Regulation of Coronatine Biosynthesis in Pseudomonas Syringae

[9316488] Bender植物毒素coronatine是由丁香假单胞菌（Pseudomonas syringae）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如何调节毒力因子。此外，细菌植物毒素生物合成所需基因的组织和功能尚未得到深入研究。本研究的长期目标是阐明紫丁香中冠状碱产生的调控机制。冠状酸具有独特的结构，源自两种不相关的生物合成途径：冠状酸（CFA）源自聚酮，而冠状酸（CMA）是源自异亮氨酸的乙基环丙基氨基酸。这两个组分是底物取食研究和互补分析，已用于确定CFA和CMA生产所需的冠状碱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，将利用Northern blot分析和转录融合来阐明冠状花序生物合成簇的转录组织和调控。cpl启动子和调控cpl转录的因子的性质将通过引物扩展、缺失分析和基因融合来鉴定。cpl蛋白产物将被大量生产，并用于体外酶活性的研究。这些结果将大大提高我们对植物毒素生产的调控和生物合成机制的理解。这项工作是国际合作努力的一部分，旨在了解丁香假单胞菌的作用方式、生物合成和调节或冠状物质的产生。这项研究将有助于我们理解为什么有些细菌能够感染植物，而另一些却不能。＊＊＊

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. ***