The Cyclic AMP-Cyclic AMP Receptor Protein Complex Regulates Activity of the traJ Promoter of the Escherichia coli Conjugative Plasmid pRK100

Abstract

ABSTRACT The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of traJ expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ-lacZ reporter system. A comparison of the enzyme activities in the wild-type Escherichia coli strain MC4100 with those in cya and crp mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the traJ promoter of the F-like plasmid pRK100. The defect in the cya mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the traJ promoter region. DNase I footprint experiments mapped the CRP binding site around position −67.5 upstream of the putative traJ promoter. Targeted mutagenesis of the traJ promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in cya or crp exhibited reduced conjugal transfer from pRK100.