SummaryThe 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'.

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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.

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 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 .

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