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Journal of Bacteriology, August 1998, p. 4192-4198, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Fnr, NarP, and NarL Regulation of Escherichia coli K-12 napF (Periplasmic Nitrate Reductase) Operon Transcription In Vitro

Andrew J. Darwin,1,dagger Eva C. Ziegelhoffer,2,Dagger Patricia J. Kiley,3 and Valley Stewart1,*

Section of Microbiology, Cornell University, Ithaca, New York 14853,1 and Departments of Bacteriology2 and Biomolecular Chemistry,3 University of Wisconsin, Madison, Wisconsin 53706

Received 16 March 1998/Accepted 17 June 1998

The expression of several Escherichia coli operons is activated by the Fnr protein during anaerobic growth and is further controlled in response to nitrate and nitrite by the homologous response regulators, NarL and NarP. Among these operons, the napF operon, encoding a periplasmic nitrate reductase, has unique features with respect to its Fnr-, NarL-, and NarP-dependent regulation. First, the Fnr-binding site is unusually located compared to the control regions of most other Fnr-activated operons, suggesting different Fnr-RNA polymerase contacts during transcriptional activation. Second, nitrate and nitrite activation is solely dependent on NarP but is antagonized by the NarL protein. In this study, we used DNase I footprint analysis to confirm our previous assignment of the unusual location of the Fnr-binding site in the napF control region. In addition, the in vivo effects of Fnr-positive control mutations on napF operon expression indicate that the napF promoter is atypical with respect to Fnr-mediated activation. The transcriptional regulation of napF was successfully reproduced in vitro by using a supercoiled plasmid template and purified Fnr, NarL, and NarP proteins. These in vitro transcription experiments demonstrate that, in the presence of Fnr, the NarP protein causes efficient transcription activation whereas the NarL protein does not. This suggests that Fnr and NarP may act synergistically to activate napF operon expression. As observed in vivo, this activation by Fnr and NarP is antagonized by the addition of NarL in vitro.

* Corresponding author. Present address: Section of Microbiology, University of California, 156 Hutchison Hall, One Shields Ave., Davis, CA 95616-8665. Phone: (530) 754-7994. Fax: (530) 752-9014. E-mail: vjstewart{at}ucdavis.edu.

dagger Present address: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110-1093.

Dagger Present address: Division of Biology 156-29, California Institute of Technology, Pasadena, CA 91125.

Journal of Bacteriology, August 1998, p. 4192-4198, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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