JOINT TRANSCRIPTIONAL CONTROL OF XPSR, THE UNUSUAL SIGNAL INTEGRATOR OF THE RALSTONIA-SOLANACEARUM VIRULENCE GENE REGULATORY NETWORK, BY A RESPONSE REGULATOR AND A LYSR-TYPE TRANSCRIPTIONAL ACTIVATOR

Ralstonia (Pseudomonas) solanacearum is a soil-borne phytopathogen tha t causes a wilting disease of many important crops. It makes large amo unts of the exopolysaccharide EPS I, which it requires for efficient c olonization, wilting, and killing of plants. Transcription of the eps operon, encoding biosynthetic enzymes for EPS I, is controlled by a un ique and complex sensory network that responds to multiple environment al signals. This network is comprised of the novel transcriptional act ivator XpsR, three distinct two-component regulatory systems (VsrAD, V srBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that the xpsR promoter (P-xpsR) is sim ultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into the eps promoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of P-xpsR to show the following. (i) PhcA primari ly interacts with a typical 14-bp LysR-type consensus around position -77, causing a sixfold activation of P-xpsR; a weaker, less-defined bi nding site between -183 and -239 likely enhances PhcA binding and acti vation via the -77 site another twofold. (ii) Full 70-fold activation of P-xpsR requires the additional interaction of the VsrD response reg ulator (or its surrogate) with a 14-bp dyadic sequence centered around -315 where it enhances activation (and possibly binding) by PhcA; how ever, VsrD alone cannot activate P-xpsR. (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect P-xpsR activation or regulation.