF or appR; for recent reviews, see references 4, 5, and 10). Expression of rpoS is induced during entry into stationary phase (7,8,11,12,15,19) and in response to an increase in medium osmolarity (8,14). Whereas starvation stimulates rpoS expression at both the transcriptional and posttranscriptional levels (8,11,12), high osmolarity influences only the posttranscriptional control of rpoS (8).In the present study, we investigated transcription of the rpoS gene. nlpD, the structural gene for a lipoprotein, is located upstream of rpoS and is transcribed in the same direction (counterclockwise on the Escherichia coli chromosome) (6, 9). While nlpD is not stationary phase induced, its two closely spaced promoters contribute to the basal level of expression of rpoS in exponentially growing cells (9). In a recent study, the subcloning of small fragments of the coding region of nlpD into a promoter probe vector was reported, and the authors suggested that at least four promoters for the expression of rpoS were present within the nlpD gene (22). In contrast, our primer extension experiments for the determination of transcriptional start sites, as well as a deletion analysis presented here, lead to the conclusion that there is only one major rpoS promoter within nlpD.The intracellular signals and the signal transduction pathways involved in the control of rpoS expression have remained largely unknown. It has been shown that relA spoT double mutants exhibit a pleiotropic phenotype very similar to that observed for rpoS mutants and contain strongly reduced levels of s protein. Therefore, guanosine-3Ј,5Ј-bispyrophosphate (ppGpp) has been implicated in the control of rpoS expression as a positively acting signal molecule, although its mechanism of action has not been studied in detail (3). In ...

The rpoS gene, which encodes a putative alternative sigma factor (sigma S), is essential for the expression of a variety of stationary-phase-induced genes as well as for stationary-phase-specific multiple-stress resistance. As previously shown for the otsA and otsB genes (R. Hengge-Aronis, W. Klein, R. Lange, M. Rimmele, and W. Boos, J. Bacteriol. 173:7918-7924, 1991), we demonstrate here that additional rpoS-controlled genes (bolA, csi-5) as well as at least 18 proteins on two-dimensional O'Farrell gels could be induced in growing cells by osmotic upshift via an rpoS-dependent mechanism. Also, rpoS-dependent thermotolerance and resistance against hydrogen peroxide could be osmotically stimulated. In contrast, the expression of glgS, while exhibiting strong stationary-phase induction, was only weakly increased by elevated osmolarity, and several rpoS-dependent proteins previously identified on two-dimensional gels were not osmotically induced. During osmotic induction of rpoS-dependent genes, rpoS transcription and the level of sigma S remained unchanged. We conclude that osmotically regulated genes represent a subfamily within the rpoS regulon that requires differential regulation in addition to that provided by sigma S.