Oxygen regulation of the Escherichia coli cytochrome d oxidase (cydAB) operon: roles of multiple promoters and the Fnr-1 and Fnr-2 binding sites.
The Escherichia coli cydAB operon encodes the high-affinity terminal oxidase of the oxygen respiratory chain, cytochrome d oxidase. The sensor-regulator pair, ArcB-ArcA, is responsible for the microaerobic activation of the cydAB operon, whereas the anaerobic regulator Fnr represses its expression in the absence of oxygen. Fnr binds in vitro at two sites within the cydAB promoter element. To discern whether these two regions have an in vivo function in the anaerobic regulation of cydAB, the Fnr-binding motifs were mutagenized individually and in combination. The effects of these mutations on in vivo gene expression were determined by lac fusion and primer extension analysis. Our results show that the Fnr-2 site is critical for Fnr-mediated anaerobic repression of the two main cydAB promoters, P1 and P2. In contrast, the Fnr-1 site has an auxiliary role in the anaerobic repression of P1, but not of P2. Transcription from P1 did not affect ArcA-mediated activation or Fnr-mediated repression of P2, indicating that oxygen regulation is exerted on both promoters in an independent fashion. In addition, three new promoters were identified in the cydAB control region, and the 5' ends of the corresponding transcripts were mapped. Two of these promoters, designated P3 and P4, are co-ordinately regulated with P1 and P2 in response to oxygen, ArcA and Fnr. The P5 promoter is not Fnr regulated and is only weakly activated by ArcA. The contribution of these three additional promoters to the overall cydAB expression is most relevant under aerobic conditions. Our results suggest a unique repression model, in which one Fnr dimer bound to one single site (Fnr-2) is sufficient to downregulate transcription from four cydAB promoters. In conclusion, transcription of the cydAB operon is driven by a complex regulatory element containing at least five promoters that act in unison to provide adequate oxygen control of gene expression.
Govantes
F
F
Department of Microbiology, Immunology and Molecular Genetics, and the Molecular Biology Institute, 1602 Molecular Sciences Building, University of California, Los Angeles, CA 90095-1489, USA.
Albrecht
J A
JA
Gunsalus
R P
RP
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics* / Q000378:metabolism*; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D001616:beta-Galactosidase; D010100:Oxygen