The YmgB-SpoT interaction triggers the stringent response in Escherichia coli.

Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent of its proposed role in the two-component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.

Protein-Ligand Interactions in Scarcity: The Stringent Response from Bacteria to Metazoa, and the Unanswered Questions.

The stringent response, originally identified in Escherichia coli as a signal that leads to reprogramming of gene expression under starvation or nutrient deprivation, is now recognized as ubiquitous in all bacteria, and also as part of a broader survival strategy in diverse, other stress conditions. Much of our insight into this phenomenon derives from the role of hyperphosphorylated guanosine derivatives (pppGpp, ppGpp, pGpp; guanosine penta-, tetra- and tri-phosphate, respectively) that are synthesized on starvation cues and act as messengers or alarmones. These molecules, collectively referred to here as (p)ppGpp, orchestrate a complex network of biochemical steps that eventually lead to the repression of stable RNA synthesis, growth, and cell division, while promoting amino acid biosynthesis, survival, persistence, and virulence. In this analytical review, we summarize the mechanism of the major signaling pathways in the stringent response, consisting of the synthesis of the (p)ppGpp, their interaction with RNA polymerase, and diverse factors of macromolecular biosynthesis, leading to differential inhibition and activation of specific promoters. We also briefly touch upon the recently reported stringent-like response in a few eukaryotes, which is a very disparate mechanism involving MESH1 (Metazoan SpoT Homolog 1), a cytosolic NADPH phosphatase. Lastly, using ppGpp as an example, we speculate on possible pathways of simultaneous evolution of alarmones and their multiple targets.

Fusion of the N-terminal 119 amino acids of RelA with the CTD domain render growth inhibitory effects of the latter, (p)ppGpp-dependent.

The guanosine nucleotide derivatives ppGpp and pppGpp are central to the remarkable capacity of bacteria to adapt to fluctuating environments and metabolic perturbations. They are synthesized by two proteins, RelA and SpoT in E. coli and the activities of each of the two enzymes are highly regulated for homeostatic control of intracellular (p)ppGpp levels. Characterization of the mutant studied here indicates that moderate level expression of RelA appreciably reduces growth of cells wherein the basal levels of (p)ppGpp are higher than in the wild type without elevating the levels further. Consistent with this result, a large part of the growth inhibition effect is reproduced by overexpression of RelA NTD-CTD fusion lacking the (p)ppGpp synthesis function. A null mutation in relA abolishes this growth inhibitory effect suggesting its requirement for basal level synthesis of (p)ppGpp. Accordingly, increase in the (p)ppGpp levels in the relA1 mutant by spoT202 mutation largely restored the growth inhibitory effects of overexpression of RelA NTD-CTD fusion. Expression of this construct consisting of 119 amino acids of the N-terminal hydrolytic domain (HD) fused in-frame with the CTD domain (±TGS domain) renders the growth inhibitory effects (p)ppGpp-responsive-inhibited growth only of spoT1 and spoT202 relA1 mutants. This finding uncovered an hitherto unrealized (p)ppGpp-dependent regulation of RelA-CTD function, unraveling the importance of RelA NTD-HD domain for its regulatory role. An incremental rise in the (p)ppGpp levels is proposed to progressively modulate the interaction of RelA-CTD with the ribosomes with possible implications in the feedback regulation of the (p)ppGpp synthesis function, a proposal that accounts for the nonlinear kinetics of (p)ppGpp synthesis and increased ratio of RelA:ribosomes, both in vitro as well as in vivo.

A hyperpromiscuous antitoxin protein domain for the neutralization of diverse toxin domains.

Toxin-antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription and translation as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.

Regulatory Themes and Variations by the Stress-Signaling Nucleotide Alarmones (p)ppGpp in Bacteria.

