Premium
Positive co‐regulation of the Escherichia coli carnitine pathway cai and fix operons by CRP and the CaiF activator
Author(s) -
Buchet Anne,
Nasser William,
Eichler Knut,
MandrandBerthelot MarieAndrée
Publication year - 1999
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1999.01622.x
Subject(s) - biology , operon , camp receptor protein , dna footprinting , promoter , footprinting , microbiology and biotechnology , transcription (linguistics) , rna polymerase , binding site , escherichia coli , activator (genetics) , dna , dna binding protein , gene , genetics , transcription factor , gene expression , linguistics , philosophy
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.