The stringent response is a conserved global regulatory mechanism that is related to the synthesis of (p)ppGpp nucleotides. Gram-positive bacteria, such as Staphylococcus aureus, possess three (p)ppGpp synthases: the bifunctional RSH (RelA/SpoT homolog) protein, which consists of a (p)ppGpp synthase and a (p)ppGpp hydrolase domain, and two truncated (p)ppGpp synthases, designated RelP and RelQ. Here, we characterized these two small (p)ppGpp synthases. Biochemical analyses of purified proteins and in vivo studies revealed a stronger synthetic activity for RelP than for RelQ. However, both enzymes prefer GDP over GTP as the pyrophosphate recipient to synthesize ppGpp. Each of the enzymes was shown to be responsible for the essentiality of the (p)ppGpp hydrolase domain of the RSH protein. The staphylococcal RSH-hydrolase is an efficient enzyme that prevents the toxic accumulation of (p)ppGpp. Expression of (p)ppGpp synthases in a hydrolase-negative background leads not only to growth arrest but also to cell death. Transcriptional analyses showed that relP and relQ are strongly induced upon vancomycin and ampicillin treatments. Accordingly, mutants lacking relP and relQ showed a significantly reduced survival rate upon treatments with cell wall-active antibiotics. Thus, RelP and RelQ are active (p)ppGpp synthases in S. aureus that are induced under cell envelope stress to mediate tolerance against these conditions.

Highlights d Crystal structure of apo B. subtilis Rel d Cryo-EM structure of the Reldstalled ribosome complex d Rel is regulated by the TGS domain in absence of the ribosome d Rel homodimerization masks the tRNA interaction interface

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.

The control of rRNA synthesis and, thereby, translation is vital for adapting to changing environmental conditions. The decrease of rRNA is a common feature of the stringent response, which is elicited by the rapid synthesis of (p)ppGpp. Here we analysed the properties and regulation of one representative rRNA operon of Staphylococcus aureus under stringent conditions and during growth. The promoters, P1 and P2, are severely downregulated at low intracellular guanosine triphosphate (GTP) concentrations either imposed by stringent conditions or in a guanine auxotroph guaBA mutant. In a (p)ppGpp(0) strain, the GTP level increased under stringent conditions, and rRNA transcription was upregulated. The correlation of the intracellular GTP levels and rRNA promoter activity could be linked to GTP nucleotides in the initiation region of both promoters at positions between +1 and +4. This indicates that not only transcriptional initiation, but also the first steps of elongation, requires high concentrations of free nucleotides. However, the severe downregulation of rRNA in post-exponential growth phase is independent of (p)ppGpp, the composition of the initiation region and the intracellular nucleotide pool. In summary, rRNA transcription in S. aureus is only partially and presumably indirectly controlled by (p)ppGpp.

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp upregulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR, katA, sodA) and the psmα1–4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

A large proportion of clinical S. aureus isolates that carry an inactive Agr system are associated with persistent infection that is difficult to treat. Once S. aureus is inside the bloodstream, it can cross the endothelial barrier and invade almost every organ in the human body. Endothelial cells can either be lysed by this pathogen or they serve as a niche for its intracellular long-term survival. Following phagocytosis, several vesicles such as phagosomes and autophagosomes, target intracellular S. aureus for elimination. S. aureus can escape from these vesicles into the host cytoplasm through the activation of phenol-soluble modulins (PSMs) αβ. Thereafter, it replicates and lyses the host cell to disseminate to adjacent tissues. Herein we demonstrate that staphylococcal strains which lack the expression of PSMs employ an alternative pathway to better persist within endothelial cells. The intracellular survival of S. aureus is associated with the co-localization of the autophagy marker LC3. In cell culture infection models, we found that the absence of psmαβ decreased the host cell lysis and increased staphylococcal long-term survival. This study explains the positive selection of agr -negative strains that lack the expression of psmαβ in chronic infection due to their advantage in surviving and evading the clearance system of the host.

DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.

Adaptation to growth condition is the key for bacterial survival, and protein phosphorylation is one of the strategies adopted to transduce extracellular signal in physiological response. In a previous work, we identified YjjJ, a putative kinase, as target of the persistence-related HipA kinase.