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

摘要

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, Rel_( Sau )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 Rel_( Sau )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 Rel_( Sau ). 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) Rel_( Sau )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, Rel_( Sau )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. Author summary The stringent response is a general stress response, which allows bacteria to survive nutrient limited conditions and to better tolerate antibiotic treatment. In the human pathogen, 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. However, a rapid and controlled inactivation of these growth inhibitory molecules is equally important for the organism. (p)ppGpp synthesis as well as hydrolysis is accomplished by a bi-functional RelA/SpoT homolog, Rel_( Sau )bearing distinct synthetase, hydrolase and sensory domains. We elucidated how the C-terminal sensory domain of Rel_(Sau)controls the intermolecular switch between hydrolase and synthetase activities in S . aureus . The switch is crucial for the appropriate response of S . aureus to adapt to changing environment encountered during infection.