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J Bacteriol, May 1998, p. 2367-2372, Vol. 180, No. 9
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites

Sudha A. Chugani,1 Matthew R. Parsek,2 and A. M. Chakrabarty1,*

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612,1 and Department of Microbiology, University of Iowa, Iowa City, Iowa 522422

Received 26 November 1997/Accepted 23 February 1998

The catBCA operon of Pseudomonas putida encodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to the catBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of the catBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured beta -galactosidase activity of catB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catB structural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

* Corresponding author. Mailing address: Department of Microbiology and Immunology (M/C 790), University of Illinois College of Medicine, 835 South Wolcott Ave., Chicago, IL 60612. Phone: (312) 996-4586. Fax: (312) 996-6415. E-mail: Ananda.Chakrabarty{at}uic.edu.

J Bacteriol, May 1998, p. 2367-2372, Vol. 180, No. 9
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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