CRP-Mediated Coherent Type-4 Feed-Forward Loop Involving Glucose-Regulated LacI in Escherichia coli

Recent studies have highlighted the importance of structure-function relationships in genetic regulatory networks, particularly in feed-forward loops (FFLs), where input-output behavior depends on both input signals and transcriptional interactions. This study elucidates the function of a CRP-LacI- lacZYA coherent type-4 FFL (Co-4 FFL) in Escherichia coli . We demonstrate that cyclic AMP receptor protein (CRP) directly represses the transcription of lacI , which encodes the Lac repressor. This finding was confirmed through multiple approaches: 1) the mRNA level of lacI decreased to one-fifth of the original level upon cAMP addition; 2) lacI expression increased 15-fold in a crp mutant; 3) DNase I footprinting identified a CRP binding site within the lacI promoter region, and a lacZ fusion assay and site-directed mutagenesis validated its functional role. Collectively, these results establish CRP as a direct repressor of lacI , thereby forming a Co-4 FFL with the lacZYA operon. Physiological studies on Co-4 FFLs in E. coli are scarce. Our model suggests that under glucose-lactose diauxic growth conditions, this circuit allows E. coli to enhance the efficiency of lactose metabolism for energy synthesis upon glucose exhaustion by repressing lacI via CRP. This work reveals how metabolic cues shape the behavior of genetic regulatory networks.