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Patacq, Clément, Nicolas Chaudet, and Fabien Létisse. "Crucial Role of ppGpp in the Resilience of Escherichia coli to Growth Disruption." mSphere 5, no. 6 (December 23, 2020): e01132-20. http://dx.doi.org/10.1128/msphere.01132-20.
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ABSTRACTBacteria grow in constantly changing environments that can suddenly become completely depleted of essential nutrients. The stringent response, a rewiring of the cellular metabolism mediated by the alarmone (p)ppGpp, plays a crucial role in adjusting bacterial growth to the severity of the nutritional stress. The ability of (p)ppGpp to trigger a slowdown of cell growth or induce bacterial dormancy has been widely investigated. However, little is known about the role of (p)ppGpp in promoting growth recovery after severe growth inhibition. In this study, we performed a time-resolved analysis of (p)ppGpp metabolism in Escherichia coli as it recovered from a sudden slowdown in growth. The results show that E. coli recovers by itself from the growth disruption provoked by the addition of serine hydroxamate, the serine analogue that we used to induce the stringent response. Growth inhibition was accompanied by a severe disturbance of metabolic activity and, more surprisingly, a transient overflow of valine and alanine. Our data also show that ppGpp is crucial for growth recovery since in the absence of ppGpp, E. coli’s growth recovery was slower. In contrast, an increased concentration of pppGpp was found to have no significant effect on growth recovery. Interestingly, the observed decrease in intracellular ppGpp levels in the recovery phase correlated with bacterial growth, and the main effect involved in the return to the basal level was identified by flux calculation as growth dilution. This report thus significantly expands our knowledge of (p)ppGpp metabolism in E. coli physiology.IMPORTANCE The capacity of microbes to resist and overcome environmental insults, known as resilience, allows them to survive in changing environments but also to resist antibiotic and biocide treatments and immune system responses. Although the role of the stringent response in bacterial resilience to nutritional stresses has been well studied, little is known about its importance in the ability of the bacteria to not just resist but also recover from these disturbances. To address this important question, we investigated growth disruption resilience in the model bacterium Escherichia coli and its dependence on the stringent response alarmone (p)ppGpp by quantifying ppGpp and pppGpp levels as growth was disrupted and then recovered. Our findings may thus contribute to understanding how ppGpp improves E. coli’s resilience to nutritional stress and other environmental insults.
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Gaca, Anthony O., Pavel Kudrin, Cristina Colomer-Winter, Jelena Beljantseva, Kuanqing Liu, Brent Anderson, Jue D. Wang, et al. "From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis." Journal of Bacteriology 197, no. 18 (June 29, 2015): 2908–19. http://dx.doi.org/10.1128/jb.00324-15.
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ABSTRACTThe 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. InEnterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolaseE. faecalisRel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation inFirmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEfsynthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEfalso 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 ofE. faecalisenzymes involved in GTP biosynthesis and, to a lesser extent, transcription ofrrnBbyEscherichia coliRNA polymerase. Activation ofE. coliRelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEfwas activated only by ppGpp. Furthermore, enzymatic activity of RelQEfis 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.IMPORTANCEAccumulation 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 ofEnterococcus faecalis(RelQEf), we found that, in addition to (p)ppGpp, RelQEfis an efficient producer of pGpp (GMP 3′-diphosphate).In vitroanalysis 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 RelQEfand suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.
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Gaca, Anthony O., Cristina Colomer-Winter, and José A. Lemos. "Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis." Journal of Bacteriology 197, no. 7 (January 20, 2015): 1146–56. http://dx.doi.org/10.1128/jb.02577-14.
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In nearly all bacterial species examined so far, amino acid starvation triggers the rapid accumulation of the nucleotide second messenger (p)ppGpp, the effector of the stringent response. While for years the enzymes involved in (p)ppGpp metabolism and the significance of (p)ppGpp accumulation to stress survival were considered well defined, a recent surge of interest in the field has uncovered an unanticipated level of diversity in how bacteria metabolize and utilize (p)ppGpp to rapidly synchronize a variety of biological processes important for growth and stress survival. In addition to the classic activation of the stringent response, it has become evident that (p)ppGpp exerts differential effects on cell physiology in an incremental manner rather than simply acting as a biphasic switch that controls growth or stasis. Of particular interest is the intimate relationship of (p)ppGpp with persister cell formation and virulence, which has spurred the pursuit of (p)ppGpp inhibitors as a means to control recalcitrant infections. Here, we present an overview of the enzymes responsible for (p)ppGpp metabolism, elaborate on the intricacies that link basal production of (p)ppGpp to bacterial homeostasis, and discuss the implications of targeting (p)ppGpp synthesis as a means to disrupt long-term bacterial survival strategies.
