Structure-based analysis of the gene network controlled by the Salmonella RcsB response regulator

RcsB is a transcriptional regulator that controls expression of numerous genes in Enterobacteriaceae. 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 a large number of genes, we performed structural and in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds the promoters of rprA and flhDC genes in a dimeric active conformation, docking the DNA-binding domains into the major groove of the DNA that preform a weak read-out of the target sequence. Interestingly, comparative structural analysis shows that DNA binding may trigger the active conformation in unphosphorylated RcsB. Furthermore, RNAseq analysis 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 regulating iron and molybdenum metabolism. Finally, we delimited the RcsB box for homodimeric active RcsB binding with sequence TN(G/A)GAN4TC(T/C)NA that can be found in promoter and intragenic regions to regulate activation or inhibition of gene transcription. mRNA profiles of S. Typhimurium expressing RcsB wild type (WT)  or RcsB mutant variants in exponential phase (OD600 ~ 0.25) were generated by deep sequencing, in triplicate, using Illumina Hiseq 400 system.