CcaR is an autoregulatory protein that binds to the ccaR and cefD-cmcI promoters of the cephamycin C-clavulanic acid cluster in Streptomyces clavuligerus.
The putative regulatory CcaR protein, which is encoded in the beta-lactam supercluster of Streptomyces clavuligerus, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in Escherichia coli, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from S. clavuligerus was able to bind DNA fragments containing the promoter regions of the ccaR gene itself and the bidirectional cefD-cmcI promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including lat, blp, claR, car-cyp, and the unlinked argR gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies. ccaR and the bidirectional cefD-cmcI promoter region were fused to the xylE reporter gene and expressed in Streptomyces lividans and S. clavuligerus. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the ccaR promoter region lacking its coding sequence in a high-copy-number plasmid in S. clavuligerus ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the cefD-cmcI bidirectional promoter region.
Santamarta
Irene
I
Area de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain.
Rodríguez-García
Antonio
A
Pérez-Redondo
Rosario
R
Martín
Juan F
JF
Liras
Paloma
P
D000591:Amino Acid Isomerases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D002513:Cephamycins / Q000096:biosynthesis*; D019818:Clavulanic Acid / Q000096:biosynthesis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005798:Genes, Bacterial; D005809:Genes, Regulator; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D001426:Bacterial Proteins; D002513:Cephamycins; C112951:ClaR protein, Streptomyces clavuligerus; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019818:Clavulanic Acid; D000591:Amino Acid Isomerases; C040886:isopenicillin N epimerase; C024925:cephamycin C