title: Positive co-regulation of the Escherichia coli carnitine pathway cai and fix operons by CRP and the CaiF activator.
abstract: Activation of the two divergent Escherichia coli cai and fix operons involved in anaerobic carnitine metabolism is co-dependent on the cyclic AMP receptor protein (CRP) and on CaiF, the specific carnitine-sensitive transcriptional regulator. CaiF was overproduced using a phage T7 system, purified on a heparin column and ran as a 15 kDa protein on SDS-PAGE. DNase I footprinting and interference experiments identified two sites, F1 and F2, with apparently comparable affinities for the binding of CaiF in the cai-fix regulatory region. These sites share a common perfect inverted repeat comprising two 11 bp half-sites separated by 13 bp, and centred at -70 and -127 from the fix transcription start site. They were found to overlap the two low-affinity binding sites, CRP2 and CRP3, determined previously for CRP. Gel shift assays and footprinting experiments suggest that CaiF and CRP bind co-operatively to the F1/CRP2 and F2/CRP3 sites of the intergenic cai-fix region. Moreover, they appeared to serve the simultaneous binding of each other, giving rise to an original multiprotein CRP-CaiF complex enabling RNA polymerase recruitment and local DNA untwisting, at least at the fix promoter. Using random mutagenesis, two CaiF mutants impaired in transcription activation were isolated. The N-terminal A27V mutation affected the structural organization of the activator, whereas the central I62N mutation was suggested to interfere with DNA binding.
authors.affiliation: Laboratoire de Génétique Moléculaire des Microorganismes et des Interactions Cellulaires, CNRS UMR 5577, Institut National des Sciences Appliquées, Bâtiment 406, 20, avenue Albert Einstein, F-69621 Villeurbanne Cedex, France.
authors.forename: A
authors.initials: A
authors.lastname: Buchet
authors.forename: W
authors.initials: W
authors.lastname: Nasser
authors.forename: K
authors.initials: K
authors.lastname: Eichler
authors.forename: M A
authors.initials: MA
authors.lastname: Mandrand-Berthelot
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002331:Carnitine / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins; D003087:Colicins; D002373:Cyclic AMP Receptor Protein / Q000235:genetics* / Q000378:metabolism; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009876:Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015534:Trans-Activators / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; C098709:CaiF protein, E coli; D002352:Carrier Proteins; D003087:Colicins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D015534:Trans-Activators; C007104:colicin immunity proteins; D012321:DNA-Directed RNA Polymerases; D002331:Carnitine