Figure 2. Growth of S. Typhimurium on succinate
as a function of pagR and tktDE. S. Typhimurium
was grown on NCE (no-carbon essential) minimal medium (Berkowitz et al.,
1968) supplemented with sodium succinate (30 mM) as the sole source of
carbon and energy. A. ThepagR1 ::kan+ strain failed to grow after
36 h of incubation at 37ºC on succinate as the sole carbon source, and
ectopic expression of pagR restores growth to wild-type. Removal
of tktDE in the pagR1 ::kan+background restores growth as well. Growth of thepagR+tktDE ::cat+ strain doesn’t significantly
differed from the parent strain. Ectopic gene expression was induced
with L-(+)-arabinose (100 µM). “pPagR” refers to plasmid pPagR-7.B. Expression of TktC, but not TktA or TktB, repressed growth
of the pagR1 ::kan+tktDE ::cat+ strain. Ectopic gene expression
was induced with L-(+)-arabinose (1 mM). The strains used werepagR + tktDE+ / vector
(JE22070, red circles), pagR1 ::kan +tktDE+ / vector (JE21566, grey triangles),pagR1 ::kan +tktDE+ / pPagR (JE21577, dark green triangles),pagR +tktDE1 ::cat+ / vector (JE25521, orange
diamonds), pagR1 ::kan +tktDE1 ::cat+ / vector (JE25523, blue
squares), pagR1 ::kan +tktDE1 ::cat+ / pTktC (JE25524, magenta
squares), pagR1 ::kan +tktDE1 ::cat+ / pTktA (JE257201, light
green squares), pagR1 ::kan +tktDE1 ::cat+ / pTktB (JE27202, yellow
squares); ‘vector’ stands for the empty cloning vector pCV1 that
contains an arabinose-inducible promoter (VanDrisse and
Escalante-Semerena, 2016). This experiment was conducted in technical
triplicate of biological duplicates three independent times. Error bars
represent one standard deviation from the mean. Error bars that are not
visible are smaller than the symbol. The statistical analysis was
performed with Prism v9 (GraphPad)
The tktD and tktE genes are part of a five-gene operon .
Bioinformatics analysis suggested that genes stm2340 (tktE),stm2341 (tktD) , stm2342 , stm2343 , andstm2344 comprised a polycistronic operon (Biocyc.org). To test
this possibility, we performed operon PCR with RNA isolated from apagR1 ::kan+ strain. The RNA was treated
with DNase, and cDNA was generated from purified total RNA. Genomic DNA,
cDNA, and RNA were PCR amplified with four primer sets to generate
products of four potential regions that overlap in neighboring putative
genes (Fig. 3A). As shown in figure 3B, the use of such primers
generated amplicons for the cDNA and the gDNA, but none amplified for
the RNA, which served as the negative control. PCR amplification of all
overlapping regions between genes stm2340 and stm2344suggested these genes were part of a polycistronic unit.