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

Ana Velic,Samuel Wagner,Marina Borisova,Gert Bange,Fabio Lino Gratani,Christoph Mayer,Danielle A. Garsin,Boris Maček,Tobias Geiger,Petra Horvatek,Wieland Steinchen,Iwan Grin,Christiane Wolz

doi:10.1371/journal.pgen.1007514

Abstract

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.

Highlights

Bacteria react to nutrient limitation via a stress response that is characterized by the synthesis of pyrophosphorylated GTP or GDP
Staphylococcus aureus, the stringent response plays an important role for virulence, phagosomal escape and antibiotic tolerance
The response is initiated by the synthesis of the nucleotide derivative (p)ppGpp which in turn leads to growth arrest and reprogramming of gene expression

Summary

Introduction

Bacteria react to nutrient limitation via a stress response that is characterized by the synthesis of pyrophosphorylated GTP (pppGpp) or GDP (ppGpp) (previously reviewed in [1,2,3,4,5, 6,7,8,9,10,11]). RelA of Escherichia coli was the first such enzyme described and has been shown to synthesize (p)ppGpp under conditions of amino acid limitation [12]. Similar to SpoT, the Rel enzymes from Firmicutes are bifunctional proteins with (p)ppGpp synthetase and hydrolase activities; similar to RelA, the synthetase activity of these enzymes is stimulated upon amino acid starvation [18,19]. The current model suggests that under non-stringent (relaxed) conditions, the interaction of the CTD with the NTD maintains the enzyme in the synthetase-OFF/hydrolase-ON conformation [21,22,23] The CTD of RelA stimulates (p)ppGpp synthesis in a ribosome-dependent manner when uncharged tRNA, as a consequence of amino acid limitation, is located in the ribosomal A-site [24,25]. The TGS motif (named after the presence in ThrRS, GTPases, and SpoT) was shown to be

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