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Journal of Bacteriology, December 1999, p. 7274-7284, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

General Nitrogen Regulation of Nitrate Assimilation Regulatory Gene nasR Expression in Klebsiella oxytoca M5al

Stephen Qitu Wu,1,dagger Weihang Chai,1,Dagger Janine T. Lin,1,§ and Valley Stewart1,2,*

Section of Microbiology, Cornell University, Ithaca, New York 14853-8101,1 and Section of Microbiology, University of California, Davis, California 95616-86652

Received 28 July 1999/Accepted 22 September 1999

Klebsiella oxytoca can assimilate nitrate and nitrite by using enzymes encoded by the nasFEDCBA operon. Expression of the nasF operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator. This paper reports our analysis of nasR gene expression. We constructed strains bearing single-copy Phi (nasR-lacZ) operon fusions within the chromosomal rhaBAD-rhaSR locus. The expression of Delta rhaBS::[Phi (nasR-lacZ)] operon fusions was induced about 10-fold during nitrogen-limited growth. Induction was reduced in both ntrC and rpoN null mutants, indicating that Ntr control of nasR gene expression requires the NtrC and sigma N (sigma 54) proteins. Sequence inspection of the nasR control region reveals an apparent sigma N-dependent promoter but no apparent NtrC protein binding sites. Analysis of site-specific mutations coupled with primer extension analysis authenticated the sigma N-dependent nasR promoter. Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of beta -galactosidase expression indistinguishable from Phi (nasR-lacZ) constructs with about 470 nt upstream. Expression was independent of the Nac protein, implying that NtrC is a direct activator of nasR transcription. Together, these results indicate that nasR gene expression does not require specific upstream NtrC-binding sequences, as previously noted for argT gene expression in Salmonella typhimurium (G. Schmitz, K. Nikaido, and G. F.-L. Ames, Mol. Gen. Genet. 215:107-117, 1988).

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

dagger Present address: Biological Research and Development, Fort Dodge Animal Health, Fort Dodge, IA 50501.

Dagger Present address: Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY 14853.

§ Present address: Department of Fungal Molecular Biology, Novo Nordisk Biotech, Inc., Davis, CA 95616.

Journal of Bacteriology, December 1999, p. 7274-7284, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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