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Liu, Kuanqing, Alycia N. Bittner, and Jue D. Wang. "Diversity in (p)ppGpp metabolism and effectors." Current Opinion in Microbiology 24 (April 2015): 72–79. http://dx.doi.org/10.1016/j.mib.2015.01.012.
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Zhang, Yong, Eva Zborníková, Dominik Rejman, and Kenn Gerdes. "Novel (p)ppGpp Binding and Metabolizing Proteins ofEscherichia coli." mBio 9, no. 2 (March 6, 2018): e02188-17. http://dx.doi.org/10.1128/mbio.02188-17.
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ABSTRACTThe alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Despite intensive studies since (p)ppGpp was discovered over 4 decades ago, (p)ppGpp binding proteins have not been systematically identified inEscherichia coli. We applied DRaCALA (differentialradialcapillaryaction ofligandassay) to identify (p)ppGpp-protein interactions. We discovered 12 new (p)ppGpp targets inE. colithat, based on their physiological functions, could be classified into four major groups, involved in (i) purine nucleotide homeostasis (YgdH), (ii) ribosome biogenesis and translation (RsgA, Era, HflX, and LepA), (iii) maturation of dehydrogenases (HypB), and (iv) metabolism of (p)ppGpp (MutT, NudG, TrmE, NadR, PhoA, and UshA). We present a comprehensive and comparative biochemical and physiological characterization of these novel (p)ppGpp targets together with a comparative analysis of relevant, known (p)ppGpp binding proteins. Via this, primary targets of (p)ppGpp inE. coliare identified. The GTP salvage biosynthesis pathway and ribosome biogenesis and translation are confirmed as targets of (p)ppGpp that are highly conserved betweenE. coliandFirmicutes. In addition, an alternative (p)ppGpp degradative pathway, involving NudG and MutT, was uncovered. This report thus significantly expands the known cohort of (p)ppGpp targets inE. coli.IMPORTANCEAntibiotic resistance and tolerance exhibited by pathogenic bacteria have resulted in a global public health crisis. Remarkably, almost all bacterial pathogens require the alarmone (p)ppGpp to be virulent. Thus, (p)ppGpp not only induces tolerance of nutritional limitations and chemical insults, including antibiotics, but is also often required for induction of virulence genes. However, understanding of the molecular targets of (p)ppGpp and the mechanisms by which (p)ppGpp influences bacterial physiology is incomplete. In this study, a systematic approach was used to uncover novel targets of (p)ppGpp inE. coli, the best-studied model bacterium. Comprehensive comparative studies of the targets revealed conserved target pathways of (p)ppGpp in both Gram-positive and -negative bacteria and novel targets of (p)ppGpp, including an alternative degradative pathway of (p)ppGpp. Thus, our discoveries may help in understanding of how (p)ppGpp increases the stress resilience and multidrug tolerance not only of the model organismE. colibut also of the pathogenic organisms in which these targets are conserved.
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Huang, Can, Wenqian Li, and Jingyu Chen. "Transcriptomic Analysis Reveals Key Roles of (p)ppGpp and DksA in Regulating Metabolism and Chemotaxis in Yersinia enterocolitica." International Journal of Molecular Sciences 24, no. 8 (April 20, 2023): 7612. http://dx.doi.org/10.3390/ijms24087612.
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The stringent response is a rapid response system that is ubiquitous in bacteria, allowing them to sense changes in the external environment and undergo extensive physiological transformations. However, the regulators (p)ppGpp and DksA have extensive and complex regulatory patterns. Our previous studies demonstrated that (p)ppGpp and DksA in Yersinia enterocolitica positively co-regulated motility, antibiotic resistance, and environmental tolerance but had opposite roles in biofilm formation. To reveal the cellular functions regulated by (p)ppGpp and DksA comprehensively, the gene expression profiles of wild-type, ΔrelA, ΔrelAΔspoT, and ΔdksAΔrelAΔspoT strains were compared using RNA-Seq. Results showed that (p)ppGpp and DksA repressed the expression of ribosomal synthesis genes and enhanced the expression of genes involved in intracellular energy and material metabolism, amino acid transport and synthesis, flagella formation, and the phosphate transfer system. Additionally, (p)ppGpp and DksA inhibited amino acid utilization (such as arginine and cystine) and chemotaxis in Y. enterocolitica. Overall, the results of this study unraveled the link between (p)ppGpp and DksA in the metabolic networks, amino acid utilization, and chemotaxis in Y. enterocolitica and enhanced the understanding of stringent responses in Enterobacteriaceae.
