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

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.

The Ps and Qs of alarmone synthesis inStaphylococcus aureus

“…Our results are consistent with those recently reported by Gratani et al [26] who noted that the deletion of the CTD of Sa-Rel led to a retention of (p)ppGpp synthesis activities (albeit at levels lower than those of the full-length Sa-Rel protein), and had negligible effects on the rate of (p)ppGpp hydrolysis. However, these authors noted considerable differences in the net balance of (p)ppGpp synthesis to hydrolysis activities when different in vivo or in vitro assays systems were utilized, highlighting the complex set of mechanisms underpinning the switch between alarmone synthesis and hydrolysis.…”

“…Background S. aureus synthesizes (pp)pGpp 'alarmones' via the combined activities of one bifunctional Rel protein (Sa-Rel) and two SAS proteins: Sa-RelP and Sa-RelQ [26,37,41,43,59,60]. Rel appears to be the major source of guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) in S. aureus cells where the stringent response has been induced [26,[60][61][62]. The rel (rsh) gene is essential for the growth of S. aureus [43], due to its alarmone hydrolyzing activities [37,60].…”

“…The Sa-RelQ (SAS1), Sa-RelP (SAS2), full-length Sa-Rel protein, and N-terminal domain of Sa-Rel (Sa-Rel trunc , residues 9392), were expressed and purified in a soluble form. The Sa-Rel trunc protein encodes the SYNTH and HD domains that are respectively responsible for the synthesis and hydrolysis of alarmones; but lacks the putative regulatory domains found in the C-terminal region (Fig 1) [16,26,27,31,[47][48][49][50]. Its composition is analogous to the previously-characterized truncated form of the Rel protein from Streptococcus dysgalactiae subsp.…”

“…However, the pGpp-synthesizing abilities of RelQ/RelP homologues from other Firmicutes species remains poorly explored.In the bacterial pathogen S. aureus, the rsh (rel) gene is essential [43] but the relP and relQ genes are dispensable [37]. The S. aureus Rel (Sa-Rel), RelP (Sa-RelP) and RelQ (SA-RelQ) proteins have the ability to synthesize ppGpp and pppGpp [26,37,41]. A recent report has shed light onto the in vivo regulation of the opposing (p)ppGpp synthetase and hydrolase activities of the Sa-Rel protein [26].…”

“…The S. aureus Rel (Sa-Rel), RelP (Sa-RelP) and RelQ (SA-RelQ) proteins have the ability to synthesize ppGpp and pppGpp [26,37,41]. A recent report has shed light onto the in vivo regulation of the opposing (p)ppGpp synthetase and hydrolase activities of the Sa-Rel protein [26]. A detailed structural and mechanistic analysis into the (p)ppGpp synthesis activities of the Sa-RelP protein has also recently been published [41].However, the detailed biochemical activities of the Sa-Rel and Sa-RelQ proteins remain to be fully established.…”