Inducible expression of (pp)pGpp synthetases inStaphylococcus aureusis associated with activation of stress response genes
Authors: Horvatek Petra , Hanna Andrew Magdy Fekry , Gratani Fabio Lino , Keinhörster Daniela , Korn Natalya , Borisova Marina , Mayer Christoph , Rejman Dominik , Mäder Ulrike , Wolz Christiane 
Published: April 2020

AbstractThe stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. InStaphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog (RSHSau) upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ, upon cell wall stress. We used RNA-Seq to compare the global effects in response to transcriptional induction of the synthetase domain of RSH (RSH-Syn), RelP or RelQ without the need to apply additional stress conditions. Enzyme expression resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than RSH-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activityin vivo. Genes involved in the SOS response, iron storage (e.g.ftnA, dps), oxidative stress response (e.g.,katA, sodA) and the thepsmα1-4 and psmß1-2operons coding for cytotoxic, phenole soluble modulins (PSMs) were highly upregulated upon (pp)pGpp synthesis. Analyses of theftnA, dpsandpsmgenes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover,psmexpression is uncoupled from expression of the quorum sensing system Agr, the main knownpsmactivator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress inS. aureusthat is likely crucial for survival upon phagocytosis.SignificanceMost bacteria make use of the second messenger (pp)pGpp to reprogram bacterial metabolism under nutrient-limiting conditions. In the human pathogenStaphylococcus aureus, (pp)pGpp plays an important role in virulence, phagosomal escape and antibiotic tolerance. Here, we analyzed the immediate consequences of (pp)pGpp synthesis upon transcriptional induction of the (pp)pGpp-producing enzymes RSH, RelP or RelQ. (pp)pGpp synthesis provokes immediate changes in the nucleotide pool and severely impacts the expression of hundreds of genes. A main consequence of (pp)pGpp synthesis inS. aureusis the induction of ROS-inducing toxic phenol-soluble modulins (PSMs) and simultaneous expression of the detoxifying system to protect the producer. This mechanism is likely of special advantage for the pathogen after phagocytosis.