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Journal of Bacteriology, June 2002, p. 3106-3113, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.3106-3113.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

CcaR Is an Autoregulatory Protein That Binds to the ccaR and cefD-cmcI Promoters of the Cephamycin C-Clavulanic Acid Cluster in Streptomyces clavuligerus

Irene Santamarta, Antonio Rodríguez-García, Rosario Pérez-Redondo, Juan F. Martín, and Paloma Liras*

Area de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain

Received 2 January 2002/ Accepted 5 March 2002

The putative regulatory CcaR protein, which is encoded in the ß-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.

* Corresponding author. Mailing address: Area de Microbiología, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain. Phone: 34 987 291504. Fax: 34 987 291506. E-mail: degplp{at}unileon.es.

Journal of Bacteriology, June 2002, p. 3106-3113, Vol. 184, No. 11
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.11.3106-3113.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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