YhaJ directly regulates yhaO expression in EHEC.
(A) Purified YhaJ was tested for its ability to bind the yhaO promoter region (pyhaO; ~300 bp region upstream of the yhaO coding sequence) by EMSA. DIG-labeled pyhaO was incubated with increasing concentrations of YhaJ that corresponded to a shift in free-DNA indicating a YhaJ-DNA complex. Specificity of the binding reaction was tested by the addition of a 100-fold excess (+) of unlabeled pyhaO probe to the binding reaction to outcompete binding of the DIG-labeled probe to YhaJ. These reaction conditions were carried out using a fragment of the kan gene as a negative control. Additionally, the unlabeled kan probe in 100-fold excess was used as a non-specific competitor for YhaJ binding to the DIG-labeled yhaO probe (pyhaO vs kan). (B) Activity of the yhaO promoter in the ΔyhaJ mutant background. A plasmid containing a GFP-yhaO promoter fusion was transformed into TUV93-0 and ΔyhaJ to monitor transcription of yhaO in RFU during growth in MEM-HEPES. Data was calculated from three biological replicates and plotted at increasing OD600 values. * denotes P ≤ 0.05.
