Molecular Genetics of Pectin Lyase Regulation in Erwinia

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Erwinia carotovora subsp. carotovora (hereafter Ecc) and most other soft-rot Erwinia spp. produce an assortment of extracellular degradative enzymes such as pectin lyase (Pnl), pectate lyase (Pel), polygalacturonase (Peh), cellulase, protease and phospholipase. A long-term goal of our research is to clarify the genetic and physiological aspects of extracellular enzyme production in soft-rot Erwinia and to elucidate the role of these enzymes in bacterial plant pathogenicity. We have shown that while pectinolytic (Pnl) and pectolytic (Pel and Peh) enzymes cause maceration of plant tissue, they are regulated differently. In contrast to pectolytic enzymes, most of which are induced by catabolic products of polygalacturonate or pectin, Pnl is induced by DNA-damaging agents such as mitomycin C, nalidixic acid, or ultraviolet light. In Ecc strain 71 (Ecc71) the induction of pnlA, the pectin lyase structural gene, by these DNA-damaging agents requires products of recA and an activator gene, digR (= regulator of damage-inducible genes). These products also appear to control bacteriocin (carotovoricin, Ctv) production and cellular lysis in this bacterium. We have reconstituted and inducible Pnl system in a RecA+ E. coli strain carrying a pnlA+ plasmid and a digR+ plasmid. The E. coli strain responds to the same regulatory signals as the Ecc71 Pnl producing system. Moreover, our recent findings indicate that digR and not pnlA is induced by DNA damaging agents in a RecA+ LexA+ E. coli. These observations suggest a cascade type of regulation wherein a regulatory gene (digR), required for the transcription of pnlA and possibly other damage-inducible genes, is controlled by DNA damaging agents. The objectives of this research are (a) to identify and characterize upstream DNA segments of pnlA that regulate transcription, (b) to characterize the activator gene, digR and to identify its product, (c) to elucidate the expression of digR, and (d) to understand the mechanism by which DigR activates pnlA transcription. In these studies we intend to utilize genetic and molecular technologies and various tools already available in E. coli and in Ecc71. The emerging knowledge should help in defining the regulatory circuit and in understanding the molecular events in the expression of a plant virulence factor by DNA-damaging agents.