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Holley, Concerta L., Xinjun Zhang, Kate R. Fortney, Sheila Ellinger, Paula Johnson, Beth Baker, Yunlong Liu, et al. "DksA and (p)ppGpp Have Unique and Overlapping Contributions to Haemophilus ducreyi Pathogenesis in Humans." Infection and Immunity 83, no. 8 (June 8, 2015): 3281–92. http://dx.doi.org/10.1128/iai.00692-15.
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The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence ofHaemophilus ducreyiin humans. Here, we constructed anH. ducreyidksAmutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation inH. ducreyi, we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28% and 17% ofH. ducreyiopen reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to thedksAmutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion ofdksAdownregulatedlspBand rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in thedksAmutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators ofH. ducreyigene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.
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Jimmy, Steffi, Chayan Kumar Saha, Tatsuaki Kurata, Constantine Stavropoulos, Sofia Raquel Alves Oliveira, Alan Koh, Albinas Cepauskas, et al. "A widespread toxin−antitoxin system exploiting growth control via alarmone signaling." Proceedings of the National Academy of Sciences 117, no. 19 (April 28, 2020): 10500–10510. http://dx.doi.org/10.1073/pnas.1916617117.
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Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin−antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS–antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.
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Brandi, Anna, Mara Giangrossi, Attilio Fabbretti, and Maurizio Falconi. "The hns Gene of Escherichia coli Is Transcriptionally Down-Regulated by (p)ppGpp." Microorganisms 8, no. 10 (October 10, 2020): 1558. http://dx.doi.org/10.3390/microorganisms8101558.
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Second messenger nucleotides, such as guanosine penta- or tetra-phosphate, commonly referred to as (p)ppGpp, are powerful signaling molecules, used by all bacteria to fine-tune cellular metabolism in response to nutrient availability. Indeed, under nutritional starvation, accumulation of (p)ppGpp reduces cell growth, inhibits stable RNAs synthesis, and selectively up- or down- regulates the expression of a large number of genes. Here, we show that the E. colihns promoter responds to intracellular level of (p)ppGpp. hns encodes the DNA binding protein H-NS, one of the major components of bacterial nucleoid. Currently, H-NS is viewed as a global regulator of transcription in an environment-dependent mode. Combining results from relA (ppGpp synthetase) and spoT (ppGpp synthetase/hydrolase) null mutants with those from an inducible plasmid encoded RelA system, we have found that hns expression is inversely correlated with the intracellular concentration of (p)ppGpp, particularly in exponential phase of growth. Furthermore, we have reproduced in an in vitro system the observed in vivo (p)ppGpp-mediated transcriptional repression of hns promoter. Electrophoretic mobility shift assays clearly demonstrated that this unusual nucleotide negatively affects the stability of RNA polymerase-hns promoter complex. Hence, these findings demonstrate that the hns promoter is subjected to an RNA polymerase-mediated down-regulation by increased intracellular levels of (p)ppGpp.
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Martínez-Costa, Oscar H., Miguel A. Fernández-Moreno, and Francisco Malpartida. "The relA/spoT-Homologous Gene inStreptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities." Journal of Bacteriology 180, no. 16 (August 15, 1998): 4123–32. http://dx.doi.org/10.1128/jb.180.16.4123-4132.1998.
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ABSTRACT Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined.S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence thatS. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of anS. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. colitruncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, arel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition,E. coli relA in an S. coelicolor reldeletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-typerel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.
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Tian, Chengzhe, Mohammad Roghanian, Mikkel Girke Jørgensen, Kim Sneppen, Michael Askvad Sørensen, Kenn Gerdes, and Namiko Mitarai. "Rapid Curtailing of the Stringent Response by Toxin-Antitoxin Module-Encoded mRNases." Journal of Bacteriology 198, no. 14 (May 2, 2016): 1918–26. http://dx.doi.org/10.1128/jb.00062-16.
