Functional characterization of RelA/SpoT homologues from oral bacteria

Abstract

Bacteria face numerous environmental stresses and rely on various intracellular signaling pathways, including those that use nucleotide-based second messengers, to adapt and survive. One such pathway: the stringent response, enables bacteria to respond to nutrient starvation by producing nucleotides called alarmones: guanosine pentaphosphate (pppGpp), guanosine tetraphosphate (ppGpp), and guanosine 5’-monophosphate 3’-diphosphate (pGpp), collectively referred to as (pp)pGpp. Cellular (pp)pGpp levels are regulated by the RSH (RelA/SpoT Homologues) superfamily of proteins, which includes small alarmone synthetases (SAS), small alarmone hydrolases (SAH), and bifunctional, multi-domain alarmone synthetase/hydrolase proteins (Rel). Here, I characterized the RSH homologues predicted to be responsible for alarmone metabolism in Treponema denticola (Tde-SAS, Tde-SAH), Fusobacterium nucleatum (Fn-Rel, Fn-SAS) and Parvimonas micra (Pm-Rel, Pm-SAS, ‘Pm-SAH’). These bacteria play important roles in various oral and/or extra-oral infections, especially periodontal disease. The biochemical activities of Tde-SAS and Tde-SAH were investigated in detail. Tde-SAS synthesized ppGpp considerably faster and more efficiently than pppGpp or pGpp. Interestingly, the presence of the C-terminal tetratricopeptide repeat (TPR) motif-domain of Tde-SAS notably inhibited the synthetic activities of the N-terminal catalytic domain. Tde-SAS also synthesized the ‘alarmone-like’ molecules adenosine tetraphosphate (ppApp) and adenosine pentaphosphate (pppApp), collectively called (p)ppApp. The biological roles of (p)ppApp are poorly understood. Tde-SAH effectively hydrolyzed (pp)pGpp and (p)ppApp, indicating its ability to degrade all alarmone (alarmone-like) nucleotides generated by Tde-SAS. The functions of Fn-Rel and Fn-SAS were investigated, to establish their involvement in (pp)pGpp metabolism within F. nucleatum. Both Fn-Rel and Fn-SAS exhibited a similar substrate preference for pppGpp over ppGpp and pGpp, suggesting pppGpp plays a major role in the stringent response in F. nucleatum. Fn-SAS also synthesized (p)ppApp, albeit at low rates. Fn-Rel efficiently hydrolyzed (pp)pGpp but not (p)ppApp. The activities of Fn-SAS were activated by Zn2+ ions. Notably, the overexpression of Fn-SAS in a ‘(p)ppGpp-zero’ E. coli relAspoT reporter strain resulted in toxicity, which was reversed by the presence of Fn-Rel. This suggested that toxicity resulted from the overproduction of (pp)pGpp by Fn-SAS. Interestingly, (pp)pGpp inhibited the activities of the adenine phosphoribosyltransferase protein (Fn-APRT), whose encoding gene lies immediately downstream of rel on the F. nucleatum genome. Biochemical investigations revealed that Pm-Rel was capable of both synthesizing and hydrolyzing (pp)pGpp. Pm-SAS synthesized all three alarmones, with ppGpp produced most efficiently. Notably, the addition of an acyl carrier protein (Pm-ACP1) stimulated the ppGpp-synthesis activities of Pm-SAS by 2-fold. This provides the first evidence supporting the involvement of SAS proteins in fatty acid metabolism. Furthermore, the P. micra small alarmone hydrolase protein, which belongs to the ‘PbcSpo’ family, displays a previously undescribed ability to hydrolyze pyrophosphate in addition to (p)ppApp. Hence, I proposed the name ‘SAHPY’ in place of ‘SAH’ (‘py’ = pyrophosphatase). In brief conclusion, I have characterized the activities of several previously-undescribed families of RSH proteins encoded within three different species of periodontal pathogens; revealing novel connectivities between the stringent response and fatty acid metabolism and purine metabolism. My research findings highlight the notable diversity in catalytic functions and regulatory mechanisms of Rel, SAS and SAH proteins encoded within diverse bacterial hosts.