Biosynthesis and Regulation of the Phytotoxin Coronatine in Pseudomonas Syringae

本德尔9603618冠状毒素是由植物病原菌紫丁香假单胞菌产生的一种植物毒素。以往的工作表明，冠状病毒的生物合成受温度的调控，并受三种调控蛋白(Corr、CORS和Corp)的控制。这项研究项目的一个目标是利用遗传方法来确定这三种调节蛋白是否直接负责体温调节，或者是否存在另一种水平的控制，并负责低温下冠状病毒基因的表达。还将研究由修改后的双组分调控系统编码的三种调控蛋白的功能作用。每个蛋白质都将被提纯，体外分析将涉及以下问题：(A)CORs作为冠状病毒生物合成的正负调控因子的作用；(B)Corr和Corp在磷酸传递中的潜在参与；以及(C)Corr作为冠状病毒基因簇中所有热调节转录物的转录激活因子的作用。先前的工作也表明，冠麻酸的生物合成需要单功能和多功能聚酮合成酶的协调作用。这项研究将证实多功能聚酮合成酶在冠麻酸生物合成中的作用。将获得冠状表面酸基因簇的完整核苷酸序列，并用于推断与冠状表面酸生物合成相关的大蛋白中催化位点的功能作用。非极性突变将在冠麻酸基因簇中构建，并用于评估环戊烯化合物参与冠麻酸生物合成的可能性。意义重大。许多由植物病原细菌丁香假单胞菌引起的疾病在较低的温度下更严重，因此这种细菌的其他特征很可能也受温度控制。此外，冠状病毒生物合成中涉及的调控措施可能具有更广泛的意义，并与其他基因表达受温度控制的细菌相关。在需要在较低温度下表达基因的情况下，识别一种对温度降低作出反应的调节系统将具有实际用途。此外，编码聚酮冠状脂肪酸的基因簇的特征将对其他致力于聚酮的研究小组，特别是那些致力于多功能聚酮合成酶的研究小组，具有极大的益处。大多数关于后一类酶的研究主要是在工业中进行的，数据通常不会向公众公布。此外，以前关于聚酮的大部分工作都是在革兰氏阳性细菌中进行的，但对革兰氏阴性细菌(如假单胞菌)的聚酮合成知之甚少。假单胞菌中聚酮基因簇的修饰具有巨大的潜力，可以增加具有改变生物合成和抗菌活性的新化合物的数量。这项研究将通过阐明冠麻酸的生物合成所涉及的基因，为实现这一目标做出贡献。冠麻酸是一种混合来源的复杂聚酮。

Bender 9603618 Coronatine is a phytotoxin produced by the plant pathogenic bacterium Pseudomonas syringae. Previous work showed that coronatine biosynthesis is regulated by temperature and controlled by three regulatory proteins (CorR, CorS, and CorP). One objective of this research project is to utilize a genetic approach to determine if the three regulatory proteins are directly responsible for thermoregulation, or if another level of control is present and responsible for coronatine gene expression at low temperatures. The functional role of the three regulatory proteins encoded by the modified two-component regulatory system will also be investigated. Each protein will be purified and in vitro assays will address the following: (a) the role of CorS as both a positive and negative regulator of coronatine biosynthesis; (b) the potential involvement of CorR and CorP in a phosphorelay; and (c) the role of CorR as a transcriptional activator of all thermally regulated transcripts in the coronatine gene cluster. Previous work has also shown that the biosynthesis of coronafacic acid, a component of coronatine, was shown to require the coordinated effort of mono- and multifunctional polyketide synthetases. This research will confirm the role of a multifunctional polyketide synthetase in the biosynthesis of coronafacic acid. The complete nucleotide sequence of the coronafacic acid gene cluster will be obtained and used to deduce the functional roles of catalytic sites within the large proteins associated with coronafacic acid biosynthesis. Nonpolar mutations will be constructed within the coronafacic acid gene cluster and used to assess the potential involvement of a cyclopentenene compound in coronafacic acid biosynthesis. Significance. Many of the diseases caused by the plant pathogenic bacterium P. syringae are more severe at cooler temperatures, so it is likely that other traits in this bacterium are also controlled by temperature. Furthermore, the regulatory controls involved in coronatine b iosynthesis are likely to have a much broader significance and be relevant in other bacteria where gene expression is controlled by temperature. The identification of a regulatory system which responds to reduced temperatures will be of practical usage in situations where gene expression at lower temperatures is desirable. Furthermore, the characterization of the gene cluster encoding the polyketide coronafacic acid will be of immense benefit to other research groups working on polyketides, but especially those working on multifunctional polyketide synthetases. Most research on the latter class of enzymes occurs largely in industry, and the data is generally not released to the public. Also, much of the previous work on polyketides has occurred with Gram-positive bacteria, but little is known about polyketide synthesis in Gram-negative bacteria such as Pseudomonas. The modification of polyketide gene clusters in Pseudomonas has enormous potential to increase the number of new compounds with altered biosynthetic and antimicrobial activities. This research will contribute towards that goal by elucidating the genes involved in the biosynthesis of coronafacic acid, a complex polyketide of mixed origins.