RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.

The RcsCDB phosphorelay system controls an extremely large regulon in Enterobacteriaceae that involves processes such as biofilm formation, flagella production, synthesis of extracellular capsules and cell division. Therefore, fine-tuning of this system is essential for virulence in pathogenic microorganisms of this group. The final master effector of the RcsCDB system is the response regulator (RR) RcsB, which activates or represses multiple genes by binding to different promoter regions. This regulatory activity of RcsB can be done alone or in combination with additional transcriptional factors in phosphorylated or dephosphorylated states. The capacity of RcsB to interact with multiple promoters and partners, either dephosphorylated or phosphorylated, suggests an extremely conformational dynamism for this RR. To shed light on the activation mechanism of RcsB and its implication on promoter recognition, we solved the crystal structure of full-length RcsB from Salmonella enterica serovar Typhimurium in the presence and absence of a phosphomimetic molecule BeF3-. These two novel structures have guided an extensive site-directed mutagenesis study at the structural and functional level that confirms RcsB conformational plasticity and dynamism. Our data allowed us to propose a β5-T switch mechanism where phosphorylation is coupled to alternative DNA binding ways and which highlights the conformational dynamism of RcsB to be so pleiotropic.