Heterologous NNR-mediated nitric oxide signaling in Escherichia coli

The transcription factor NMR fi-om Paracoccus denitrificans was expressed i n 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 anaerobi c 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 incl usion of reduced hemoglobin in culture media, because of the ability of hem oglobin to sequester nitric oxide and nitrite. The ability of nitrate and n itrite to activate NNR is likely due to the formation of NO (or related spe cies) during nitrate and nitrite respiration. Amino acids potentially invol ved in NNR activity were replaced by site-directed mutagenesis, and the act ivities of NNR derivatives were tested in the E. coli reporter system. Subs titutions 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 ret ained the ability to repress an FNR-repressible promoter, so these mutation s 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 activ ity, and the protein was undetectable in cell extracts. In contrast, NNR in which Ser-96 was replaced with threonine retained full activity.