Functional Characterization of a Small Alarmone Hydrolase in Corynebacterium glutamicum

The (pp)pGpp metabolism is an important component of bacterial physiology as it is involved in various stress responses and mechanisms of cell homeostasis, e.g., the regulation of growth. However, in order to better understand the (pp)pGpp associated regulation, it is crucial to study the molecular mechanisms of (pp)pGpp metabolism. In recent years, bioinformatic analyses of the RelA/SpoT homolog (RSH) superfamily have led to the discovery of small monofunctional RSH derivatives in addition to the well-known bifunctional Rel proteins. These are also referred to as small alarmone synthetases (SASs) or small alarmone hydrolases (SAHs). In this study, the ORF cg1485 from C. glutamicum was identified as a putative SAH encoding gene, based on a high similarity of the corresponding amino acid sequence with the (pp)pGpp hydrolysis domain. The characterization of its gene product, designated as RelH_Cg, represents the first functional investigation of a bacterial representative of the SAH subfamily. The predicted pyrophosphohydrolase activity was demonstrated in vivo by expression in two E. coli strains, characterized by different alarmone basal levels, as well as by in vitro analysis of the purified protein. During the assay-based analysis of hydrolysis activity in relation to the three known alarmone species, both RelH_Cg and the bifunctional RSH enzyme Rel_Cg were found to exhibit a pronounced substrate inhibition for alarmone concentrations of more than 0.75 mM. This characteristic of (pp)pGpp hydrolases could be an important mechanism for realizing the bistable character of the (pp)pGpp metabolism between a (pp)pGpp basal level and stress-associated alarmone production. The deletion of relH_Cg caused only a minor effect on growth behavior in both wild-type background and deletion mutants with deletion of (pp)pGpp synthetases. Based on this observation, the protein is probably only present or active under specific environmental conditions. The independent loss of the corresponding gene in numerous representatives of the genus Corynebacterium, which was found by bioinformatic analyses, also supports this hypothesis. Furthermore, growth analysis of all possible deletion combinations of the three active C. glutamicum RSH genes revealed interesting functional relationships which will have to be investigated in more detail in the future.