ABSTRACT
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.
Subject(s)
Bacterial Proteins , Coenzymes , Escherichia coli Proteins , Escherichia coli/genetics , Nitric Oxide/metabolism , Paracoccus denitrificans/genetics , Transcription Factors/genetics , Amino Acid Substitution , Cloning, Molecular , Culture Media , DNA-Binding Proteins/genetics , Escherichia coli/metabolism , Genetic Vectors , Hemoglobins , Metalloproteins/metabolism , Molybdenum Cofactors , Mutagenesis, Site-Directed , Mutation , Nitrates , Nitrites , Nitroprusside , Pteridines/metabolism , Signal Transduction , Trans-Activators/genetics , Transcription Factors/biosynthesisABSTRACT
The gua promoter (guaP) of Escherichia coli resembles those for ribosomal RNA (rrn) operons and lies in a close back-to-back arrangement with the promoter for xseA (xseP). Transcription from guaP is subject to stringent control and growth-rate-dependent regulation, and to repression by DnaA and PurR. In addition, transcription from guaP is regulated by the cyclic AMP receptor protein (CRP). Plasmid-borne promoter fusions to the receptor gene for chloramphenicol acetyl transferase were used to assess the role of CRP in controlling transcription from guaP and xseP following a downshift of cultures from rich into minimal medium. CRP is required to activate guaBA transcription and repress xseA transcription following downshift. Bandshift assays with a DNA fragment carrying the divergent promoters revealed specific binding of CRP. We propose that CRP, binding to a near-consensus site centred at -117.5, activates transcription from guaP and obstructs transcription from the xseA promoter.
