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Journal of Bacteriology, November 2000, p. 6434-6439, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Heterologous NNR-Mediated Nitric Oxide Signaling in Escherichia coli

Matthew I. Hutchings,1 Neil Shearer,1 Sarah Wastell,1 Rob J. M. van Spanning,2 and Stephen Spiro1,*

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom,1 and Department of Molecular Cell Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, NL-1081 HV Amsterdam, The Netherlands2

Received 11 July 2000/Accepted 5 September 2000

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melR promoter to lacZ, with a consensus FNR-binding site 41.5 bp upstream of the transcription start site. This promoter was activated by NNR under anaerobic growth conditions in media containing nitrate, nitrite, or the NO+ donor sodium nitroprusside. Activation by nitrate was abolished by a mutation in the molybdenum cofactor biosynthesis pathway, indicating a requirement for nitrate reductase activity. Activation by nitrate was modulated by the inclusion of reduced hemoglobin in culture media, because of the ability of hemoglobin to sequester nitric oxide and nitrite. The ability of nitrate and nitrite to activate NNR is likely due to the formation of NO (or related species) during nitrate and nitrite respiration. Amino acids potentially involved in NNR activity were replaced by site-directed mutagenesis, and the activities of NNR derivatives were tested in the E. coli reporter system. Substitutions at Cys-103 and Tyr-35 significantly reduced NNR activity but did not abolish the response to reactive nitrogen species. Substitutions at Phe-82 and Tyr-93 severely impaired NNR activity, but the altered proteins retained the ability to repress an FNR-repressible promoter, so these mutations have a "positive control" phenotype. It is suggested that Phe-82 and Tyr-93 identify an activating region of NNR that is involved in an interaction with RNA polymerase. Replacement of Ser-96 with alanine abolished NNR activity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.

* Corresponding author. Mailing address: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. Phone: 44 1603 593222. Fax: 44 1603 592250. E-mail: s.spiro{at}uea.ac.uk.

Journal of Bacteriology, November 2000, p. 6434-6439, Vol. 182, No. 22
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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