Comparative Functional Analysis of Bacterial Small Alarmone Synthase Proteins

Objective: During periods of nutritional or environmental stress, bacteria synthesize phosphorylated guanosine nucleotide derivatives known as alarmones. There are two major alarmones: guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp). The (p)ppGpp molecules modulate a variety of intracellular processes in a coordinated manner known as the stringent response, to help the cell conserve and recycle essential resources. To characterize and compare the biochemical activities of a diverse set of proteins belonging to the ‘small alarmone synthase’ (SAS) family, to establish their putative roles in the production of (p)ppGpp alamone molecules. Method: Several SAS protein homologues from a variety of oral and systemic bacterial pathogens were cloned, expressed and purified. This included proteins encoded by relP and relQ genes from Fusobacterium nucleatum subsp. nucleatum, Staphylococcus aureus, Entereococcus faecalis and Streptococcus mutans. Their biochemical activities were characterized in vitro; focusing on their respective abilities to synthesize pppGpp, ppGpp and pGpp alarmones. Result:  RelP and RelQ proteins from S. aureus, E. faecalis and S. mutans all possessed the ability to synthesize pppGpp, ppGpp and pGpp alamones; albeit with varying efficiencies. The single SAS encoded by F. nucleatum(RelQ, FN0926) primarily synthezied ppGpp, and to a lesser extent ppGpp, but lacked the ability to produce pGpp. All proteins lacked alarmone hydrolase activities. Conclusion:  The biochemical activities of RelQ and RelP small alarmone synthase protein homologues vary considerably, suggesting that the major alamone molecules ppGpp and pppGpp are produced and utilized to differing extents within different bacterial species.