Bacterial stress-signaling alarmones are important components of a protective network against diverse stresses such as nutrient starvation and antibiotic assault. pppGpp and ppGpp, collectively (p)ppGpp, have well-documented regulatory roles in gene expression and protein translation. Recent work has highlighted another key function of (p)ppGpp: inducing rapid and coordinated changes in cellular metabolism by regulating enzymatic activities, especially those involved in purine nucleotide synthesis. Failure of metabolic regulation by (p)ppGpp results in the loss of coordination between metabolic and macromolecular processes, leading to cellular toxicity. In this review, we document how (p)ppGpp and newly characterized nucleotides pGpp and (p)ppApp directly regulate these enzymatic targets for metabolic remodeling. We examine targets' common determinants for alarmone interaction as well as their evolutionary diversification. We highlight classical and emerging themes in nucleotide signaling, including oligomerization and allostery along with metabolic interconversion and crosstalk, illustrating how they allow optimized bacterial adaptation to their environmental niches.

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

(p)ppGpp synthetases are required for the pathogenicity of Salmonella Pullorum in chickens.

Salmonella Pullorum is a pathogen specific to birds that can cause Pullorum disease in young chickens and lead to considerable economic losses in the poultry industry. During transmission and infection, S. Pullorum will encounter various environmental stresses and host defenses. The stringent response is an important adaptation response induced by (p)ppGpp, and in Salmonella, (p)ppGpp is synthesized by two (p)ppGpp synthetases, RelA and SpoT. To investigate the role of (p)ppGpp synthetases in the adaptation and pathogenicity of S. Pullorum, a (p)ppGpp synthetases mutant (ΔrelAΔspoT) was constructed, and its physiological phenotypes and pathogenicity, as well as transcription profiling, were compared with the parent strain. The ΔrelAΔspoT mutant showed decreased ability to form biofilms, and reduced resistance to acidic, alkaline, high osmolarity and H2O2 conditions. The internalization of the ΔrelAΔspoT mutant into host cells in vitro and its lethality and colonization abilities within young chickens were also significantly reduced. RNA sequencing showed that the (p)ppGpp synthetases did not only affect the classic stringent response, such as inhibition of DNA replication and protein synthesis, but also controlled the expression of many virulence factors, in particular, the Salmonella pathogenicity island 1 (SPI-1) and SPI-2 type III secretion systems (T3SSs), and adhesion factors. These results suggest that the (p)ppGpp synthetases are required for the pathogenicity of S. Pullorum by affecting its stress response and the expression of the virulence factors.

(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes.

The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenesIMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2'-OH of GTP.

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3'-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 2'-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogs substituted with different chemical moieties at the 2' position suggest a clear role for 2'-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2'-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.

The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile.

The human pathogen Clostridioides difficile is increasingly tolerant of multiple antibiotics and causes infections with a high rate of recurrence, creating an urgent need for new preventative and therapeutic strategies. The stringent response, a universal bacterial response to extracellular stress, governs antibiotic survival and pathogenesis in diverse organisms but has not previously been characterized in C. difficile Here, we report that the C. difficile (p)ppGpp synthetase RSH is incapable of utilizing GTP or GMP as a substrate but readily synthesizes ppGpp from GDP. The enzyme also utilizes many structurally diverse metal cofactors for reaction catalysis and remains functionally stable at a wide range of environmental pHs. Transcription of rsh is stimulated by stationary-phase onset and by exposure to the antibiotics clindamycin and metronidazole. Chemical inhibition of RSH by the ppGpp analog relacin increases antibiotic susceptibility in epidemic C. difficile R20291, indicating that RSH inhibitors may be a viable strategy for drug development against C. difficile infection. Finally, transcriptional suppression of rsh also increases bacterial antibiotic susceptibility, suggesting that RSH contributes to C. difficile antibiotic tolerance and survival.IMPORTANCEClostridioides difficile infection (CDI) is an urgent public health threat with a high recurrence rate, in part because the causative bacterium has a high rate of antibiotic survival. The (p)ppGpp-mediated bacterial stringent response plays a role in antibiotic tolerance in diverse pathogens and is a potential target for development of new antimicrobials because the enzymes that metabolize (p)ppGpp have no mammalian homologs. We report that stationary-phase onset and antibiotics induce expression of the clostridial ppGpp synthetase RSH and that both chemical inhibition and translational suppression of RSH increase C. difficile antibiotic susceptibility. This demonstrates that development of RSH inhibitors to serve as adjuvants to antibiotic therapy is a potential approach for the development of new strategies to combat CDI.

