Abstract
During the stringent response, bacteria synthesize guanosine-3’,5’-bis(diphosphate) (ppGpp) and guanosine-5’-triphosphate 3’-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp ‘alarmones’ are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode ‘small alarmone synthetase’ (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5’-phosphate 3’-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10-fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) and Streptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically modulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species.
Highlights
The stringent response is a coordinated, multifaceted physiological response that bacterial cells initiate in response to encountering certain adverse extracellular conditions, such as nutrient deprivation
It should be noted that the recombinant 763 aa Sa-Rel protein studied here corresponds to residues 8–736 of the predicted coding DNA sequence for the S. aureus Rel protein [54], and includes a 34 aa N-terminal affinity tag encoded by the pET28a vector
The Sa-Reltrunc 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, 21, 55,56,57]
Summary
The stringent response is a coordinated, multifaceted physiological response that bacterial cells initiate in response to encountering certain adverse extracellular conditions, such as nutrient deprivation. RelA proteins, such as the Escherichia coli RelA protein, are termed monofunctional or ‘synthase-only’, in that they contain an active alarmone synthesis (SYNTH) domain, but have a catalytically-inactive form of the Histidine-Aspartate (HD) domain responsible for hydrolytic activities, and only catalyze (p)ppGpp production [15, 16] This synthase activity involves the transfer of a diphosphate unit from ATP to the 3’hydroxyl group of GTP or GDP in a Mg2+-dependent manner to produce pppGpp or ppGpp, respectively, with the concomitant release of AMP [17]. SpoT proteins (in species such as E. coli that possess an additional RelA homologue) have potent (p)ppGpp hydrolyzing activities, but weak (p)ppGpp synthesizing activities in the absence of activating factors [4, 24, 25]
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