Claude Gutierrez

RcsCDB His-Asp phosphorelay system negatively regulates the flhDC operon in Escherichia coli

The genes involved in flagellum synthesis, motility and chemotaxis in Escherichia coli are expressed in a hierarchical fashion. At the top of the hierarchy lies the master regulator FlhDC, required for the expression of the whole set of genes. The operon flhDC is controlled by numerous regulators including H‐NS, CRP, EnvZ/OmpR, QseBC and LrhA. In the present work, we report that the flhDC operon is also negatively regulated by the His‐Asp phosphorelay system RcsCDB. The regulation is potentiated by the RcsB cofactor RcsA. Genetic analysis indicates that an RcsAB box, located downstream of the promoter, is required for the regulation. The binding of RcsB and RcsA to this site was demonstrated by gel retardation and DNase I protection assays. In addition, mutation analysis suggests that RcsA‐specific determinants lie in the right part of the ‘RcsAB box’.

Acid stress response in Escherichia coli: mechanism of regulation of gadA transcription by RcsB and GadE

Escherichia coli can survive extreme acid stress for several hours. The most efficient acid resistance system is based on glutamate decarboxylation by the GadA and GadB decarboxylases and the import of glutamate via the GadC membrane protein. The expression of the corresponding genes is controlled by GadE, the central activator of glutamate-dependent acid resistance (GDAR). We have previously shown by genetic approaches that as well as GadE, the response regulator of the Rcs system, RcsB is absolutely required for control of gadA/BC transcription. In the presence of GadE, basal activity of RcsB stimulates the expression of gadA/BC, whereas activation of RcsB leads to general repression of the gad genes. We report here the results of various in vitro assays that show RcsB to regulate by direct binding to the gadA promoter region. Furthermore, activation of gadA transcription requires a GAD box and binding of an RcsB/GadE heterodimer. In addition, we have identified an RcsB box, which lies just upstream of the −10 element of gadA promoter and is involved in repression of this operon.

Regulation of osmC Gene Expression by the Two-Component System rcsB-rcsC in Escherichia coli

The Escherichia coli osmC gene encodes an envelope protein of unknown function whose expression depends on osmotic pressure and growth phase. The gene is transcribed from two overlapping promoters, osmCp(1) and osmCp(2). Several factors regulating these promoters have been reported. The leucine-responsive protein Lrp represses osmCp(1) and activates osmCp(2), the nucleoid-associated protein H-NS represses both promoters, and the stationary-phase sigma factor sigma(s) specifically recognizes osmCp(2). This work reports the identification of an additional regulatory element, the two-component system rcsB-rcsC, affecting positively the distal promoter osmCp(1). The response regulator of the system, RcsB, does not affect expression of the proximal promoter osmCp(2). Deletion analysis located the site necessary for RcsB activation just upstream of osmCp(1). In vitro transcription experiments and gel mobility shift assays demonstrated that RcsB stimulates RNA polymerase binding at osmCp(1).

Interplay between global regulators of Escherichia coli : effect of RpoS, Lrp and H-NS on transcription of the gene osmC

The transcription of the osmC gene of Escherichia coli is regulated as a function of the phase of growth. It is induced during the decelerating phase, before entry into stationary phase. osmC expression is directed by two overlapping promoters, osmCp1 and osmCp2. osmCp2 is mainly transcribed by E‐σs, the RNA polymerase using the σs (RpoS) sigma factor, and is responsible for the growth phase regulation. Transcription from osmCp1 is independent of σs. The leucine‐responsive protein (Lrp) has been shown to bind the osmC promoter region in band shift experiments. In vivo analysis using osmC–lacZ transcriptional fusions demonstrated that Lrp affects the expression of both promoters. It represses the transcription of osmCp1 and activates the transcription of osmCp2 by E‐σs. An absence of Lrp results in an increase in the amount of RpoS during exponential growth in minimal medium. The nucleoid‐associated protein H‐NS also represses osmC transcription from both promoters. However, this happens through different mechanisms. The effect on osmCp2 is probably mediated by the increase in σs concentration in the cytoplasm of hns− mutants, while the effect on osmCp1 is independent of σs. No binding of H‐NS to the promoter region DNA could be detected, indicating that the effect on osmCp1 could also be indirect.

Involvement of differential efficiency of transcription by esigmas and esigma70 RNA polymerase holoenzymes in growth phase regulation of the Escherichia coli osmE promoter.

