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The alarmone species ppGpp and pppGpp are elementary components of bacterial physiology as they both coordinate the bacterial stress response and serve as fine-tuners of general metabolism during conditions of balanced growth. Since the regulation of (p)ppGpp metabolism and the effects of (p)ppGpp on cellular processes are highly complex and show massive differences between bacterial species, the underlying molecular mechanisms have so far only been insufficiently investigated for numerous microorganisms. In this study, (p)ppGpp physiology in the actinobacterial model organism Corynebacterium glutamicum was analyzed by phenotypic characterization and RNAseq-based transcriptome analysis. Total nutrient starvation was identified as the most effective method to induce alarmone production, whereas traditional induction methods such as the addition of serine hydroxamate (SHX) or mupirocin did not show a strong accumulation of (p)ppGpp. The predominant alarmone in C. glutamicum represents guanosine tetraphosphate, whose stress-associated production depends on the presence of the bifunctional RSH enzyme Rel. Interestingly, in addition to ppGpp, another substance yet not identified accumulated strongly under inducing conditions. A C. glutamicum triple mutant (Δrel,ΔrelS,ΔrelH) unable to produce alarmones [(p)ppGpp0 strain] exhibited unstable growth characteristics and interesting features such as an influence of illumination on its physiology, production of amino acids as well as differences in vitamin and carotenoid production. Differential transcriptome analysis using RNAseq provided numerous indications for the molecular basis of the observed phenotype. An evaluation of the (p)ppGpp-dependent transcriptional regulation under total nutrient starvation revealed a complex interplay with the involvement of ribosome-mediated transcriptional attenuation, the stress-responsive sigma factors σB and σH and transcription factors such as McbR, the master regulator of sulfur metabolism. In addition to the differential regulation of genes connected with various cell functions, the transcriptome analysis revealed conserved motifs within the promoter regions of (p)ppGpp-dependently and independently regulated genes. In particular, the representatives of translation-associated genes are both (p)ppGpp-dependent transcriptionally downregulated and show a highly conserved and so far unknown TTTTG motif in the −35 region, which is also present in other actinobacterial genera.

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 RelHCg, 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 RelHCg and the bifunctional RSH enzyme RelCg 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 relHCg 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.

The hyperphosphorylated guanosine derivatives ppGpp and pppGpp represent global regulators of the bacterial stress response, as they act as central elements of the stringent response system. Although it was assumed that both, (p)ppGpp synthesis and hydrolysis, are catalyzed by one bifunctional RSH-protein in the actinobacterial model organism Corynebacterium glutamicum ATCC 13032, two putative short alarmone synthetases (SASs) were identified by bioinformatic analyses. The predicted sequences of both enzymes, designated as RelP*Cg and RelSCg, exhibit high similarities to the conserved (p)ppGpp synthetase catalytic domain. In the context of sequence analysis, significant differences were found between the RelP variants of different C. glutamicum isolates. In contrast to the bifunctional RelA/SpoT homolog (RSH) protein RelCg, whose gene deletion results in a reduced growth rate, no change in growth characteristics were observed for deletion mutants of the putative SAS proteins under standard growth conditions. The growth deficit of the Δrel strain could be restored by the additional deletion of the gene encoding RelSCg, which clearly indicates a functional relationship between both enzymes. The predicted pyrophosphokinase activity of RelSCg was demonstrated by means of genetic complementation of an Escherichia coli ΔrelAΔspoT strain. For the expression of RelP*Cg, as well as the slightly differing variant RelPCg from C. glutamicum AS1.542, no complementation was observed, concluding that both RelP versions possess no significant pyrophosphokinase activity in vivo. The results were confirmed by in vitro characterization of the corresponding proteins. In the course of this investigation, the additional conversion of GMP to pGpp was determined for the enzyme RelSCg. Since the SAS species analyzed extend both the network of stringent response related enzymes and the number of substances involved, the study of this class of enzymes is an important component in understanding the bacterial stress response. In addition to the comprehension of important biological processes, such as growth rate regulation and the survival of pathogenic species in the host organism, SAS enzymes can be used to produce novel hyperphosphorylated nucleotide species, such as pGpp.