title: Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters.
abstract: Two transcriptional regulators, the FadR activator and the FabR repressor, control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was blocked in the absence of exogenous unsaturated fatty acids in a ΔfadR strain and found that the rates of transcription of fabA and fabB were unaffected by the lack of unsaturated thioesters. To examine the discrepancy between our in vivo results and the prior in vitro results we obtained active, natively folded forms of the E. coli and Vibrio cholerae FabRs by use of an in vitro transcription-translation system. We report that FabR bound the intact promoter regions of both fabA and fabB in the absence of unsaturated acyl thioesters, but bound the two promoters differently. Native FabR bound the fabA promoter region provided that the canonical FabR binding site is extended by inclusion of flanking sequences that overlap the neighbouring FadR binding site. In contrast, although binding to the fabB operator also required a flanking sequence, a non-specific sequence could suffice. However, unsaturated thioesters did allow FabR binding to the minimal FabR operator sites of both promoters which otherwise were not bound. Thus unsaturated thioester ligands were not essential for FabR/target DNA interaction, but acted to enhance binding. The gel mobility shift data plus in vivo expression data indicate that despite the remarkably similar arrangements of promoter elements, FadR predominately regulates fabA expression whereas FabR is the dominant regulator of fabB expression. We also report that E. coli fabR expression is not autoregulated. Complementation, qRT-PCR and fatty acid composition analyses demonstrated that V. cholerae FabR was a functional repressor of unsaturated fatty acid synthesis. However, in contrast to E. coli, gel mobility shift assays indicated that neither E. coli nor V. cholerae FabRs bound the V. cholerae fabB promoter, although both proteins efficiently bound the V. cholerae fabA promoter. This asymmetry was shown to be due to the lack of a FabR binding site within the V. cholerae fabB promoter region.
authors.affiliation: Department of Microbiology, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Youjun
authors.initials: Y
authors.lastname: Feng
authors.forename: John E
authors.initials: JE
authors.lastname: Cronan
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D019295:Computational Biology; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000235:genetics / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis* / Q000378:metabolism; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C458522:FabR protein, E coli; D005231:Fatty Acids, Unsaturated; D014157:Transcription Factors; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; C490369:FabB protein, E coli; D006836:Hydro-Lyases; C047118:3-hydroxyacyl-(acyl-carrier-protein) dehydratase; D054889:Fatty Acid Synthase, Type II