Heterologous NNR-mediated nitric oxide signaling in Escherichia coli

M I Hutchings, N. Shearer, S Wastell, R J van Spanning, S. Spiro

Research output: Contribution to JournalArticleAcademicpeer-review


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.

Original languageEnglish
Pages (from-to)6434-9
Number of pages6
JournalJournal of Bacteriology
Issue number22
Publication statusPublished - Nov 2000


  • Amino Acid Substitution
  • Bacterial Proteins
  • Cloning, Molecular
  • Coenzymes
  • Culture Media
  • DNA-Binding Proteins
  • Escherichia coli
  • Escherichia coli Proteins
  • Genetic Vectors
  • Hemoglobins
  • Metalloproteins
  • Mutagenesis, Site-Directed
  • Mutation
  • Nitrates
  • Nitric Oxide
  • Nitrites
  • Nitroprusside
  • Paracoccus denitrificans
  • Pteridines
  • Signal Transduction
  • Trans-Activators
  • Transcription Factors
  • Journal Article
  • Research Support, Non-U.S. Gov't


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