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). In 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

Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Escherichia coli

The Rcs phosphorelay is a two-component signal transduction system that is induced by cell envelope stress. RcsB, the response regulator of this signaling system, is a pleiotropic transcription regulator, which is involved in the control of various stress responses, cell division, motility, and biofilm formation. RcsB regulates transcription either as a homodimer or together with auxiliary regulators, such as RcsA, BglJ, and GadE in Escherichia coli. In this study, we show that RcsB in addition forms heterodimers with MatA (also known as EcpR) and with DctR. Our data suggest that the MatA-dependent transcription regulation is mediated by the MatA-RcsB heterodimer and is independent of RcsB phosphorylation. Furthermore, we analyzed the relevance of amino acid residues of the active quintet of conserved residues, and of surface-exposed residues for activity of RcsB. The data suggest that the activity of the phosphorylation-dependent dimers, such as RcsA-RcsB and RcsB-RcsB, is affected by mutation of residues in the vicinity of the phosphorylation site, suggesting that a phosphorylation-induced structural change modulates their activity. In contrast, the phosphorylation-independent heterodimers BglJ-RcsB and MatA-RcsB are affected by only very few mutations. Heterodimerization of RcsB with various auxiliary regulators and their differential dependence on phosphorylation add an additional level of control to the Rcs system that is operating at the output level

YjjQ Represses Transcription of flhDC and Additional Loci in Escherichia coli

The presumptive transcriptional regulator YjjQ has been identified as being virulence associated in avian pathogenic Escherichia coli (APEC). In this work, we characterize YjjQ as transcriptional repressor of the flhDC operon, encoding the master regulator of flagellar synthesis, and of additional loci. The latter include gfc (capsule 4 synthesis), ompC (outer membrane porin C), yfiRNB (regulated c-di-GMP synthesis), and loci of poorly defined function (ybhL and ymiA-yciX). We identify the YjjQ DNA-binding sites at the flhDC and gfc promoters and characterize a DNA-binding sequence motif present at all promoters found to be repressed by YjjQ. At the flhDC promoter, the YjjQ DNA-binding site overlaps the RcsA-RcsB DNA-binding site. RcsA-RcsB likewise represses the flhDC promoter, but the repression by YjjQ and that by RcsA-RcsB are independent of each other. These data suggest that YjjQ is an additional regulator involved in the complex control of flhDC at the level of transcription initiation. Furthermore, we show that YjjQ represses motility of the E. coli K-12 laboratory strain and of uropathogenic E. coli (UPEC) strains CFT073 and 536. Regulation of flhDC, yfiRNB, and additional loci by YjjQ may be features relevant for pathogenicity. IMPORTANCE Escherichia coli is a commensal and pathogenic bacterium causing intra-and extraintestinal infections in humans and farm animals. The pathogenicity of E. coli strains is determined by their particular genome content, which includes essential and associated virulence factors that control the cellular physiology in the host environment. However, the gene pools of commensal and pathogenic E. coli are not clearly differentiated, and the function of virulence-associated loci needs to be characterized. In this study, we characterize the function of yjjQ, encoding a transcription regulator that was identified as being virulence associated in avian pathogenic E. coli (APEC). We characterize YjjQ as transcriptional repressor of flagellar motility and of additional loci related to pathogenicity