Figure 5. Expression analysis of the operon containingtktDE and of pagR, as a function of PagR. A.Effect of PagR on the expression of stm2343 , tktD , andtktE as shown by RT-qPCR. The experiment was performed in biological and technical triplicates. B. Activity of thepagR promoter (in Miller Units) without and withpagR + expressed in trans (red vs blue bars, respectively). * = p ≤ 0.05; ** = p < 0.01, ***, p ≤ 0.001, **** = p ≤ 0.0001. Cultures were grown in LB, and details of the RT-qPCR and β-galactosidase assay conditions used can be found in the Experimental proceduressection. Vector = pCV1 (VanDrisse and Escalante-Semerena, 2016).
PagR protects two binding sites in the stm2344pagR promoter region. DNase I footprinting was performed using a 291-nt DNA fragment spanning from 2,457,048 to 2,457,339 nt of the chromosome to define the DNA sequence bound by PagR. Results of an EMSA comparing PagR binding to this fragment with the one used in figure 4 suggested that the shorter probe used here contained all the relevant PagR binding sites (Fig. S1). Figure 6 shows the digestion results for the coding strand of the operon containing tktD and tktE(left to right going towards stm2344 ). The presence of PagR (Fig. 6, black trace) clearly protected two 21-bp regions of thestm2344 - pagR promoter region that were not protected without PagR (Fig. 6, orange trace). A nearly identical sequence was observed in both (Fig. 6, red underlines). The underlined sequence in the protected region on the left is a perfect 14 base pair palindrome, 5’-TGATAGCGCTATCA-3’, and the protected region on the right differs at only 2 bases (5’-TGG TAGCGCTATCT -3’, differences underlined). These data supported our conclusion that PagR was a LacI family regulator, since LacI family repressors typically bind palindromic or inverted repeat DNA (Weickert and Adhya, 1992).