The Ps and Qs of alarmone synthesis inStaphylococcus aureus

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. However, 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. 10fold, 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) andStreptococcus 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 stimulated 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. intracellular messengers that directly or indirectly up-regulate cellular processes that promote the conservation, recycling and biosynthesis of important molecular building-blocks; initiate physiological mechanisms that preserve cellular integrity and tolerance in response to certain cytotoxic exogenous agents or harsh extracellular conditions; and down-regulate processes that are superfluous or non-essential for short-term viability (reviewed in [4][5][6][7][8][9][10][11][12]).Alarmones are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT-Homologue (RSH) family [13,14] RelA proteins, such as the Escherichia coli RelA protein, are termed monofunctional or 'synthase-only', in that they lack a catalytic 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 Mg 2+ -dependent manner to pr...