Mutations that increase expression of the rpoS gene and decrease its dependence on hfq function in Salmonella typhimurium

Abstract

The RpoS transcription factor (also called S or 38 ) is required for the expression of a number of stationary-phase and osmotically inducible genes in enteric bacteria. RpoS is also a virulence factor for several pathogenic species, including Salmonella typhimurium. The activity of RpoS is regulated in response to many different signals, at the levels of both synthesis and proteolysis. Previous work with rpoS-lac protein fusions has suggested that translation of rpoS requires hfq function. The product of the hfq gene, host factor I (HF-I), is a ribosome-associated, site-specific RNA-binding protein originally characterized for its role in replication of the RNA bacteriophage Q␤ of Escherichia coli. In this study, the role of HF-I was explored by isolating suppressor mutations that map to the region directly upstream of rpoS. These mutations increase rpoS-lac expression in the absence of HF-I and also confer substantial independence from HF-I. DNA sequence analysis of the mutants suggests a model in which the RNA secondary structure near the ribosome binding site of the rpoS mRNA plays an important role in limiting expression in the wild type. Genetic tests of the model confirm its predictions, at least in part. It seems likely that the mutations analyzed here activate a suppression pathway that bypasses the normal HF-I-dependent route of rpoS expression; however, it is also possible that some of them identify a sequence element with an inhibitory function that is directly counteracted by HF-I.The rpoS gene encodes a specificity factor for RNA polymerase (44, 46, 59) which is required for the transcription of many genes expressed during the onset of stationary phase. RpoS-dependent adaptations to nutrient limitation and starvation identified so far in Escherichia coli include not only shifts in metabolic pathways but also resistance mechanisms protective against life-threatening stresses such as high osmolarity, heat shock, elevated H 2 O 2 , and UV light (reviewed in references 26 and 37). RpoS is also a virulence factor for Salmonella typhimurium (16) and other enteric bacteria. The regulation of this regulatory protein is itself complex: RpoS abundance can be increased by a variety of inducing treatments (37). Control of RpoS can occur both at the level of synthesis and by proteolysis (27,34,65); in this respect it is reminiscent of heat shock sigma factor (RpoH) regulation (66). It has also been demonstrated that RpoS abundance is positively regulated by ppGpp (23), and this may provide a unifying mechanism for the multitude of RpoS inducers. An hns mutant, lacking the abundant DNA-binding protein H-NS, has an increased level of RpoS in exponential phase, and this effect is remarkable because it occurs through increases in both translation and protein stability (2, 65). Genetic evidence suggests that RpoS is degraded by the energy-dependent ClpXP protease (52) with the help of other factors (42,48,58).The E. coli hfq gene product, host factor I (HF-I), was discovered through its role in the in vitro replicati...

A trans-acting RNA as a control switch in Escherichia coli : DsrA modulates function by forming alternative structures

“…DsrA acts at rpoS by enhancing RpoS translation (9) via RNA-RNA interactions that antagonize the rpoS translational operator (4,5,10). Here we show that DsrA overproduction also increases rpoS mRNA stability (Fig.…”

“…Thus, DsrA has opposite effects on these two targets, both mediated by RNA-RNA interactions, with global regulatory consequences for the transcriptional state of the cell. Whereas the mechanism of DsrA action at hns is not known, DsrA binds the translational operator of rpoS (4,5) to open a stable stem-loop of rpoS RNA (10), enabling access to the Shine-Dalgarno sequence and thus enhancing translation.…”

Interactions of the Non-coding RNA DsrA and RpoS mRNA with the 30 S Ribosomal Subunit

“…Even if DsrA had no secondary structure of its own, it is not perfectly complementary to the RpoS mRNA leader sequence and therefore has a lower affinity for the mRNA than a perfectly complementary sequence would have. In order to make a stronger hybrid complex and to eliminate the requirement for denaturing the DsrA structure, we designed an oligonucleotide, AS- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), that perfectly complements the RpoS mRNA sequence. The effect of increasing intermolecular complementarity and decreasing self-hybridization is that we have made a molecule that hybridizes to renatured RpoS mRNA with an affinity of ϳ9.5 ϫ 10 6 M Ϫ1 at 0°C.…”

Regulation of RpoS proteolysis in Escherichia coli : The response regulator RssB is a recognition factor that interacts with the turnover element in RpoS

“…In addition, RpoS exhibited stationary phase induction when expressed from pRpoS18 (data not shown). Translational regulation of RpoS, however, is altered with the pRpoS18 construct, because the extended 5Ј untranslated regions present in wild-type rpoS mRNA, which have been implicated in secondary structure formation and translational control (30)(31)(32), are not present in pRpoS18. Therefore, mutations within the rpoS part on pRpoS18 that affect RpoS levels most likely influence RpoS proteolysis.…”