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ABSTRACTEscherichia coliregulates its metabolism to adapt to changes in the environment, in particular to stressful downshifts in nutrient quality. Such shifts elicit the so-called stringent response, coordinated by the alarmone guanosine tetra- and pentaphosphate [(p)ppGpp]. On sudden amino acid (aa) starvation, RelA [(p)ppGpp synthetase I] activity is stimulated by binding of uncharged tRNAs to a vacant ribosomal site; the (p)ppGpp level increases dramatically and peaks within the time scale of a few minutes. The decrease of the (p)ppGpp level after the peak is mediated by the decreased production of mRNA by (p)ppGpp-associated transcriptional regulation, which reduces the vacant ribosomal A site and thus constitutes negative feedback to the RelA-dependent (p)ppGpp synthesis. Here we showed that on sudden isoleucine starvation, this peak was higher in anE. colistrain that lacks the 10 known mRNase-encoding toxin-antitoxin (TA) modules present in the wild-type (wt) strain. This observation suggested that toxins are part of the negative-feedback mechanism to control the (p)ppGpp level during the early stringent response. We built a ribosome trafficking model to evaluate the fold increase in RelA activity just after the onset of aa starvation. Combining this with a feedback model between the (p)ppGpp level and the mRNA level, we obtained reasonable fits to the experimental data for both strains. The analysis revealed that toxins are activated rapidly, within a minute after the onset of starvation, reducing the mRNA half-life by ∼30%.IMPORTANCEThe early stringent response elicited by amino acid starvation is controlled by a sharp increase of the cellular (p)ppGpp level. Toxin-antitoxin module-encoded mRNases are activated by (p)ppGpp through enhanced degradation of antitoxins. The present work shows that this activation happens over a very short time scale and that the activated mRNases negatively affect the (p)ppGpp level. The proposed mathematical model of (p)ppGpp regulation through the mRNA level highlights the importance of several feedback loops in early (p)ppGpp regulation.
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Sivapragasam, Smitha, and Anne Grove. "The Link between Purine Metabolism and Production of Antibiotics in Streptomyces." Antibiotics 8, no. 2 (June 6, 2019): 76. http://dx.doi.org/10.3390/antibiotics8020076.
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Stress and starvation causes bacterial cells to activate the stringent response. This results in down-regulation of energy-requiring processes related to growth, as well as an upregulation of genes associated with survival and stress responses. Guanosine tetra- and pentaphosphates (collectively referred to as (p)ppGpp) are critical for this process. In Gram-positive bacteria, a main function of (p)ppGpp is to limit cellular levels of GTP, one consequence of which is reduced transcription of genes that require GTP as the initiating nucleotide, such as rRNA genes. In Streptomycetes, the stringent response is also linked to complex morphological differentiation and to production of secondary metabolites, including antibiotics. These processes are also influenced by the second messenger c-di-GMP. Since GTP is a substrate for both (p)ppGpp and c-di-GMP, a finely tuned regulation of cellular GTP levels is required to ensure adequate synthesis of these guanosine derivatives. Here, we discuss mechanisms that operate to control guanosine metabolism and how they impinge on the production of antibiotics in Streptomyces species.
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Geiger, Tobias, Christiane Goerke, Michaela Fritz, Tina Schäfer, Knut Ohlsen, Manuel Liebeke, Michael Lalk, and Christiane Wolz. "Role of the (p)ppGpp Synthase RSH, a RelA/SpoT Homolog, in Stringent Response and Virulence of Staphylococcus aureus." Infection and Immunity 78, no. 5 (March 8, 2010): 1873–83. http://dx.doi.org/10.1128/iai.01439-09.
