Molecular Genetics of Pectin Lyase Regulation in Erwinia

胡萝卜欧文氏菌亚种Carotovora(以下简称ECC)和大多数其他软腐欧文氏菌(Erwinia spp.产生各种胞外降解酶，如果胶裂解酶(PNL)、果胶裂解酶(PEL)、多聚半乳糖醛酸酶(PEH)、纤维素酶、蛋白酶和磷脂酶。我们研究的一个长期目标是阐明软腐欧文氏菌胞外酶产生的遗传和生理方面，并阐明这些酶在细菌植物致病性中的作用。我们已经证明，虽然果胶分解酶(PNL)和果胶分解酶(PEL和PEH)会导致植物组织的浸渍，但它们的调控方式不同。与大多数由聚半乳糖醛酸酯或果胶的分解产物诱导的果胶水解酶不同，PNL是由DNA损伤剂诱导的，如丝裂霉素C、奈利地克酸或紫外线。在ECC株71(Ecc71)中，这些DNA损伤剂诱导果胶裂解酶结构基因pnlA的产生需要recA和激活基因DigR(=损伤诱导基因的调节)的产物。这些产品似乎也控制细菌素(胡萝卜素，CTV)在这种细菌中的产生和细胞裂解。我们在携带pnlA+和DigR+两种pnlA+质粒的RecA+大肠杆菌中重组并诱导了PnL系统。该大肠杆菌菌株对与Ecc71 PNL生产系统相同的调控信号做出反应。此外，我们最近的发现表明，在RecA+LexA+E.Coli中，DIGR而不是pnlA是由DNA损伤剂诱导的。这些观察表明了一种级联类型的调控，其中pnlA和可能的其他损伤诱导基因的转录所需的调控基因(DIGR)由DNA损伤剂控制。本研究的目的是(A)鉴定和鉴定pnlA的上游调控转录的DNA片段，(B)鉴定激活基因DigR并鉴定其产物，(C)阐明DigR的表达，以及(D)了解DigR激活pnlA转录的机制。在这些研究中，我们打算利用基因和分子技术以及在大肠杆菌和ECC71中已有的各种工具。新出现的知识应该有助于确定调控回路，并有助于理解DNA损伤剂表达植物毒力因子的分子事件。

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.