Transcriptional regulation of flhDC by QseBC and σ²⁸ (FliA) in enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, the causative agent of haemorrhagic colitis, has been shown to utilize a cell-to-cell signalling system to regulate gene expression. We have previously reported that the quorum sensing E. coli regulators B and C (QseBC) may act as a two-component system in EHEC to transcriptionally regulate the expression of flagella and motility through flhDC, the master regulator of flagella and motility genes. Here, we performed deletion analyses using the flhDC promoter in order to determine the minimal promoter regions necessary for QseBC transcriptional activation. We also performed electrophoretic mobility shift assays, competition experiments and DNasel footprints, which suggest that QseB directly binds the flhDC promoter at high- and low-affinity binding sites. These analyses have allowed us to determine the potential consensus sequence to which QseB binds in order to regulate transcription. Additionally, we mapped the transcriptional start site of flhDC responsive to QseBC, leading to the identification of a conserved FliA (σ²⁸) consensus sequence. These results suggest that FliA (σ²⁸), a class 2 flagellar gene, may be aiding in the transcriptional initiation of class 1 genes (flhDC) in EHEC. In order to further characterize the role of FliA (σ28) in transcription of the flhDC promoter, we constructed a fliA isogenic mutant in EHEC. The flhDC::lacZ transcriptional fusion showed decreased activity in the fliA mutant compared with wild-type and complemented strains. Taken together, these results indicate that transcriptional initiation at the flhDC promoter by QseBC appears to be complex and dependent on the presence of FliA (σ²⁸).