Data_Sheet_2_Dynamic Metabolic Response to (p)ppGpp Accumulation in Pseudomonas putida.xlsx

The stringent response is a ubiquitous bacterial reaction triggered by nutrient deprivation and mediated by the intracellular concentrations of ppGpp and pppGpp. These alarmones, jointly referred to as (p)ppGpp, control gene transcription, mRNA translation and protein activity to adjust the metabolism and growth rate to environmental changes. While the ability of (p)ppGpp to mediate cell growth slowdown and metabolism adaptation has been demonstrated in Escherichia coli, it’s role in Pseudomonas putida remains unclear. The aims of this study were therefore to determine which forms of (p)ppGpp are synthetized in response to severe growth inhibition in P. putida, and to decipher the mechanisms of (p)ppGpp-mediated metabolic regulation in this bacterium. We exposed exponentially growing cells of P. putida to serine hydroxamate (SHX), a serine analog known to trigger the stringent response, and tracked the dynamics of intra- and extracellular metabolites using untargeted quantitative MS and NMR-based metabolomics, respectively. We found that SHX promotes ppGpp and pppGpp accumulation few minutes after exposure and arrests bacterial growth. Meanwhile, central carbon metabolites increase in concentration while purine pathway intermediates drop sharply. Importantly, in a ΔrelA mutant and a ppGpp0 strain in which (p)ppGpp synthesis genes were deleted, SHX exposure inhibited cell growth but led to an accumulation of purine pathway metabolites instead of a decrease, suggesting that as observed in other bacteria, (p)ppGpp downregulates the purine pathway in P. putida. Extracellular accumulations of pyruvate and acetate were observed as a specific metabolic consequence of the stringent response. Overall, our results show that (p)ppGpp rapidly remodels the central carbon metabolism and the de novo purine biosynthesis pathway in P. putida. These data represent a hypothesis-generating resource for future studies on the stringent response.