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 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.
Rangaswamy
V
V
Department of Plant Pathology, Oklahoma State University, Stillwater 74078-3032, USA.
Ullrich
M
M
Jones
W
W
Mitchell
R
R
Parry
R
R
Reynolds
P
P
Bender
C L
CL
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
D000596:Amino Acids; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007192:Indenes; C058487:coronatine; D019732:Amide Synthases; C092402:Cfl protein, Pseudomonas syringae