Analysis of transcriptional regulation by RcsB homo- and heterodimers in Escherichia coli

The FixJ/NarL-type transcription factor RcsB is the response regulator of the Rcs phosphorelay, a complex signal transduction system that senses perturbations of the bacterial cell envelope. RcsB regulates expression of multiple loci related to motility, biofilm formation, and various stress responses. The activity of RcsB is controlled by two mechanisms. First, the Rcs phosphorelay controls RcsB activity by phosphorylating a conserved aspartate residue within its receiver domain. Second, RcsB activity is modulated by interaction with auxiliary proteins, such as RcsA (regulation of capsule synthesis), BglJ (pleiotropic regulator, activating bgl and leuO), and GadE (acid stress response). These auxiliary regulators likewise belong to the FixJ/NarL transcription factor family and their activity depends on RcsB. Previously, RcsB was demonstrated to interact with two additional transcriptional regulators of the FixJ/NarL-family, MatA (control of the Mat pili expression) and DctR (encoded in the acid stress island). \nIn this work, determinants for transcriptional activation by RcsB homo- and heterodimers were analyzed. To this end, suitable reporter systems for RcsB homodimers and RcsB heterodimers with RcsA, and MatA were established. The results show that MatA requires RcsB as a dimerization partner for activating the matA promoter of UPEC strain CFT073 and that activation is independent of RcsB phosphorylation. In addition, it was shown that MatA- RcsB is able to repress the motility of E. coli K-12. Moreover, the results confirmed that transcriptional activation by RcsA-RcsB and RcsB-RcsB is phosphorylation dependent. This work also identified particular residues of the RcsB receiver domain being relevant for transcriptional activation by a specific dimer where RcsB homodimers and RcsA-RcsB heterodimers that are depending on RcsB phosphorylation possess the most similar properties. All relevant amino acids are located close to the active site, suggesting an important role for the structural change that is elicited by phosphorylation. Finally, data respective the mechanism of transcriptional activation, suggest that at some promoters BglJ- RcsB activates transcription by direct contacts to the RNA polymerase in a pre-recruitment mechanism.