Functional characterization of proteins involved in bacterial polyphosphate and alarmone metabolism

Abstract

To adapt and survive within harsh environments, bacteria rely on a range of conserved physiological processes to adjust their growth, metabolism or cellular characteristics in response to nutritional starvation or other extracellular stresses. Polyphosphate (polyP) metabolism and the stringent response (SR) are two such processes. In most bacteria, polyP is synthesized by polyphosphate kinase family 1 (PPK1) and family 2 (PPK2), and hydrolyzed by exopolyphosphatase (PPX) proteins. Guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) mediate the SR, but have differing biological effects. These intracellular signaling molecules are synthesized and/or degraded by RelA/SpoT-homologue (RSH) family proteins including long-RSH (Rel), small alarmone synthetases (SAS) and small alarmone hydrolases (SAH). Here, I investigated the biochemical links between polyP metabolism and the SR in diverse (oral) bacterial species. In particular, I examined the respective abilities of diverse PPX homologues to hydrolyze pppGpp to ppGpp, and the degree to which their polyP hydrolysis activities were inhibited by pppGpp. Specifically, I characterized the biochemical properties of proteins putatively responsible for polyphosphate and/or alarmone metabolism in Veillonella parvula (Vp-PPXa, Vp-PPXb, Vp-PPK1), Capnocytophaga gingivalis (Cg-PPX, Cg-RSH), Bacteroides fragilis (Bf-PPX, Bf-PPK1, Bf-PPK2), Zymomona mobilis (Zm-PPK2), Bacillus cereus (Bc-PPK2) and Treponema socranskii (Tsoc-SAH, Tsoc-SAS, Tsoc-RSH), and others. Vp-PPK1 preferentially synthesized long-chain polyP from ATP. Vp-PPXb was catalytically inactive, but physically associated with Vp-PPK1, inhibiting its activities. Vp-PPXa hydrolysed polyP but could not hydrolyze pppGpp to ppGpp. Its exopolyphosphatase activities were not inhibited by pppGpp. Gallein, a recently-identified PPK1 inhibitor, inhibited polyP synthesis by Vp-PPK1 (IC50 value ca. 25 micromolar). Gallein inhibited biofilm formation by V. parvula, as well as V. parvula-Streptococcus co-cultures, suggesting polyP accumulation promotes biofilm formation. Cg-PPX, Ch-PPX and Bf-PPX efficiently hydrolyzed long and short-chain polyP, and were modestly inhibited by (0.1-0.4 mM) pppGpp. Whilst Cg-PPX and Ch-PPX hydrolyzed pppGpp to ppGpp, Bf-PPX lacked this ability. Bf-PPK1 efficiently synthesized polyP from ATP, whilst Bf-PPK2 (class III PPK2 family) preferentially utilized polyP for converting ADP to ATP. Gallein effectively inhibited Bf-PPK1 and Bf-PPK2 with IC50 values of ca. 5 micromolar and 90 micromolar, respectively. Zm-PPK2 and Bc-PPK2 (both class II PPK2) preferentially utilized polyP for phosphorylating AMP to ADP. Zm-PPK2 was more ‘catalytically versatile’ and could phosphorylate adenosine 3’,5’-bisphosphate (pAp) and guanosine 3’,5’-bisphosphate (pGp) at the 5’- and 3’-positions to a variety of ‘alarmone-like’ nucleotides. Zm-PPK2 was modestly inhibited by gallein (IC50 ca. 500 micromolar). The long-RSH (Rel) protein of T. socranskii (Tsoc-RSH) preferentially synthesized pppGpp over ppGpp and pGpp, but entirely lacked alarmone hydrolysis activities. Its alarmone synthesizing activities were allosterically stimulated by pppGpp. Tsoc-SAS preferentially synthesized ppGpp over pppGpp and pGpp, but unlike Treponema denticola SAS, could not synthesize ‘toxic’ adenosine tetraphosphate (ppApp) nucleotides. Tsoc-SAH effectively hydrolyzed (pp)pGpp and (p)ppApp. In summary, PPX homologues should not be assumed to convert pppGpp to ppGpp. Furthermore, (p)ppGpp production during SR should not be assumed to promote polyP accumulation by inhibiting the exopolyphosphatase activities of all PPX homologues. Gallein may be invaluable for studying the putative relationships between polyphosphate synthesis by PPK1 (and PPK2) and mono-/mixed-species biofilm formation.