Molecular Mechanism of Regulation of the Purine Salvage Enzyme XPRT by the Alarmones pppGpp, ppGpp, and pGpp.

The alarmones pppGpp and ppGpp mediate starvation response and maintain purine homeostasis to protect bacteria. In the bacterial phyla Firmicutes and Bacteroidetes, xanthine phosphoribosyltransferase (XPRT) is a purine salvage enzyme that produces the nucleotide XMP from PRPP and xanthine. Combining structural, biochemical, and genetic analyses, we show that pppGpp and ppGpp, as well as a third newly identified alarmone pGpp, all directly interact with XPRT from the Gram-positive bacterium Bacillus subtilis and inhibit XPRT activity by competing with its substrate PRPP. Structural analysis reveals that ppGpp binds the PRPP binding motif within the XPRT active site. This motif is present in another (p)ppGpp target, the purine salvage enzyme HPRT, suggesting evolutionary conservation in different enzymes. However, XPRT oligomeric interaction is distinct from HPRT in that XPRT forms a symmetric dimer with two (p)ppGpp binding sites at the dimer interface. (p)ppGpp's interaction with an XPRT bridging loop across the interface results in XPRT cooperatively binding (p)ppGpp. Also, XPRT displays differential regulation by the alarmones as it is potently inhibited by both ppGpp and pGpp, but only modestly by pppGpp. Lastly, we demonstrate that the alarmones are necessary for protecting GTP homeostasis against excess environmental xanthine in B. subtilis, suggesting that regulation of XPRT is key for regulating the purine salvage pathway.

The stringent response regulator (p) ppGpp mediates virulence gene expression and survival in Erwinia amylovora.

BACKGROUND: The nucleotide second messengers, i.e., guanosine tetraphosphate and pentaphosphate [collectively referred to as (p) ppGpp], trigger the stringent response under nutrient starvation conditions and play an essential role in virulence in the fire blight pathogen Erwinia amylovora. Here, we present transcriptomic analyses to uncover the overall effect of (p) ppGpp-mediated stringent response in E. amylovora in the hrp-inducing minimal medium (HMM). RESULTS: In this study, we investigated the transcriptomic changes of the (p) ppGpp0 mutant under the type III secretion system (T3SS)-inducing condition using RNA-seq. A total of 1314 differentially expressed genes (DEGs) was uncovered, representing more than one third (36.8%) of all genes in the E. amylovora genome. Compared to the wild-type, the (p) ppGpp0 mutant showed down-regulation of genes involved in peptide ATP-binding cassette (ABC) transporters and virulence-related processes, including type III secretion system (T3SS), biofilm, and motility. Interestingly, in contrast to previous reports, the (p) ppGpp0 mutant showed up-regulation of amino acid biosynthesis genes, suggesting that it might be due to that these amino acid biosynthesis genes are indirectly regulated by (p) ppGpp in E. amylovora or represent specific culturing condition used. Furthermore, the (p) ppGpp0 mutant exhibited up-regulation of genes involved in translation, SOS response, DNA replication, chromosome segregation, as well as biosynthesis of nucleotide, fatty acid and lipid. CONCLUSION: These findings suggested that in HMM environment, E. amylovora might use (p) ppGpp as a signal to activate virulence gene expression, and simultaneously mediate the balance between virulence and survival by negatively regulating DNA replication, translation, cell division, as well as biosynthesis of nucleotide, amino acid, fatty acid, and lipid. Therefore, (p) ppGpp could be a promising target for developing novel control measures to fight against this devastating disease of apples and pears.

The Absence of (p)ppGpp Renders Initiation of Escherichia coli Chromosomal DNA Synthesis Independent of Growth Rates.

The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase.IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.

The Ps and Qs of alarmone synthesis in Staphylococcus aureus.

During the stringent response, bacteria synthesize guanosine-3',5'-bis(diphosphate) (ppGpp) and guanosine-5'-triphosphate 3'-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp 'alarmones' are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode 'small alarmone synthetase' (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5'-phosphate 3'-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10-fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) and Streptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically modulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species.

(p)ppGpp and CodY Promote Enterococcus faecalis Virulence in a Murine Model of Catheter-Associated Urinary Tract Infection.

