The ancestral stringent response potentiator, DksA has been adapted throughout Salmonella evolution to orchestrate the expression of metabolic, motility, and virulence pathways

DksA is a conserved RNA polymerase-binding protein known to play a key role in the stringent response of proteobacteria species, including many gastrointestinal pathogens. Here, we used RNA-sequencing of Escherichia coli, Salmonella bongori and Salmonella enterica serovar Typhimurium, together with phenotypic comparison to study changes in the DksA regulon, during Salmonella evolution. Comparative RNA-sequencing showed that under non-starved conditions, DksA controls the expression of 25%, 15%, and 20% of the E. coli, S. bongori, and S. enterica genes, respectively, indicating that DksA is a pleiotropic regulator, expanding its role beyond the canonical stringent response. We demonstrate that DksA is required for the growth of these three enteric bacteria species in minimal medium and controls the expression of the TCA cycle, glycolysis, pyrimidine biosynthesis, and quorum sensing. Interestingly, at multiple steps during Salmonella evolution, the type I fimbriae and various virulence genes encoded within SPIs 1, 2, 4, 5, and 11 have been transcriptionally integrated under the ancestral DksA regulon. Consequently, we show that DksA is necessary for host cells invasion by S. Typhimurium and S. bongori and for intracellular survival of S. Typhimurium in bone marrow-derived macrophages (BMDM). Moreover, we demonstrate regulatory inversion of the conserved motility-chemotaxis regulon by DksA, which acts as a negative regulator in E. coli, but activates this pathway in S. bongori and S. enterica. Overall, this study demonstrates the regulatory assimilation of multiple horizontally acquired virulence genes under the DksA regulon and provides new insights into the evolution of virulence genes regulation in Salmonella spp.