The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli.
Pseudomonas aeruginosa is a well-known pathogen in chronic respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli-based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix-turn-helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro. Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro, suggesting that DNR responds specifically to NO.
Castiglione
Nicoletta
N
Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, Rome, Italy.
Rinaldo
Serena
S
Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, Rome, Italy.
Giardina
Giorgio
G
Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, Rome, Italy.
Cutruzzolà
Francesca
F
Consorzio INBB, 00136 Rome, Italy.|Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, Rome, Italy.
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D002248:Carbon Monoxide / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D017930:Genes, Reporter; D006418:Heme / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011552:Pseudomonas Infections / Q000378:metabolism / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000378:metabolism / Q000472:pathogenicity; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014774:Virulence
D001426:Bacterial Proteins; C095475:DNR protein, Pseudomonas aeruginosa; D014157:Transcription Factors; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide