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.

Metformin inhibits digestive proteases and impairs protein digestion in mice.

Metformin is among the most prescribed medications worldwide and the first-line therapy for type 2 diabetes. However, gastrointestinal side effects are common and can be dose limiting. The total daily metformin dose frequently reaches several grams, and poor absorption results in high intestinal drug concentrations. Here, we report that metformin inhibits the activity of enteropeptidase and other digestive enzymes at drug concentrations predicted to occur in the human duodenum. Treatment of mouse gastrointestinal tissue with metformin reduces enteropeptidase activity; further, metformin-treated mice exhibit reduced enteropeptidase activity, reduced trypsin activity, and impaired protein digestion within the intestinal lumen. These results indicate that metformin-induced protein maldigestion could contribute to the gastrointestinal side effects and other impacts of this widely used drug.

The nucleotide messenger (p)ppGpp is an anti-inducer of the purine synthesis transcription regulator PurR in Bacillus.

The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here, we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis gene expression upon amino acid starvation. We first identified PurR as a receptor of (p)ppGpp in Bacillus anthracis. A co-structure with Bacillus subtilis PurR reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of phosphoribosyltransferase enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant in B. subtilis fails to downregulate purine synthesis genes upon amino acid starvation. Our work establishes the precedent of (p)ppGpp as an effector of a classical transcription repressor and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from soil bacteria to pathogens.

Treatment of food allergy: Oral immunotherapy, biologics, and beyond.

The prevalence of food allergy (FA) has been increasing globally and comes with a heavy burden not just economically, but also on quality of life. Although oral immunotherapy (OIT) is effective at inducing desensitization to food allergens, it has several limitations that weaken its success. Limitations include a long duration of build-up, especially when used for multiple allergens, and a high rate of reported adverse events. Furthermore, OIT may not be effective in all patients. Efforts are underway to identify additional treatment options, either as monotherapy or in combination, to treat FA or enhance the safety and efficacy of OIT. Biologics such as omalizumab and dupilumab, which already have US Food and Drug Administration approval for other atopic conditions have been the most studied, but additional biologics and novel strategies are emerging. In this review, we discuss therapeutic strategies including immunoglobulin E inhibitors, immunoglobulin E disruptors, interleukin-4 and interleukin-13 inhibitors, antialarmins, JAK1 and BTK inhibitors, and nanoparticles, and the data surrounding their application in FA and highlighting their potential.

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis.

UNLABELLED: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE: Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

Health and wealth in Mesoamerica: findings from Salud Mesomérica 2015.

BACKGROUND: Individual income and poverty are associated with poor health outcomes. The poor face unique challenges related to access, education, financial capacity, environmental effects, and other factors that threaten their health outcomes. METHODS: We examined the variation in the health outcomes and health behaviors among the poorest quintile in eight countries of Mesoamerica using data from the Salud Mesomérica 2015 baseline household surveys. We used multivariable logistic regression to measure the association between delivering a child in a health facility and select household and maternal characteristics, including education and measures of wealth. RESULTS: Health indicators varied greatly between geographic segments. Controlling for other demographic characteristics, women with at least secondary education were more likely to have an in-facility delivery compared to women who had not attended school (OR: 3.20, 95 % confidence interval [CI]: 2.56-3.99, respectively). Similarly, women from households with the highest expenditure were more likely to deliver in a health facility compared to those from the lowest expenditure households (OR 3.06, 95 % CI: 2.43-3.85). Household assets did not impact these associations. Moreover, we found that commonly-used definitions of poverty do not align with the disparities in health outcomes observed in these communities. CONCLUSIONS: Although poverty measured by expenditure or wealth is associated with health disparities or health outcomes, a composite indicator of health poverty based on coverage is more likely to focus attention on health problems and solutions. Our findings call for the public health community to define poverty by health coverage measures rather than income or wealth. Such a health-poverty metric is more likely to generate attention and mobilize targeted action by the health communities than our current definition of poverty.

Salud Mesoamérica 2015 Initiative: design, implementation, and baseline findings.

