THE PECT REPRESSOR INTERACTS WITH REGULATORY REGIONS OF PECTATE LYASEGENES IN ERWINIA-CHRYSANTHEMI

Erwinia chrysanthemi is a broad host range phytopathogenic enterobacte rium responsible for soft-rat disease of many plant species. The peer gene encodes a repressor that negatively regulates the expression of v irulence factors, such as pectinases, motility or exopolysaccharide sy nthesis. The cloned peer gene was overexpressed using a phage T7 syste m. The purification of PecT involved the use of a TSK-heparin column a nd delivered the PecT protein that was purified to near homogeneity. T he purified repressor displayed a 34 kDa apparent molecular mass. Gel- filtration experiments revealed that the PecT protein is a dimer. Band -shift assays demonstrated that the tetramer of the PecT protein could specifically bind in vitro to the regulatory regions of the pectate l yase genes with variable affinities. In addition, we demonstrated that PecT represses its own synthesis by interacting independently with tw o 200 bp regions, R1 and R2, located from -382 to -632 and -17 to -234 , respectively, from the distal P1 promoter and from -465 to -715 and -100 to -317 from the P2 proximal promoter. We propose a model that ex plains the regulation exerted by PecT on its target genes and that int egrates the phenotype obtained with a PecT overproducing pec(-1) mutan t or a pecT mutant. (C) 1998 Elsevier Science B.V. All rights reserved .