Abstract
The 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. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau 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 induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn 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 Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1–4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. 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 in S. aureus that is likely crucial for survival upon phagocytosis.
Highlights
In the human pathogen Staphylococcus aureus,pGpp plays an important role in virulence, phagosomal escape and antibiotic tolerance
A main consequence ofpGpp synthesis in S. aureus is the induction of reactive oxygen species (ROS)-inducing toxic phenol soluble modulins (PSMs) and simultaneous expression of the detoxifying system to protect the producer
The stringent response is characterized by the synthesis of the alarmones pGpp, ppGpp and pppGpp, here collectively namedpGpp.pGpp interferes with many cellular processes, including transcription, replication and translation [1,2,3,4,5,6,7,8,9,10]
Summary
The stringent response is characterized by the synthesis of the alarmones pGpp, ppGpp and pppGpp, here collectively named (pp)pGpp. (pp)pGpp interferes with many cellular processes, including transcription, replication and translation [1,2,3,4,5,6,7,8,9,10]. The stringent response is characterized by the synthesis of the alarmones pGpp, ppGpp and pppGpp, here collectively named (pp)pGpp. Long RSH enzymes are present in most bacteria and show a conserved molecular architecture composed of a C-terminal sensory region and an N-terminal enzymatic region with distinct (pp)pGpp hydrolase and synthetase domains [12]. Firmicutes, such as Staphylococcus aureus, possess one long RSH enzyme, RelSau and in addition two small alarmone synthetases (SAS), RelP and RelQ. Amino acid limitation is the only condition known to induce a RelSau-mediated stringent response phenotype [13]. The strong hydrolase activity of RelSau makes the enzyme essential for the detoxification of (pp)pGpp produced by RelP or RelQ [13]
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