Heterologous NNR-Mediated Nitric Oxide Signaling in Escherichia coli

Hutchings, Matthew I., Shearer, Neil, Wastell, Sarah, van Spanning, Rob J. M. and Spiro, Stephen (2000) Heterologous NNR-Mediated Nitric Oxide Signaling in Escherichia coli. Journal of Bacteriology, 182 (22). pp. 6434-6439. ISSN 0021-9193

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Abstract

The transcription factor NNR from Paracoccus denitrificans was expressed in a strain of Escherichia coli carrying a plasmid-borne fusion of the melRpromoter 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.

Item Type: Article
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 2731 not found.
Date Deposited: 18 Jan 2012 11:44
Last Modified: 28 Oct 2019 15:47
URI: https://ueaeprints.uea.ac.uk/id/eprint/36239
DOI: 10.1128/JB.182.22.6434-6439.2000

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