Author/Editor		Starčič, Marjanca; Žgur-Bertok, Darja; Jordi, Bart JAM; Woesten, Marc MSM; Gaastra, Wim; van Putten, Jos PM
Title		The cyclic AMP-cyclic AMP receptor protein complex regulates activity of the traJ promoter of the Escherichia coli conjugative plasmid pRK100
Type		članek
Source		J Bacteriol
Vol. and No.		Letnik 185, št. 5
Publication year		2003
Volume		str. 1616-23
Language		eng
Abstract		The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of traJ expression, we studied tbe possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in traJ transcription using a traJ IacZ 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 tbat there was direct binding of CRP to the traJ promoter region. DNase I footprint experlments mapped tbe 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 tbe demonstrated regulation of TraJ by the cAMP-CRP complex, mutants wit6 defects in cya or crp exhibited reduced conjugal transfer from pRK100.
Descriptors		CYCLIC AMP CYCLIC AMP RECEPTOR PROTEIN ESCHERICHIA COLI PROMOTER REGIONS (GENETICS) PLASMIDS F FACTOR POLYMERASE CHAIN REACTION BETA-GALACTOSIDASE DNA FOOTPRINTING BINDING SITES