Open AccessExpression and analysis of coronafacate ligase, a thermoregulated gene required for production of the phytotoxin coronatine in Pseudomonas syringae
Author(s) -
Rangaswamy V,
Ullrich M,
Jones W,
Mitchell R,
Parry R,
Reynolds P,
Bender C.L
Publication year - 1997
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1997.tb12625.x
Subject(s) - pseudomonas syringae , coronatine , biology , adenylylation , phytotoxin , biochemistry , gene , dna ligase , microbiology and biotechnology , biosynthesis , mutant , toxin , arabidopsis
Coronafacic acid, the polyketide component of the phytotoxin coronatine, is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the c orona f acate l igase ( cfl ) gene product. In the present study, cfl was fused to the carboxy terminus of malE , which encodes the maltose‐binding protein (MBP), and overexpressed in Escherichia coli . Immunoblot analysis indicated that Cfl contained an ATP‐binding region, a motif conserved in enzymes which 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 fusion of the cfl promoter to a promoterless glucuronidase gene were used to follow the temporal expression of coronafacate ligase. The results indicated that transcription of cfl is temperature‐sensitive. Furthermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.