Molecular basis of regulation of galacturonic acid transport by the regulatory protein exuR in Erwinia chrysanthemi EC16
Erwinia chrysanthemi causes soft-rot disease in crop plants by secreting large amounts of extracellular plant cell wall degrading enzymes. Pectate lyases, enzymes that depolymerize pectinaceous cementing material of plant cell wall, play a predominant role in causing the maceration and destruction of plant tissue. Degradation of pectin, a polymer of a-l,4-linked D-galacturonic acid (GalUA), results in the formation of monomers and multimers of GalUA residues. Bacterial uptake of pectin degradation products provides inducers for the synthesis of higher levels of pectate lyases and therefore important for disease initiation and progression. Transport of the GalUA monomer is mediated by the exuT gene product. Mutants in exuT have been observed to have a significantly lower capability to macerate potato tuber tissue than the wild type parent. The focus of this research was to characterize the mechanism of regulation of the exuT geuQ expression to better understand its role in the vimlence potential of E. chrysanthemi. Preliminary genetic analyses have indicated the role of a repressor protein encoded by the exuR gene in the regulation of exuT. An exuR mutant was therefore constructed to study its role in the regulation of ^xwr expression. Northem blot analysis indicated that the exuR mutation resulted in constitutive transcription of the exuT gene. A detailed profile of exwr transcription was obtained both in vivo and in planta using an exuT-luciferase reporter gene fusion. In vivo, exuT-lux expression was inducible in wild type cells but constitutive in the exuR mutant indicating the loss of a repressor function. In planta, exuT-lux expression was markedly higher in the exuR mutant compared to the parental strain. [14CjGalUA uptake ability was also deregulated and constitutive in the exuR mutant. These results strongly suggest that ExuR is the major regulatory protein negatively controlling exuT expression. Furthermore, the ExuR protein was overexpressed, purified and used in electrophoretic mobility shift assays with the exuT 5' upstream region. Purified ExuR was capable of specifically interacting with the exuT 5' upstream region. This is important evidence for the direct role of ExuR in the regulation of exwr transcription. The present study provides a better understanding of the coordinated regulation ofexuTdind virulence gene expression in E. chrysanthemi EC 16.