title: Expression and analysis of coronafacate ligase, a thermoregulated gene required for production of the phytotoxin coronatine in Pseudomonas syringae.
abstract: 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 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), 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.
authors.affiliation: Department of Plant Pathology, Oklahoma State University, Stillwater 74078-3032, USA.
authors.forename: V
authors.initials: V
authors.lastname: Rangaswamy
authors.forename: M
authors.initials: M
authors.lastname: Ullrich
authors.forename: W
authors.initials: W
authors.lastname: Jones
authors.forename: R
authors.initials: R
authors.lastname: Mitchell
authors.forename: R
authors.initials: R
authors.lastname: Parry
authors.forename: P
authors.initials: P
authors.lastname: Reynolds
authors.forename: C L
authors.initials: CL
authors.lastname: Bender
mesh_terms: D019732:Amide Synthases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism*; D000918:Antibody Specificity; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D015151:Immunoblotting; D007192:Indenes / Q000032:analysis / Q000378:metabolism*; D016296:Mutagenesis; D010641:Phenotype; D017434:Protein Structure, Tertiary; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007192:Indenes; C058487:coronatine; D019732:Amide Synthases; C092402:Cfl protein, Pseudomonas syringae