Fine-tuned regulation by oxygen and nitric oxide of the activity of a semi-synthetic FNR-dependent promoter and expression of denitrification enzymes in Paracoccus denitrificans

• Published in: Microbiology (Society for General Microbiology) 2003-12, Vol.149 (12), p.3405-3412
• Main Authors: Mazoch, Jiri, Kunak, Michal, Kucera, Igor, van Spanning, Rob J. M
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; beta-Galactosidase - genetics; Biological and medical sciences; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial - drug effects; Gene Expression Regulation, Enzymologic - drug effects; Genes, Bacterial; Genes, Reporter; Lac Operon; Microbiology; Miscellaneous; Mutation; Nitric Oxide - metabolism; Nitroprusside - pharmacology; Oxidoreductases - metabolism; Oxygen - metabolism; Paracoccus denitrificans - drug effects; Paracoccus denitrificans - genetics; Paracoccus denitrificans - metabolism; Promoter Regions, Genetic; Trans-Activators - genetics; Trans-Activators - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.26546-0

1 Department of Biochemistry, Faculty of Science, Masaryk University, Kotlá ská 2, CZ-61137 Brno, Czech Republic 2 Department of Molecular Cell Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, NL-1081 HV Amsterdam, The Netherlands Correspondence Igor Ku era ikucera{at}chemi.muni.cz In Paracoccus denitrificans at least three fumarate and nitrate reductase regulator (FNR)-like proteins [FnrP, nitrite and nitric oxide reductases regulator (NNR) and NarR] control the expression of several genes necessary for denitrifying growth. To gain more insight into this regulation, -galactosidase activity from a plasmid carrying the lacZ gene fused to the Escherichia coli melR promoter with the consensus FNR-binding (FF) site was examined. Strains defective in the fnrP gene produced only very low levels of -galactosidase, indicating that FnrP is the principal activator of the FF promoter. Anoxic -galactosidase levels were much higher relative to those under oxic growth and were strongly dependent on the nitrogen electron acceptor used, maximal activity being promoted by N 2 O. Additions of nitrate or nitroprusside lowered -galactosidase expression resulting from an oxic to micro-oxic switch. These results suggest that the activity of FnrP is influenced not only by oxygen, but also by other factors, most notably by NO concentration. Observations of nitric oxide reductase (NOR) activity in a nitrite-reductase-deficient strain and in cells treated with haemoglobin provided evidence for dual regulation of the synthesis of this enzyme, partly independent of NO. Both regulatory modes were operative in the FnrP-deficient strain, but not in the NNR-deficient strain, suggesting involvement of the NNR protein. This conclusion was further substantiated by comparing the respective NOR promoter activities. Abbreviations: FNR, fumarate and nitrate reductase regulator ( E. coli ); FnrP, NarR, nitrate reductase regulators ( P. denitrificans ); FF site, FNR-binding site; NAR, nitrate reductase; NIR, nitrite reductase (cytochrome cd 1 ); NNR, nitrite and nitric oxide reductases regulator ( P. denitrificans ); NOR, membrane-bound nitric oxide reductase (cytochrome bc ); SNP, sodium nitroprusside