BACKGROUND: Health has improved markedly in Mesoamerica, the region consisting of southern Mexico and Central America, over the past decade. Despite this progress, there remain substantial inequalities in health outcomes, access, and quality of medical care between and within countries. Poor, indigenous, and rural populations have considerably worse health indicators than national or regional averages. In an effort to address these health inequalities, the Salud Mesoamérica 2015 Initiative (SM2015), a results-based financing initiative, was established. METHODS: For each of the eight participating countries, health targets were set to measure the progress of improvements in maternal and child health produced by the Initiative. To establish a baseline, we conducted censuses of 90,000 households, completed 20,225 household interviews, and surveyed 479 health facilities in the poorest areas of Mesoamerica. Pairing health facility and household surveys allows us to link barriers to care and health outcomes with health system infrastructure components and quality of health services. RESULTS: Indicators varied significantly within and between countries. Anemia was most prevalent in Panama and least prevalent in Honduras. Anemia varied by age, with the highest levels observed among children aged 0 to 11 months in all settings. Belize had the highest proportion of institutional deliveries (99%), while Guatemala had the lowest (24%). The proportion of women with four antenatal care visits with a skilled attendant was highest in El Salvador (90%) and the lowest in Guatemala (20%). Availability of contraceptives also varied. The availability of condoms ranged from 83% in Nicaragua to 97% in Honduras. Oral contraceptive pills and injectable contraceptives were available in just 75% of facilities in Panama. IUDs were observed in only 21.5% of facilities surveyed in El Salvador. CONCLUSIONS: These data provide a baseline of much-needed information for evidence-based action on health throughout Mesoamerica. Our baseline estimates reflect large disparities in health indicators within and between countries and will facilitate the evaluation of interventions and investments deployed in the region over the next three to five years. SM2015's innovative monitoring and evaluation framework will allow health officials with limited resources to identify and target areas of greatest need.

New biologics for food allergy.

PURPOSE OF REVIEW: This review aims to explore role of emerging biologics, including ligelizumab, UB-221, dupilumab, and antialarmins, in food allergy management. With a focus on recent developments, we evaluate their promise in mitigating adverse events during oral immunotherapy (OIT), reducing allergic reactions, and addressing the limitations of current therapeutic options. RECENT FINDINGS: Antiimmunoglobulin E mAbs, exemplified by omalizumab, demonstrate efficacy in desensitization and safety improvement during multiallergen OIT. Next-generation antibodies like ligelizumab and UB-221 exhibit enhanced potency and unique mechanisms, holding promise for food allergy treatment. Dupilumab, targeting IL-4 receptor alpha, presents potential benefits in decreasing allergen-specific IgE and modifying the atopic march. Exploration of antialarmins, specifically anti-IL-33 (etokimab) and anti-TSLP (tezepelumab), reveals encouraging results, with etokimab showing early success in peanut allergy trials. SUMMARY: Biologics hold promising potential for food allergy treatment. Tailoring therapeutic approaches based on shared decision-making becomes pivotal. While omalizumab remains a significant option, next-generation anti-IgE antibodies and agents targeting alarmins exhibit unique strengths. Dupilumab, despite limited success as monotherapy, shows promise as an adjunct for OIT. Careful consideration of treatment goals, patient preferences, and the evolving landscape of biologics will shape future clinical practice, offering allergists an expanded toolbox for personalized food allergy management.

The Road to Reintegration: Status and Continuing Support of the U.S. Air Force's Wounded, Ill, and Injured.

The U.S. Air Force asked RAND Project AIR FORCE (PAF) to help assess the well-being of its wounded members and the quality of services provided to facilitate their recovery and reintegration. RAND PAF fielded a survey in the fall of 2016 to assess wounded airmen's functioning in the domains of physical health, mental health, interpersonal relationships, unemployment, and financial status, as well as their utilization and perceptions of Air Force nonmedical programs for wounded airmen. The authors of this study invited all 713 wounded airmen enrolled in the Air Force Wounded Warrior program to complete the survey, and 270 airmen (38 percent) completed it. One-third of airmen reported difficulty obtaining care for physical or mental health conditions, and one-quarter expressed dissatisfaction with coordination of care. Similar proportions of airmen reported barriers to care for physical and mental health conditions. Difficulty scheduling appointments was the most commonly endorsed barrier for both types of conditions. Small but notable proportions of airmen reported potential social support deficits, unemployment, and financial problems. For many of the Air Force's programs for wounded airmen, over 80 percent of program users reported overall program satisfaction. The authors recommend that the Air Force consider focusing on improving care coordination, increasing health care system capacity, continuing employment assistance, and improving marketing of programs with low uptake.

Allosteric Regulation of Pyruvate Kinase Enables Efficient and Robust Gluconeogenesis by Preventing Metabolic Conflicts and Carbon Overflow.

Glycolysis and gluconeogenesis are reciprocal metabolic pathways that utilize different carbon sources. Pyruvate kinase catalyzes the irreversible final step of glycolysis, yet the physiological function of its regulation is poorly understood. Through metabolomics and enzyme kinetics studies, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis in the soil bacterium Bacillus subtilis . This regulation involves an extra C-terminal domain (ECTD) of pyruvate kinase, which is essential for autoinhibition and regulation by metabolic effectors. Introducing a pyruvate kinase mutant lacking the ECTD into B. subtilis resulted in defects specifically under gluconeogenic conditions, including inefficient carbon utilization, slower growth, and decreased resistance to the herbicide glyphosate. These defects are not caused by the phosphoenolpyruvate-pyruvate-oxaloacetate futile cycle. Instead, we identified two significant metabolic consequences of pyruvate kinase dysregulation during gluconeogenesis: increased carbon overflow into the medium and failure to expand glycolytic intermediates such as phosphoenolpyruvate (PEP). In silico analysis revealed that in wild-type cells, an expanded PEP pool enabled by pyruvate kinase regulation is critical for the thermodynamic feasibility of gluconeogenesis. Our findings underscore the importance of allosteric regulation during gluconeogenesis in coordinating metabolic flux, efficient energy utilization, and antimicrobial resistance.

Transitioning from epicutaneous to oral peanut immunotherapy.

Introduction: Epicutaneous immunotherapy (EPIT) has been tested in clinical trials for children with peanut allergy (PA) for its safety and efficacy in inducing desensitization. Aside from peanut avoidance and symptom management, oral immunotherapy (OIT) is another option for PA patients. However, OIT can be associated with adverse events and pose safety concerns to children and their caregivers. Methods: This study assessed 27 children who successfully completed a peanut EPIT trial. 18 of them transitioned to peanut OIT with starting doses ranging from 10-600 mg of peanut protein. Our aim was to learn more about the EPIT to OIT experience through descriptive survey responses and to gather information that may support the sequential use of the two immunotherapies for safe and positive outcomes that may not be achieved by either alone. Results: Overall, children and their caregivers had less anxiety about starting OIT after having had peanut exposure through EPIT. Most children who transitioned from EPIT to OIT had no or minor symptoms initially, with symptoms lessening later in OIT. Most were also able to maintain or increase their peanut dose over time, achieving maintenance doses of 60-2,000 mg. Discussion: In comparison with current literature on OIT for PA in children, the reported symptoms appeared less severe and less prevalent in the EPIT to OIT group. However, there were 3 participants who withdrew from OIT due to the development of intolerable symptoms. This study provides initial data in support of EPIT to OIT, and larger randomized controlled trials assessing effectiveness of the two therapies together are warranted.

Cancer prevention, screening, and survivorship ECHO: A pilot experience with an educational telehealth program.

INTRODUCTION: The American Cancer Society, Inc. (ACS) estimates that 37,940 Indiana residents were diagnosed with cancer in 2020, which remains the leading cause of death in the state. Across the cancer continuum, national goals have been established targeting recommended benchmarks for states in prevention, screening, treatment, and survivorship. Indiana consistently falls below most goals for each of these targeted categories. METHODS: To address these disparities, we implemented Project ECHO (Extension for Community Healthcare Outcomes) as a virtual telehealth educational platform targeted at local healthcare providers. ECHO programs utilize a novel tele-mentoring approach to the education of clinicians in a hub/spoke design. Sessions occurred twice monthly from September 2019 to September 2020 and consisted of a traditional didactic lecture and a case-based discussion led by participating providers. RESULTS: During the pilot year there were a total of 22 ECHO sessions with 140 different participants. On average, 15.5 spokes attended each session with increasing participation at the end of the year. Post-session surveys suggested generally favorable perception with 72% of respondents finding the quality "excellent." DISCUSSION: Given the increasing rate of recurrent participation toward the end of the pilot year in conjunction with the favorable survey responses following each session, it was felt that the program was overall successful and warranted continued implementation. CONCLUSION: The Project ECHO platform is a validated telehealth education platform that has the potential to impact cancer care at multiple points along the cancer continuum at the regional level.

Performance characteristics of the ARK diagnostics gabapentin immunoassay.

BACKGROUND: Gabapentin is an antiepileptic drug used as adjunct therapy in the treatment of seizures. Absorption is saturable, and drug clearance can be reduced if patients have impaired renal function. Therapeutic drug monitoring can be useful for optimizing the dose in patients with impaired renal function, for evaluating individual patient absorption thresholds, and for monitoring compliance. Although chromatographic techniques have historically been used to support gabapentin monitoring, an immunoassay was recently introduced by ARK Diagnostics, for use with open channel chemistry analyzers. Here, we evaluated the immunoassay on a random access instrument. METHODS: The ARK gabapentin assay was validated using a Beckman AU400e automated chemistry analyzer. Imprecision was assessed with 5 replicates of 3 concentrations (2.5, 8.0, and 25.0 mg/L), analyzed for 4 days. The analytical measurement range was evaluated with duplicate measurements of a prepared sample (40.0 mg/L) that was serially diluted. Patients' results were compared with the results generated with a previously validated ultra-high performance liquid chromatography coupled to tandem mass spectrometry method (n = 45, range, 1.5-45.6 mg/L). RESULTS: The within-run and between-run coefficients of variation were ≤8.1%. The analytical measurement range was confirmed to be 1.5-40.0 mg/L, as stated by the manufacturer. The Deming regression for the results of 45 patients produced a correlation coefficient of 0.9987, a linear regression slope of 1.01, and an intercept of 0.24 when compared with the ultra-high performance liquid chromatography coupled to tandem mass spectrometry assay. CONCLUSIONS: The ARK immunoassay is suitable for the clinical use of monitoring gabapentin in serum or plasma on the Beckman AU400e.

