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.