FIG 2.

Direct regulation of eepR by cAMP-CRP. (A) Expression of the eepR promoter measured from a chromosomal lacZ reporter integrated at eepR in a WT and crp mutant background. The WT strain is CMS376, and the crp mutant is CMS786. (B) As described for panel A but at an OD₆₀₀ of 4.0. (C) qPCR analysis of eepR expression from the WT and crp mutant measured at an OD₆₀₀ of 1.5. The WT strain is CMS376, and the crp mutant is CMS1687. (D) EMSA of His₈-CRP interaction with the biotin-labeled eepR predicted promoter (P_eepR; 2 ng) in vitro. His₈-CRP produced a gel shift of labeled P_eepR that could be inhibited by an excess of unlabeled P_eepR (P_eepR-UL) but not by a nonspecific unlabeled amplicon (Nonspecific-UL), a 360-bp internal region of eepR. The gel shift required cAMP; whereas 0 and 0.1 μM did not support binding, 10 μM was sufficient. (E) Chromatin affinity purification of His₈-CRP suggests binding of the eepR promoter in vivo. PCR amplification of the eepR promoter was elevated from ChAP purification of CRP-bound DNA in the WT strain containing a plasmid expressing His₈-CRP (+CRP) compared to the WT strain with the empty vector (−CRP). The flhDC promoter was included as a positive control and the oxyR promoter as a negative control.