Aortic root spontaneous echocardiographic contrast due to haemodynamic support with a percutaneous left ventricular assist device (TandemHeart).

Intra-procedural transoesophageal echocardiography (TEE) is useful in guiding device placement and monitoring for procedural complications in the interventional lab. We report a case of spontaneous echo contrast (SEC) formation in the aortic root immediately following initiation of haemodynamic support with a percutaneous left ventricular (LV) assist device for cardiogenic shock. With TEE guidance, assist device flow rates were adjusted, resulting in resolution of SEC. This case illustrates another potential use for intra-procedural TEE during LV assist device placement.

Oral Immunotherapy in Children: Clinical Considerations and Practical Management.

Oral immunotherapy (OIT) in pediatric patients provides an alternative option to the current standard of care in food allergy, which is allergen avoidance and reactive treatment. Because patients are exposed to one or more food allergens during treatment, OIT is associated with adverse events and can be a cumbersome process for children, their caregivers, and clinicians. However, there have been an overwhelming number of studies that show high efficacy in both single- and multi-allergen OIT, and that quality of life is greatly improved for both patients and their families after undergoing immunotherapy. This review discusses clinical considerations for OIT in pediatrics, including efficacy and safety, practical management, and future directions of treatment.

A Common Pathway for Activation of Host-Targeting and Bacteria-Targeting Toxins in Human Intestinal Bacteria.

Human gut microbes exhibit a spectrum of cooperative and antagonistic interactions with their host and also with other microbes. The major Bacteroides host-targeting virulence factor, Bacteroides fragilis toxin (BFT), is produced as an inactive protoxin by enterotoxigenic B. fragilis strains. BFT is processed by the conserved bacterial cysteine protease fragipain (Fpn), which is also encoded in B. fragilis strains that lack BFT. In this report, we identify a secreted antibacterial protein (fragipain-activated bacteriocin 1 [Fab1]) and its cognate immunity protein (resistance to fragipain-activated bacteriocin 1 [RFab1]) in enterotoxigenic and nontoxigenic strains of B. fragilis. Although BFT and Fab1 share no sequence identity, Fpn also activates the Fab1 protoxin, resulting in its secretion and antibacterial activity. These findings highlight commonalities between host- and bacterium-targeting toxins in intestinal bacteria and suggest that antibacterial antagonism may promote the conservation of pathways that activate host-targeting virulence factors. IMPORTANCE The human intestine harbors a highly complex microbial community; interpersonal variation in this community can impact pathogen susceptibility, metabolism, and other aspects of health. Here, we identified and characterized a commensal-targeting antibacterial protein encoded in the gut microbiome. Notably, a shared pathway activates this antibacterial toxin and a host-targeting toxin. These findings highlight unexpected commonalities between host- and bacterium-targeting toxins in intestinal bacteria.

C3 R102G polymorphism increases risk of age-related macular degeneration.

Inflammation has long been suspected to play a role in the pathogenesis of age-related macular degeneration (AMD). Association of variants in the complement factor H (CFH) and complement factor B (CFB) genes has targeted the search for additional loci to the alternative complement cascade, of which C3 is a major component. Two non-synonymous coding polymorphisms within C3, R102G and L314P, have previously been strongly associated with increased risk. These variants are in strong linkage disequilibrium (LD), making the contribution of this locus to AMD even more difficult to ascertain. We sought to determine whether the C3 association resulted primarily from only one of these two variants or from a combined effect of both in 223 families and an independent dataset of 701 cases and 286 unrelated controls. The C3 polymorphisms were in strong LD (r(2) = 0.85), and both were associated in the family-based and case-control datasets (R102G genoPDT P = 0.02, case-control genotypic P = 0.004; L314P genoPDT P = 0.001, case-control genotypic P = 0.04). In conditional analyses in the case-control dataset, R102G remained associated with disease in the L314P risk allele carriers (P = 0.01), but there was no effect of L314P in the R102G risk allele carriers (P = 0.2). After adjusting for age, smoking, CFH Y402H, LOC387715 A69S, and CFB R32Q, the effect of R102G remained strong [P = 0.015, odds ratio = 1.55, 95% confidence interval 1.09 to 2.21, adjusted PAR(population attributable risk) = 0.17]. Therefore, while the strong LD between R102G and L314P makes it difficult to disentangle their individual effects on disease risk, the R102G polymorphism acting alone provides the best model for disease in our data.

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 nucleotide pGpp acts as a third alarmone in Bacillus, with functions distinct from those of (p) ppGpp.

The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5'-monophosphate-3'-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp.