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.