Regulation of Coronatine Biosynthesis in Pseudomonas Syringae

9316488冠状植物毒素是由植物病原菌紫丁香假单胞菌引起的几种植物病害的重要毒力因子。了解致病性的分子基础的基础是确定毒力因子是如何调节的。此外，细菌植物毒素生物合成所需基因的组织和功能还没有得到深入的研究。这项研究的一个长期目标是阐明调节紫丁香树冠状病毒产生的机制。冠花碱具有独特的结构，来源于两条互不相关的生物合成途径：冠花酸(CFA)是聚酮类化合物，而冠花酸(CMA)是异亮氨酸衍生的乙基环丙基氨基酸。这两个组分是底物馈送研究，互补分析已被用于确定产生CFA和CMA所需的冠状生物合成簇的区域以及催化酰胺键形成的偶联(CPL)基因。在这项工作中，Northern印迹分析和转录融合将被用来阐明冠状病毒生物合成簇的转录组织和调控。Cpl启动子的性质和调控Cpl转录本的因素将通过引物延伸、缺失分析和基因融合来鉴定。Cpl的蛋白产物将被大量生产，并用于其体外酶活性的研究。这些结果将大大加深我们对植物毒素生产中的调控和生物合成机制的理解。这项工作是一项国际合作努力的一部分，目的是了解丁香松的作用方式、生物合成和调节或冠状生成。这项研究将有助于我们理解一些细菌如何能够感染植物，而另一些细菌则不能。***

9316488 Bender The phytotoxin coronatine is an important virulence factor in several plant disease caused by the phytopathogenic bacterium, Pseudomonas syringae. Fundamental to understanding the molecular basis of pathogenicity is to determine how virulence factors are modulated. Furthermore, the organization and function of genes required for biosynthesis of bacterial phytotoxins has not been intensively studied. A long-term goal of this research is to elucidate mechanisms that regulate coronatine production in P. syringae. Coronatine has a unique structure derived from two unrelated biosynthetic pathways: coronafacic acid (CFA) is of polyketide origin, whereas coronamic acid (CMA), is an ethylcyclopropyl amino acid derived from isoleucine. These two components are substrate feeding studies and complementation analyses have been used to identify regions of the coronatine biosynthetic cluster required for CFA and CMA production and the coupling (cpl) gene which catalyze amide bond formation. In this work, Northern blot analyses and transcriptional fusions will be utilized to elucidate the transcriptional organization and regulation of the coronatine biosynthetic cluster. The nature of the cpl promoter and factors which regulated the cpl transcript will be identified using primer extension, deletion analysis and gene fusions. The protein product of cpl will be overproduced and used to study its enzymatic activity in vitro. The results will significantly enhance our understanding of the regulatory controls and biosynthetic mechanisms employed in phytotoxin production. This work is part of an international collaborative effort to understand the mode of action, biosynthesis, and regulation or coronatine production in P. syringae. %%% This research will aid in our understanding of how some bacteria are able to infect plants while others cannot. ***