Transcription of the gene osmE of Escherichia coli is inducible by elevated osmotic pressure and during the decelerating phase of growth. osmE expression is directed by a single promoter, osmEp. Decelerating phase induction of osmEp is dependent on the σs (RpoS) factor, whereas its osmotic induction is independent of σs. Purified Eσs and Eσ70 were both able to transcribe osmEpin vitro on supercoiled templates. In the presence of rpoD800, a mutation resulting in a thermosensitive σ70 factor, a shift to non‐permissive temperature abolished induction of osmEp after an osmotic shock during exponential phase, but did not affect the decelerating phase induction. Point mutations affecting osmEp activity were isolated. Down‐promoter mutations decreased transcription in both the presence and the absence of σs, indicating that the two forms of RNA polymerase holoenzyme recognize very similar sequence determinants on the osmE promoter. Three up‐promoter mutations brought osmEp closer to the consensus of Eσ70‐dependent promoters. The two variant promoters exhibiting the highest efficiency became essentially independent of σsin vivo. Our data suggest that Eσs transcribes wild‐type osmEp with a higher efficiency than Eσ70. A model in which an intrinsic differential recognition contributes to growth phase‐dependent regulation is proposed. Generalization of this model to other σs‐dependent promoters is discussed.

Growth-phase-dependent expression of the osmotically inducible gene osmC of Escherichia coli K-12.

The transcription of the osmotically inducible gene osmC of Escherichia coli is initiated by two overlapping promoters, osmCp1 and osmCp2. The existence of these two promoters was confirmed by site‐directed mutagenesis. osmC transcription is regulated by the growth phase. In a medium of low osmotic pressure, expression of osmC is induced at the onset of decelerating phase and continues during the beginning of stationary phase. At elevated osmotic pressure, the induction occurs somewhat earlier during growth. Both promoters are repressed during early exponential phase. osmCp2 is induced during entry into stationary phase. Transcription from osmCp1, which is approximately 10‐fold lower than that of osmCp2 in rich medium, starts during the mid‐log phase and stops in early stationary phase. In the absence of σS, the stationary‐phase sigma factor encoded by rpoSosmCp2 expression is much reduced while expression of osmCp1 is unaffected. As a consequence, the regulation of osmC as a function of growth is at least partially independent of σS

The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

NhaR and RcsB Independently Regulate the osmCp1 Promoter of Escherichia coli at Overlapping Regulatory Sites

Transcription of the Escherichia coli osmC gene is induced by several stress conditions. osmC is expressed from two overlapping promoters, osmCp1 and osmCp2. The proximal promoter, osmCp2, is transcribed at the entry into the stationary phase by the sigma(s) sigma factor. The distal promoter, osmCp1, is activated by NhaR and RcsB. NhaR is a positive regulator of the LysR family and is known to be an activator of the nhaA gene encoding an Na(+)/H(+) antiporter. RcsB is the response regulator of the RcsCDB His-Asp phosphorelay signal transduction system. Genetic data indicated that activation of osmCp1 by both NhaR and RcsB requires the same short sequences upstream of the -35 region of the promoter. Accordingly, DNase I footprint analysis indicated that both activators protect an overlapping region close to the -35 box of the promoter and suggested that the regulatory effect is direct. Despite the overlap of the binding sites, each activator acts independent of the other and is specific for a particular stress. NhaR can stimulate osmCp1 in response to an osmotic signal even in the absence of RcsB. RcsB is responsible for the induction of osmCp1 by alteration of the cell envelope, even in the absence of NhaR. osmCp1 as an example of multiple-stress-responsive promoter is discussed in light of a comparison of the NhaR and RcsB target regions in the Enterobacteriaceae.

Osmotic Regulation of the Escherichia coli bdm (Biofilm-Dependent Modulation) Gene by the RcsCDB His-Asp Phosphorelay

The RcsCDB His-Asp phosphorelay is shown to positively regulate the bdm (biofilm-dependent modulation) and sra (stationary-phase-induced ribosome-associated protein) genes in Escherichia coli. The regulation is direct and requires an RcsB box next to the bdm -35 element. In addition, bdm is shown to be activated by osmotic shock in an Rcs-dependent way.

Multistress Regulation in Escherichia coli: Expression of osmB Involves Two Independent Promoters Responding either to σS or to the RcsCDB His-Asp Phosphorelay

Transcription of the Escherichia coli osmB gene is induced by several stress conditions. osmB is expressed from two promoters, osmBp1 and osmBp2. The downstream promoter, osmBp2, is induced after osmotic shock or upon entry into stationary phase in a sigma(S)-dependent manner. The upstream promoter, osmBp1, is independent of sigma(S) and is activated by RcsB, the response regulator of the His-Asp phosphorelay signal transduction system RcsCDB. RcsB is responsible for the induction of osmBp1 following treatment with chlorpromazine. Activation of osmBp1 by RcsB requires a sequence upstream of its -35 element similar to the RcsB binding site consensus, suggesting a direct regulatory role. osmB appears as another example of a multistress-responsive gene whose transcription involves both a sigma(S)-dependent promoter and a second one independent of sigma(S) but controlled by stress-specific transcription factors.