Regulation of the carnitine pathway in Escherichia coli: investigation of the cai-fix divergent promoter region.
The divergent structural operons caiTABCDE and fixABCX of Escherichia coli are required for anaerobic carnitine metabolism. Transcriptional monocopy lacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an fnr mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at -41.5 of the cai transcriptional start site, plays a direct role in the sole cai activation. In contrast, mutation in CRP site 2, positioned at -69.5 of the fix promoter, caused only a threefold reduction in fix expression. Thus, the role of the third CRP site, located at -126.5 of fix, might be to reinforce the action of site 2. A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.
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Laboratoire de Génétique Moléculaire des Microorganismes et des Interactions Cellulaires, CNRS UMR 5577, Institut National des Sciences Appliquées, F-69621 Villeurbanne Cedex, France. lgmm@cismibm.uni-lyon1.fr
Eichler
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Mandrand-Berthelot
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D000217:Acyltransferases; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002331:Carnitine / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005420:Flavoproteins; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D006836:Hydro-Lyases / Q000235:genetics; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016415:Sequence Alignment
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; C098624:FixA protein, E coli; D005420:Flavoproteins; C068361:fixB protein, E coli; C091403:CaiA protein, E coli; D010088:Oxidoreductases; D000217:Acyltransferases; C060278:crotonobetainyl-CoA carnitine coenzyme A transferase; D006836:Hydro-Lyases; D002331:Carnitine