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.