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ABSTRACT In most bacteria, nutrient limitations provoke the stringent control through the rapid synthesis of the alarmones pppGpp and ppGpp. Little is known about the stringent control in the human pathogen Staphylococcus aureus, partly due to the essentiality of the major (p)ppGpp synthase/hydrolase enzyme RSH (RelA/SpoT homolog). Here, we show that mutants defective only in the synthase domain of RSH (rsh syn) are not impaired in growth under nutrient-rich conditions. However, these mutants were more sensitive toward mupirocin and were impaired in survival when essential amino acids were depleted from the medium. RSH is the major enzyme responsible for (p)ppGpp synthesis in response to amino acid deprivation (lack of Leu/Val) or mupirocin treatment. Transcriptional analysis showed that the RSH-dependent stringent control in S. aureus is characterized by repression of genes whose products are predicted to be involved in the translation machinery and by upregulation of genes coding for enzymes involved in amino acid metabolism and transport which are controlled by the repressor CodY. Amino acid starvation also provoked stabilization of the RNAs coding for major virulence regulators, such as SaeRS and SarA, independently of RSH. In an animal model, the rsh syn mutant was shown to be less virulent than the wild type. Virulence could be restored by the introduction of a codY mutation into the rsh syn mutant. These results indicate that stringent conditions are present during infection and that RSH-dependent derepression of CodY-regulated genes is essential for virulence in S. aureus.
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Nascimento, Marcelle M., José A. Lemos, Jacqueline Abranches, Vanessa K. Lin, and Robert A. Burne. "Role of RelA of Streptococcus mutans in Global Control of Gene Expression." Journal of Bacteriology 190, no. 1 (October 19, 2007): 28–36. http://dx.doi.org/10.1128/jb.01395-07.
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ABSTRACT The production of (p)ppGpp by Streptococcus mutans UA159 is catalyzed by three gene products: RelA, RelP, and RelQ. Here, we investigate the role of the RelA (Rel) homologue of S. mutans in the stringent response and in the global control of gene expression. RelA of S. mutans was shown to synthesize pppGpp in vitro from GTP and ATP in the absence of added ribosomes, as well as in vivo in an Escherichia coli relA-spoT mutant. Mupirocin (MUP) was shown to induce high levels of (p)ppGpp production in S. mutans in a relA-dependent manner, with a concomitant reduction in GTP pools. Transcription profiling after MUP treatment of S. mutans revealed that 104 genes were upregulated and 130 were downregulated (P ≤ 0.001); mainly, genes for macromolecular biosynthesis, translation, and energy metabolism were downregulated. When a derivative of UA159 carrying a complete deletion of the relA gene was treated with MUP, 72 genes were upregulated and 52 were downregulated (P ≤ 0.001). The expression of 50 genes (P ≤ 0.001) was commonly affected by MUP treatment in the two strains, suggesting that S. mutans can mount a relA-independent response to MUP. Consistent with the gene expression profiling, RelA was shown to play major roles in the regulation of phenotypic traits that are required for establishment, persistence, and virulence expression by this oral pathogen. Thus, RelA is the major (p)ppGpp synthase controlling the stringent response in S. mutans, and it coordinates the expression of genes and phenotypes that contribute to the pathogenic potential of the organism.
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Battesti, Aurélia, and Emmanuelle Bouveret. "Bacteria Possessing Two RelA/SpoT-Like Proteins Have Evolved a Specific Stringent Response Involving the Acyl Carrier Protein-SpoT Interaction." Journal of Bacteriology 191, no. 2 (November 7, 2008): 616–24. http://dx.doi.org/10.1128/jb.01195-08.
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ABSTRACT Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation.
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Gupta, Kuldeepkumar Ramnaresh, Priyanka Baloni, Shantinath S. Indi, and Dipankar Chatterji. "Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis." Journal of Bacteriology 198, no. 9 (February 22, 2016): 1414–22. http://dx.doi.org/10.1128/jb.00126-16.
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ABSTRACTThe alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. InMycobacterium smegmatis, both (p)ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins RelMsmand DcpA, encoded byrelMsmanddcpAgenes, respectively. We have previously shown that the ΔrelMsmand ΔdcpAknockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571–2578, 2015,http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p)ppGpp and c-di-GMP in mycobacteria. We have shown that both (p)ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated ΔrelMsmand ΔdcpAcells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p)ppGpp and c-di-GMP affect some common phenotypes inM. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria.IMPORTANCEOur work has expanded the horizon of (p)ppGpp and c-di-GMP signaling in Gram-positive bacteria. We have come across a novel observation thatM. smegmatisneeds (p)ppGpp and c-di-GMP for cold tolerance. We had previously shown that the ΔrelMsmand ΔdcpAstrains are defective in biofilm formation. In this work, the overproduction of (p)ppGpp and c-di-GMP encasedM. smegmatisin a biofilm-like matrix, which shows that both (p)ppGpp and c-di-GMP are needed for biofilm formation. The regulation of cell length and cell division by (p)ppGpp was known in mycobacteria, but our work shows that c-di-GMP also affects the cell size and cell division in mycobacteria. This is perhaps the first report of c-di-GMP regulating cell division in mycobacteria.
