Conserved protein families involved in bacterial polyphosphate and alarmone metabolism

Abstract

Bacteria employ a variety of conserved physiological responses that enable them to survive within hostile environments, or to alter their metabolism or cellular properties to adapt to nutrient deficiencies or periods of extracellular ‘stress’. Inorganic polyphosphate is involved in a wide variety of fundamentally-important biochemical and biological processes, including ones that promote bacterial survival and cellular adaptation. Two intracellular signaling molecules: guanosine 5’,3’-diphosphate (ppGpp) and guanosine 5’-triphosphate 3’-diphosphate (pppGpp), collectively referred to as (p)ppGpp or ‘alarmones’, mediate a coordinated cellular stress response known as the stringent response. In this thesis, I investigated the biochemical activities of three highly-conserved protein families that mediate the metabolism of polyphosphate and alarmone molecules: polyphosphate kinase 2 (PPK2), exopolyphosphatase (polyphosphate phosphohydrolase)/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) and small alarmone synthetase (SAS). In chapter 3, the biochemical activities of a PPX/GPPA homologue from Zymomonas mobilis (ZmPPX, Za10_0559) were characterized. Two mutant forms: ZmPPX30-508 and ZmPPX30-508(E137A) were studied in parallel. Exopolyphosphatase activity in ZmPPX30-508 was not drastically affected, but guanosine pentaphosphate phosphohydrolase (pppGpp-ase) were completely abrogated. Replacement of the highly-conserved glutamate-137 residue by alanine caused a near-complete loss of all activities. (p)ppGpp non-competitively inhibited exopolyphosphatase activity of ZmPPX. Inhibition constants (Ki) indicated that ppGpp was a more potent inhibitor than pppGpp, suggesting that stringent response in Z. mobilis may operate in a subtly different mechanism, compared to other bacterial species studied to date. In chapter 4, the biochemical activities of a PPK2 homologue from Zymomonas mobilis (ZmPPK2, ZZ6_0566) were characterized in detail, and compared/contrasted with those of PPK2 homologues from Vibrio cholera (VC0728), Laribacter hongkongensis (LHK_00958) and Mycobacterium smegmatis, (MSMEG_0891), as well as Z. mobilis polyphosphate kinase 1 (ZmPPK1, ZZ6_0570). ZmPPK2 was a tetrameric one-domain PPK2 possessing polyphosphate-dependent nucleotide monophosphate (NMP) kinase activities that were selective for purine-based nucleoside monophosphates (e.g. AMP, GMP). ZmPPK2 lacked detectable polyphosphate synthesis activities. Unlike the majority of PPK1 proteins studied to date, ZmPPK1 was devoid of polyphosphate synthesis activities. Most notably, ZmPPK2 possessed polyphosphate-dependent ppGpp and pGpp (guanosine 5’-phosphate, 3’-diphosphate) kinase activities. All other PPK2 homologues tested lacked this ppGpp/pGpp-kinase activity. In chapter 5, the activities of five SAS homologues were characterized; four from Treponema species: Treponema denticola (TdeSAS, TDE1711), Treponema putidum (Tput_SAS, JO40_10655), ‘Treponema sinensis’ (Tsin_SASA, JO41_00560; Tsin_SASB, JO41_10760), and one from Fusobacterium nucleatum (FnSAS, Fn0926). Phylogenetic analysis revealed three distinct treponeme SAS lineages (subgroups): Trep_SASI (ca. 400aa), Trep_SASII (ca. 250aa) and Trep_SASIII (ca. 350aa). Trep_SASI formed an independent subgroup; Trep_SASII was closely related to RelP, and Trep_SASIII clustered with various unclassified SASs. The respective treponeme SAS homologues possessed notably different substrate preferences for alarmone synthesis. Notably, the pGpp, ppGpp and pppGpp synthetase activities of Tsin_SASB were allosterically-stimulated by (p)ppGpp. Unlike RelP/RelQ-family SAS proteins, FnSAS synthesized pppGpp most efficiently. In conclusion, the results presented in this thesis greatly add to our general understanding of the biochemical activities of several highly-conserved protein families involved in bacterial polyphosphate and alarmone metabolism. Most notably a novel biochemical activity for a PPK2 protein was identified, which further interconnects polyphosphate and alarmone metabolic pathways.