Characterising the interaction between (p)ppGpp and ribosome-associated GTPases in Staphylococcus aureus

Staphylococcus aureus is an important human pathogen, responsible for a range of superficial and invasive infections worldwide. During invasion and colonisation of the host, the ability of S. aureus to adapt to stressful conditions is paramount for survival and the success and continuation of infection. These stresses trigger production of two small nucleotide alarmones, guanosine 3′,5′-bis(diphosphate) and guanosine 3′-diphosphate 5′-triphosphate, known as ppGpp and pppGpp respectively, which function as the effector molecules of the highly conserved signalling network known as the stringent response to enable cell adaptation and survival. Previously, screening for (p)ppGpp interacting proteins in S. aureus identified four Ribosome Associated (RA)-GTPases, RsgA, Era, RbgA and HflX, each of which are cofactors in ribosome assembly where they cycle between the GTP-bound ON state and the GDP-bound OFF state to govern their function. While bound to (p)ppGpp, the GTPase activity of these proteins is inhibited. In this study, we sought to determine the mechanistic effect of (p)ppGpp on RA-GTPase interactions with the ribosome by examining the affinity and kinetics of this binding in different nucleotide-bound states. We show that RA-GTPases bind with higher affinity to 5′-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a mechanism of regulation of RA-GTPase activity within the cell. (p)ppGpp binding also reduced stable complex formation between RA-GTPases and the ribosomal subunits when compared to GTP binding. Structural studies of RsgA revealed that the ppGpp-bound state is conformationally reflective of the GDP-bound OFF state in which the switch I loop necessary for catalysis adopts a conformation which is incompatible with ribosome association and enzymatic activity, leading to a decrease in 70S ribosome biogenesis, translation rate and growth. Altogether, we characterise and highlight the inhibition of RA-GTPase activity by (p)ppGpp as a major mechanism of control of ribosome biogenesis and cell growth during the stringent response in S. aureus.