Overlapping repressor binding sites result in additive regulation of Escherichia coli FadH by FadR and ArcA.
Escherichia coli fadH encodes a 2,4-dienoyl reductase that plays an auxiliary role in beta-oxidation of certain unsaturated fatty acids. In the 2 decades since its discovery, FadH biochemistry has been studied extensively. However, the genetic regulation of FadH has been explored only partially. Here we report mapping of the fadH promoter and document its complex regulation by three independent regulators, the fatty acid degradation FadR repressor, the oxygen-responsive ArcA-ArcB two-component system, and the cyclic AMP receptor protein-cyclic AMP (CRP-cAMP) complex. Electrophoretic mobility shift assays demonstrated that FadR binds to the fadH promoter region and that this binding can be specifically reversed by long-chain acyl-coenzyme A (CoA) thioesters. In vivo data combining transcriptional lacZ fusion and real-time quantitative PCR (qPCR) analyses indicated that fadH is strongly repressed by FadR, in agreement with induction of fadH by long-chain fatty acids. Inactivation of arcA increased fadH transcription by >3-fold under anaerobic conditions. Moreover, fadH expression was increased 8- to 10-fold under anaerobic conditions upon deletion of both the fadR and the arcA gene, indicating that anaerobic expression is additively repressed by FadR and ArcA-ArcB. Unlike fadM, a newly reported member of the E. coli fad regulon that encodes another auxiliary beta-oxidation enzyme, fadH was activated by the CRP-cAMP complex in a manner similar to those of the prototypical fad genes. In the absence of the CRP-cAMP complex, repression of fadH expression by both FadR and ArcA-ArcB was very weak, suggesting a possible interplay with other DNA binding proteins.
Feng
Youjun
Y
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.
Cronan
John E
JE
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D019295:Computational Biology; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; C057119:FadR protein, Bacteria; D012097:Repressor Proteins; C041610:arcA protein, E coli; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase