Multicomponent Transcriptional Regulation at the Complex Promoter of the Exopolysaccharide I Biosynthetic Operon of Ralstonia solanacearum

Abstract

ABSTRACT High-level transcription of eps , an operon encoding biosynthesis of an exopolysaccharide virulence factor of the phytopathogen Ralstonia ( Pseudomonas ) solanacearum , requires the products of at least seven regulatory genes ( phcA , phcB , xpsR , vsrA-vsrD , and vsrB-vsrC ), which are organized in three converging signal transduction cascades. Because xpsR and the vsrB-vsrC two-component system are the most downstream cascade components required for activation of eps , we explored how these components control transcription from the eps promoter (P eps ). Deletion and PCR mutagenesis identified an upstream region of P eps (nucleotides −82 to −62) that is critical for transcription activation by VsrB-VsrC and XpsR and also is required for negative control of P eps by the putative eps regulator EpsR. Using PCR mutagenesis we generated the vsrC1 allele that encodes a response regulator that constitutively activates P eps in the absence of its cognate sensor, VsrB. However, activation of P eps by vsrC1 still required xpsR . Unexpectedly, the amino acid substitution conferring the constitutive phenotype on VsrC1 is 12 residues from its C terminus, outside the known functional domains of response regulators. Finally, a modified DNase I footprinting method was used to demonstrate specific binding of both VsrC1 and VsrC to the −72 to −62 upstream region of P eps .