In Firmicutes, the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infection (CAUTI) mouse model and used global transcriptional analysis to investigate the relationship of (p)ppGpp and CodY. The absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence, supporting previous evidence that the association of (p)ppGpp with enterococcal virulence is not dependent on the activation of the stringent response. Inactivation of codY in the (p)ppGpp0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicates that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may play a role in enterococcal CAUTI. Collectively, data from this study identify the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and support that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism under adverse conditions.IMPORTANCE Catheter-associated urinary tract infections (CAUTIs) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently causes complicated CAUTI is the bacterium Enterococcus faecalis, a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice.

tRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.

Growth suppression by altered (p)ppGpp levels results from non-optimal resource allocation in Escherichia coli.

Understanding how bacteria coordinate gene expression with biomass growth to adapt to various stress conditions remains a grand challenge in biology. Stress response is often associated with dramatic accumulation of cellular guanosine tetra- or penta-phosphate (p)ppGpp (also known as 'magic spot'), which is a key second messenger participating in regulating various biochemical and physiological processes of bacteria. Despite of the extensive studies on the mechanism of gene regulation by (p)ppGpp during stringent response, the connection between (p)ppGpp and bacterial steady-state exponential growth remains elusive. Here, we establish a versatile genetic approach to systematically perturb the (p)ppGpp level of Escherichia coli through titrating either the single-function (p)ppGpp synthetase or the singe-function (p)ppGpp hydrolase and quantitatively characterize cell growth and gene expression. Strikingly, increased and decreased (p)ppGpp levels both cause remarkable growth suppression of E. coli. From a coarse-grained insight, we demonstrate that increased (p)ppGpp levels limit ribosome synthesis while decreased (p)ppGpp levels limit the expression of metabolic proteins, both resulting in non-optimal resource allocation. Our study reveals a profound role of (p)ppGpp in regulating bacterial growth through governing global resource allocation. Moreover, we highlight the Mesh1 (p)ppGpp hydrolase from Drosophila melanogaster as a powerful genetic tool for interrogating bacterial (p)ppGpp physiology.

The Stringent Response Determines the Ability of a Commensal Bacterium to Survive Starvation and to Persist in the Gut.

In the mammalian gut, bacteria compete for resources to maintain their populations, but the factors determining their success are poorly understood. We report that the human gut bacterium Bacteroides thetaiotaomicron relies on the stringent response, an intracellular signaling pathway that allocates resources away from growth, to survive carbon starvation and persist in the gut. Genome-scale transcriptomics, 13C-labeling, and metabolomics analyses reveal that B. thetaiotaomicron uses the alarmone (p)ppGpp to repress multiple biosynthetic pathways and upregulate tricarboxylic acid (TCA) cycle genes in these conditions. During carbon starvation, (p)ppGpp triggers accumulation of the metabolite alpha-ketoglutarate, which itself acts as a metabolic regulator; alpha-ketoglutarate supplementation restores viability to a (p)ppGpp-deficient strain. These studies uncover how commensal bacteria adapt to the gut by modulating central metabolism and reveal that halting rather than accelerating growth can be a determining factor for membership in the gut microbiome.

Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift in Escherichia coli.

Bacteria respond to nutritional stresses by changing the cellular concentration of the alarmone (p)ppGpp. This control mechanism, called the stringent response, depends on two enzymes, the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT in Escherichia coli and related bacteria. Because SpoT is the only enzyme responsible for (p)ppGpp hydrolysis in these bacteria, SpoT activity needs to be tightly regulated to prevent the uncontrolled accumulation of (p)ppGpp, which is lethal. To date, however, no such regulation of SpoT (p)ppGpp hydrolase activity has been documented in E. coli In this study, we show that Rsd directly interacts with SpoT and stimulates its (p)ppGpp hydrolase activity. Dephosphorylated HPr, but not phosphorylated HPr, of the phosphoenolpyruvate-dependent sugar phosphotransferase system could antagonize the stimulatory effect of Rsd on SpoT (p)ppGpp hydrolase activity. Thus, we suggest that Rsd is a carbon source-dependent regulator of the stringent response in E. coli.