Biosynthesis and Regulation of the Phytotoxin Coronatine in Pseudomonas Syringae

Bender 9603618冠菌素是由植物病原细菌假单胞菌产生的植物毒素。以前的工作表明冠菌素的生物合成受温度的调节，并由三种调节蛋白（CorR，CorS和CorP）控制。该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的冠菌素基因表达。还将研究由经修改的双组分调控系统编码的三种调控蛋白的功能作用。将纯化每种蛋白质，体外试验将解决以下问题：（a）CorS作为冠菌素生物合成的正调节剂和负调节剂的作用;（B）CorR和CorP在磷酸化中的潜在参与;以及（c）CorR作为冠菌素基因簇中所有热调节转录物的转录激活剂的作用。先前的工作还表明，冠菌素的一种组分冠酸的生物合成需要单和多功能聚酮化合物合成酶的协同作用。本研究将证实多功能聚酮合成酶在冠伐酸生物合成中的作用。将获得冠状酸基因簇的完整核苷酸序列，并用于推断与冠状酸生物合成相关的大蛋白质内的催化位点的功能作用。非极性突变将在冠状酸基因簇内构建，并用于评估环戊烯化合物在冠状酸生物合成中的潜在参与。 意义许多由植物病原细菌P. lichingae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也可能受到温度的控制。此外，参与冠菌素B合成的调控可能具有更广泛的意义，并且与基因表达受温度控制的其他细菌相关。鉴定对降低的温度作出响应的调节系统将在需要在较低温度下基因表达的情况下具有实际用途。此外，编码聚酮冠酸的基因簇的表征将对其他研究聚酮的研究小组，特别是那些研究多功能聚酮合成酶的研究小组有巨大的好处。大多数关于后一类酶的研究主要发生在工业中，并且数据通常不向公众发布。此外，许多以前的工作对聚酮化合物发生与革兰氏阳性细菌，但很少有人知道在革兰氏阴性细菌，如假单胞菌的聚酮化合物的合成。假单胞菌中聚酮基因簇的修饰在增加具有改变的生物合成和抗微生物活性的新化合物的数量方面具有巨大的潜力。这项研究将有助于实现这一目标，阐明基因参与的生物合成的coronafacic酸，一个复杂的聚酮化合物的混合来源。

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.