Molecular Genetics of Pectin Lyase Regulation in Erwinia

9419403卡特吉欧文氏胡萝卜亚种食胡萝卜菌和大多数软腐欧文氏菌都能产生多种胞外降解酶，如果胶Iyase(PNL)、果胶Iyase(PEL)、多聚半乳糖醛酸酶(Peh)、纤维素酶(Cel)、蛋白酶(Prt)和磷脂酶。本研究的一个长期目标是阐明软腐欧文氏菌产生胞外酶的遗传和生理方面，并阐明这些酶在细菌植物致病性中的作用。提纯的PNL，如PEL和PEH，可以浸泡植物组织。遗传数据还表明，PNL参与了ECC的毒力。然而，我们的研究在NSF的支持下发现，PNL的调控方式与PEL、Peh、Cel和PRT不同。在我的实验室中使用的ECC模型菌株Ecc71以及许多其他欧文氏菌中，PNL的产生是在对DNA损伤剂施加的压力做出反应时被激活的。这种将毒力因子的产生与DNA损伤联系在一起的不同寻常的反应，除了RecA之外，还受到一个独特的调控系统的控制，其中包括新发现的RDG基因(RDG=损伤诱导基因的调节因子)。2.1kb的RDG区包含两个调控基因rdgA和rdgB，它们被组织为单独的转录单位。RdgA和RdgB中都存在螺旋-转角-螺旋基序，表明它们可能与DNA结合。RdgA与噬菌体抑制子、RdgB与转录激活子之间有很高的同源性。RdgA和rdgB中可能的操纵子的这些特征和结构上的相似性促使人们假设RdgA作为一种抑制因子，可能调节自己的表达，也可能调节rdgB的表达。通过启动子的转换，已经确定rdgB产物激活了大肠杆菌中PNL的产生，但RdG+DNA的缺失分析表明，当rdgB由自身的启动子驱动时，rdgA和rdgB的功能都是诱导丝裂霉素C(MC)产生PNL所必需的。随后发现，rdgB转录的激活需要MC和功能性的recA和rdgA，这暗示了RecA处理的RdgA衍生物在rdgB表达中的作用。因此，RdgA被认为扮演着两个关键角色：在天然状态下，RdgA发挥抑制因子的作用，而经RecA处理的RdgA则充当转录激活因子。该项目的主要目标是检验这一假说，并阐明RdgB激活ECC71中PNL产生的机制。具体目标是(A)分离RdgA，(B)鉴定由RecA*产生的RdgA衍生物(即具有蛋白酶活性的激活的RecA)，(C)通过报告基因和DNA结合分析来阐明rdgA的调控，(D)鉴定激活rdgB转录所需的RecA*衍生的RdgA产物，以及(E)检测RdgB与果胶水解酶结构基因PnlA上游序列的结合。这项研究应该阐明一种新的调控电路控制细菌植物毒力因子产生的分子机制。这可能会带来更好的方法来抗击农作物的细菌性疾病。***

9419403 Chatterjee Erwinia carotovora subsp. carotovora (hereafter Ecc) and most other soft-rotting Erwinia species produce an assortment of extracellular degradative enzymes such as pectin Iyase (Pnl), pectate Iyase (Pel), polygalacturonase (Peh), cellulase (Cel), protease (Prt) and phospholipase. A long-term goal of this research is to clarify the genetic and physiological aspects of extracellular enzyme production in soft-rotting Erwinia and to elucidate the roles of these enzymes in bacterial plant pathogenicity. Purified Pnls, like Pel and Peh, can macerate plant tissues. Genetic data also indicate that Pnl contributes to virulence of Ecc. However, our research, supported by NSF, has revealed that Pnl is regulated differently from Pel, Peh, Cel and Prt. In Ecc71, the model Ecc strain used in my laboratory, as well as in many other Erwinia, Pnl production is activated in response to stress imposed by DNA-damaging agents. This unusual response, which links the production of a virulence factor with DNA damage, is controlled by a unique regulatory system involving, in addition to RecA, the newly discovered rdg genes (rdg = regulator of damage-inducible gene). The 2.1 kb rdg region contains two regulatory genes, rdgA and rdgB, organized as separate transcriptional units. The presence of helix-turn-helix motifs in both RdgA and RdgB indicates that they probably can bind DNA. There is high homology between RdgA and bacteriophage repressors and between RdgB and transcriptional activators. These features and structural similarity between the putative operators in rdgA and rdgB prompted the hypothesis that RdgA, as a repressor, may regulate its own expression and possibly also rdgB expression. By promoter switching, it has been determined that rdgB product activated Pnl production in E. coli, yet deletion analysis of rdg+ DNA indicated that when rdgB was driven by its own promoter, functions of both rdgA and rdgB were necessary for the induction of Pnl production by mitomycin C (MC). The activation of rdgB transcription was subsequently found to require MC and functional recA and rdgA, implicating a role of a RecA processed RdgA derivative in rdgB expression. Thus, RdgA is postulated to play two key roles: in the native state RdgA functions as a repressor whereas the RecA processed RdgA serves as a transcriptional activator. The major goals of this project are to test this hypothesis and to elucidate the mechanism by which RdgB activates Pnl production in Ecc71. The specific objectives are (a) to fractionate RdgA, (b) to characterize RdgA derivatives produced by RecA* (i.e., activated RecA with protease activity), (c) to elucidate the regulation of rdgA by the use of reporter genes and DNA binding assays, (d) to identify the RecA* derived RdgA product required for the activation of rdgB transcription, and (e) to examine binding of RdgB to the sequences upstream of PnlA, the structural gene for pectin Iyase. %%% This research should elucidate the molecular mechanisms by which a novel regulatory circuit controls the production of a bacterial plant virulence factor. It could lead to better ways to combat bacterial diseases of crop plants. ***