Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios

• DOI: 10.1038/srep14921
• Publication Date: 2015-10-07T06:41:16Z
• Authors: Rui Wu; Meng Zhao; Jing Li; He Gao; Biao Kan; Weili Liang
• Keywords: 0303 health sciences; Binding Sites; Cyclic AMP Receptor Protein; Base Sequence; Transcription, Genetic; Molecular Sequence Data; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Bacterial; DNA Transformation Competence; Article; 03 medical and health sciences; Bacterial Proteins; Sequence Homology, Nucleic Acid; Cyclic AMP; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Vibrio

AbstractTfoX (Sxy) and CRP are two important competence activators. The link betweentfoXand CRP has been shown inH. influenzabut lacking evidence of direct interaction. Recently a Sxy-dependent CRP (CRP-S) site autoregulating Sxy was reported inE. coli. Here, we show that the cAMP-CRP complex transcriptionally regulatestfoXexpression through multiple canonical CRP (CRP-N) sites inVibrios.This conclusion is supported by an analysis of thetfoXmRNA levels andtfoXtranscriptional reporter fusions. The reduced expression oftfoXVCwas restored by trans-complementation ofcrpin ∆crpand by exogenous cAMP in ∆cya. A promoter deletion analysis and the site-directed mutagenesis of the putative CRP-N sites revealed the presence of two functional CRP-N sites. The direct binding of cAMP-CRP to thetfoXVCpromoter was demonstrated by EMSA assays. Additionally, the transcriptional start site (TSS) oftfoXVFinV. fluvialiswas determined and −10/−35 regions were predicted. Further comparison of thetfoXpromoter inVibriosrevealed the existence of similar −10 motifs and putative CRP-N sites, indicating the conserved mechanism of CRP regulation ontfoX. Our study demonstrates the direct binding of the cAMP-CRP complex totfoXpromoter and broadens the understanding of the molecular mechanism regulatingtfoXinVibrios.