The twists and turns of alarmone synthesis in Treponema denticola

Abstract

Upon encountering certain environmental stresses, such as amino acid or carbon nutrient starvation, bacteria synthesize two hyper-phosphorylated guanine nucleotide ‘alarmone’ molecules: guanosine 5’-triphosphate, 3’-diphosphate (ppGpp) and guanosine 5’,3’-bisdisphosphate (ppGpp), in a process called the stringent response. Alarmones act as intracellular signaling and effector molecules, which modulate various aspects of cellular physiology including transcription, translation, DNA replication and nucleotide metabolism. Collectively, this promotes bacterial survival, persistence, and virulence in pathogenic species. Proteins belonging to the RelA/SpoT homologue (RSH) family catalyze the synthesis and/or hydrolysis of alarmones. The oral spirochete Treponema denticola appears unique in the bacterial world by encoding one small alarmone synthetase (SAS) protein (Tde-SAS) that has an unusual two-domain architecture, one small alarmone hydrolase (SAH) protein (Tde-SAH), but lacking a ‘long-RSH’ protein. Here, we report the biochemical characterization of the Tde-SAS and Tde-SAH proteins, and compare and contrast their activities with those of diverse SAS, SAH and long-RSH proteins encoded by other (oral) bacterial taxa. We show that Tde-SAS preferentially synthesizes ppGpp (from GDP+ATP) over pppGpp (from GTP+ATP). Unlike RelQ family SAS proteins, alarmones do not allosterically stimulate Tde-SAS synthetic activities. The removal of the ca. 200 amino acid regulatory C-terminal domain of Tde-SAS results in a >10-fold up-regulation in alarmone synthesis activities, suggesting this domain negatively modulates the catalytic activities of the N-terminal ca. 200 amino acid synthetic domain. Tde-SAH efficiently hydrolyzes pppGpp, ppGpp and pGpp to GTP, GDP and GMP + diphosphate, respectively; exhibiting little selectivity between alarmone substrates. In conclusion, our data indicate that Tde-SAS and Tde-SAH are respectively responsible for (pp)pGpp synthesis and hydrolysis in T. denticola. Further biochemical, structural and biological investigations are ongoing, and we present and discuss our preliminary results within the broader context of what is currently known about the metabolism and functions of alarmones within diverse bacterial systems.