Figure 4. PagR binds the region between pagR and the five-gene operon that includes tktD and tktE. Representative results of an experiment designed to determine whether PagR could bind to the intergenic region between pagR and the five-gene operon containing genes encoding TktC. In this experiment we used 0.5 pmol of probe. The experiment was repeated three times and the amount of free probe (406 nt) decreased as a function of the amount of PagR added to the reaction mixture. More details about how the experiment was performed can be found under Experimental procedures.
PagR represses the expression of the operon containingtktDE, and its own gene . Given the proximity of pagR to the operon containing the tktD and tktE genes (Fig. 3A) and the data presented in figure 2, we investigated the possibility that PagR repressed the expression of the operon containing tktD andtktE . We isolated RNA from three strains, namelypagR + / vector,pagR1 ::kan+ / vector, andpagR1 ::kan+ / pPagR, and quantified the amount of stm2343 , tktD, and tktE mRNA using RT-qPCR. For this purpose, cells were grown on lysogeny broth (LB) plus ampicillin and L-(+)-arabinose (100 µM), and RNA was isolated when cultures reached an OD600 of 0.5. As shown in figure 5A, expression of stm2343 , tktD and tktE increased ~10 fold in thepagR1 ::kan+ / vector strain (red bars) when compared to the pagR+ / vector and thepagR1 ::kan+ / pPagR strains (black and blue bars, respectively). To assess whether PagR regulated its own expression, we constructed a lacZY transcriptional fusion with the chromosomal pagR gene and assayed β-galactosidase activity with and without ectopic expression of pagR . Cells were grown to an OD600 of ~0.5 in LB plus ampicillin and L-(+)-arabinose (100 µM), then quantified β-galactosidase activity following a described protocol (Miller and Hershberger, 1984). The β-galactosidase activity of thepagR3 ::lacZY+ strain was significantly lower in the strain expressing pagR than in the vector control (Fig. 5B, blue vs red bars). Together, these data suggested that PagR repressed expression of the operon containing tktDE, and its own gene. This conclusion was consistent with its annotated homology to the LacI family of repressors.