Characterization of the genes controlling the biosynthesis of the polyketide phytotoxin coronatine including conjugation between coronafacic and coronamic acid

Abstract

Pseudomonas syringae pv. glycinea PG4180 produces a chlorosis-inducing phytotoxin, coronatine (COR), which consists of a polyketide component, coronafacic acid (CFA), which is coupled via amide bond formation to coronamic acid (CMA), an ethylcyelopropyl amino acid (aa) derived from isoleucine. P. syringae pv. syringae strains PS51 and PS61, which do not synthesize coronafacoyl compounds (conjugates between CFA and aa), acquired the ability to produce CFA and COR when transformed with p4180A, a 90-kb indigenous plasmid in PG4180. Tn5 mutagenesis indicated that the COR biosynthetic genes in PG4180 are clustered within a 30-kb region on p4180A. The phenotype of selected COR-defective mutants was determined by supplying them with CFA and CMA and by complementation studies with cloned DNA from the COR biosynthetic cluster. Using this approach, the regions encoding CFA and CMA synthesis and coupling activity were localized to the 24-, 12.5- and 2.3-kb regions of the cluster, respectively. Mutants in a 6-kb region required the addition of both CFA and CMA for COR synthesis, which may indicate a regulatory role for this part of the cluster. PS51 and PS61 transconjugants containing cloned DNA from the coupling region produced COR when supplied with CFA and CMA, indicating that coupling activity was cloned and expressed in bacteria lacking the COR biosynthetic cluster.