Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus

PLoS Genet. 2018 Jul 9;14(7):e1007514. doi: 10.1371/journal.pgen.1007514. eCollection 2018 Jul.


The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptation, Physiological / genetics
  • Amino Acid Motifs / physiology
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Gene Expression Regulation, Bacterial*
  • Hydrolases / genetics*
  • Hydrolases / metabolism
  • Ligases / genetics*
  • Ligases / metabolism
  • Ribosomes / metabolism
  • Staphylococcus aureus / physiology*
  • Stress, Physiological / physiology


  • Bacterial Proteins
  • Hydrolases
  • Ligases
  • guanosine 3',5'-polyphosphate synthetases

Grants and funding

This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG), TR34/B1 to CW; DFG SFB766/A15, and GRK1708/B2 to CM; SFB766/Z02 to BM; SPP1879 to GB, SFB766 to SW. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of manuscript.