Effect of (pp)pGpp on stress responses in Staphylococcus aureus

Abstract

The stringent response is a conserved regulatory mechanism among a variety of bacterial species and is characterized by the synthesis of the alarmones pGpp, ppGpp and pppGpp, also collectively called (pp)pGpp. The activating signal of the stringent response differs among species and results in a variety of physiological alterations. In Staphylococcus aureus three enzymes regulate the intracellular level of (pp)pGpp and react to different stress signals. The RelA-SpoT homologue RSH from Staphylococcus aureus synthesizes (pp)pGpp in response to amino acid deprivation. In contrast, the small alarmone synthetase RelP and RelQ are transcriptionally activated upon cell wall stress. To analyze the nucleotide pool and downstream consequences of the three enzymes, we transcriptionally induced a truncated RSH with an inactive hydrolase, RelP and RelQ to ensure results are (pp)pGpp-mediated. Comparison of the nucleotide pool by direct induction of (pp)pGpp versus stress mimicking conditions via mupirocin resulted in similar changes. Stringent response related changes of the nucleotide pool are characterized by lowering of the GTP-Pool, increased ATP pool and the detection of pGpp, ppGpp and pppGpp. These changes were detectable for RSH and RelP but minor in RelQ, indicating a limited synthetase activity in vivo. We next used RNA-Seq to compare global transcriptional changes between RSH and RelQ-mediated (pp)pGpp synthesis. Genes involved in iron storage and oxidative stress response were highly upregulated in response to RSH-induction. Furthermore, (pp)pGpp-mediated expression of phenol soluble modulins (PSMs) consequently resulted in an increased formation of reactive oxygen species (ROS). Furthermore, we demonstrated a protective role of (pp)pGpp for survival under PSM-mediated or exogenous ROS and identified a new link between (pp)pGpp-dependent regulation of oxidative stress response and activation of PSMs.