It’s all Rel-ative. Comparative analysis of bacterial (pp)pGpp alarmone synthesizing proteins

Microbial Physiology, Metabolism and Molecular Mechanisms ForumBackground During the ‘stringent response' bacteria produce phosphorylated guanine nucleotides known as ‘alarmones' (pppGpp, ppGpp and pGpp). These alarmones modulate bacterial cell physiology to promote survival. However, the precise activities of the protein families implicated in the synthesis and/or degradation of (pp)pGpp alarmones remain to be fully-elucidated. Objectives To compare and contrast the biochemical properties of RelA/SpoT Homologue (RSH) and small alarmone synthase (SAS) proteins from diverse bacterial species of relevance to human health. Methods Selected SAS and RSH protein homologues encoded by Staphylococcus aureus, Enterococcus faecalis, Fusobacterium nucleatum, Actinomyces gerencseriae and several other bacterial species were cloned, expressed, purified and biophysically-characterized using standard procedures. Their respective abilities to synthesize and degrade alarmones were systematically characterized. Enzymatic kinetic parameters were established by quantifying reaction products using liquid chromatography. Results The tetrameric SAS homologues preferentially utilized GDP over GTP or GMP as the acceptor for alarmone synthesis, with ATP solely-utilized as diphosphate donor. SAS proteins also accepted inosine nucleotides (ITP, IDP, IMP) as acceptors to differing extents. The alarmone-synthesizing activities of some SAS proteins were not notably stimulated by pppGpp or ppGpp. Regarding the RSH proteins, all RSH homologues tested rapidly hydrolyzed pppGpp, ppGpp and pGpp; with activities essentially-dependent on Mn(II) ions. Notably, certain RSH proteins could utilize GMP as substrate for the production of pGpp. Conclusions Certain species of bacteria may employ both RSH and SAS proteins for the synthesis of pppGpp, ppGpp or pGpp. Furthermore, RSH proteins have the capability to efficiently hydrolyze any (pp)pGpp formed within the cell