EXPRESSION AND ANALYSIS OF CORONAFACATE LIGASE, A THERMOREGULATED GENE REQUIRED FOR PRODUCTION OF THE PHYTOTOXIN CORONATINE IN PSEUDOMONAS-SYRINGAE

Coronafacic acid, the polyketide component of the phytotoxin coronatin e, is activated and coupled to coronamic acid via amide bond formation , a biosynthetic step presumably catalyzed by the coronafacate ligase (cfl) gene product. In the present study, cfl was fused to the carboxy terminus of malE, which encodes the maltose-binding protein (MBP), an d overexpressed in Escherichia coli. Immunoblot analysis indicated tha t Cn contained an ATP-binding region, a motif conserved in enzymes whi ch activate their substrates by adenylation. MBP-Cfl was overproduced and purified from Pseudomonas syringae and the protein fusion was used to generate antisera. Anti-MBP-Cfl antibodies and a transcriptional f usion of the cfl promoter to a promoterless glucuronidase gene were us ed to follow the temporal expression of coronafacate ligase. The resul ts indicated that transcription of cfl is temperature-sensitive. Furth ermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.