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Shields, Robert C., Jeong Nam Kim, Sang-Joon Ahn, and Robert A. Burne. "Peptides encoded in the Streptococcus mutans RcrRPQ operon are essential for thermotolerance." Microbiology 166, no. 3 (March 1, 2020): 306–17. http://dx.doi.org/10.1099/mic.0.000887.
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The MarR-like transcriptional regulator and two ABC transporters encoded by the rcrRPQ operon in the dental caries pathogen Streptococcus mutans have important regulatory roles related to oxidative stress tolerance, genetic competence and (p)ppGpp metabolism. A unique feature of the rcrRPQ operon, when compared to other bacteria, is the presence of two peptides, designated Pep1 and Pep2, encoded in alternative reading frames at the 3′ end of rcrQ. Here, we show that the rcrRPQ operon, including Pep1 and 2, is essential for S. mutans to survive and maintain viability at elevated temperatures. No major changes in the levels of the heat shock proteins DnaK or GroEL that could account for the thermosensitivity of rcrRPQ mutants were observed. By introducing a single amino acid substitution into the comX gene that deletes an internally encoded peptide, XrpA, we found that XrpA is a contributing factor to the thermosensitive phenotype of a ΔrcrR strain. Overexpression of XrpA on a plasmid also caused a significant growth defect at 42 °C. Interestingly, loss of the gene for the RelA/SpoT homologue (RSH) enzyme, relA, restored growth of the ΔrcrR strain at 42 °C. During heat stress and when a stringent response was induced, levels of (p)ppGpp were elevated in the ΔrcrR strain. Deletion of relA in the ΔrcrR strain lowered the basal levels of (p)ppGpp to those observed in wild-type S. mutans . Thus, (p)ppGpp pools are dysregulated in ΔrcrR, which likely leads to aberrant control of transcriptional/translational processes and the thermosensitive phenotype. In summary, the genes and peptides encoded in the rcrRPQ operon are critical for thermotolerance, and in some strains these phenotypes are related to altered (p)ppGpp metabolism and increased production of the XrpA peptide.
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Ronneau, Séverin, and Régis Hallez. "Make and break the alarmone: regulation of (p)ppGpp synthetase/hydrolase enzymes in bacteria." FEMS Microbiology Reviews 43, no. 4 (March 27, 2019): 389–400. http://dx.doi.org/10.1093/femsre/fuz009.
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ABSTRACTBacteria use dedicated mechanisms to respond adequately to fluctuating environments and to optimize their chances of survival in harsh conditions. One of the major stress responses used by virtually all bacteria relies on the sharp accumulation of an alarmone, the guanosine penta- or tetra-phosphate commonly referred to as (p)ppGpp. Under stressful conditions, essentially nutrient starvation, these second messengers completely reshape the metabolism and physiology by coordinately modulating growth, transcription, translation and cell cycle. As a central regulator of bacterial stress response, the alarmone is also involved in biofilm formation, virulence, antibiotics tolerance and resistance in many pathogenic bacteria. Intracellular concentrations of (p)ppGpp are determined by a highly conserved and widely distributed family of proteins called RelA-SpoT Homologs (RSH). Recently, several studies uncovering mechanisms that regulate RSH activities have renewed a strong interest in this field. In this review, we outline the diversity of the RSH protein family as well as the molecular devices used by bacteria to integrate and transform environmental cues into intracellular (p)ppGpp levels.
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Wehmeier, L., A. Schafer, A. Burkovski, R. Kramer, U. Mechold, H. Malke, A. Puhler, and J. Kalinowski. "The role of the Corynebacterium glutamicum rel gene in (p)ppGpp metabolism." Microbiology 144, no. 7 (July 1, 1998): 1853–62. http://dx.doi.org/10.1099/00221287-144-7-1853.
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Gupta, Kuldeepkumar Ramnaresh, Sanjay Kasetty, and Dipankar Chatterji. "Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis." Applied and Environmental Microbiology 81, no. 7 (January 30, 2015): 2571–78. http://dx.doi.org/10.1128/aem.03999-14.
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ABSTRACTThe bacterial second messengers (p)ppGpp and bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, aPseudomonas aeruginosastrain lacking (p)ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whetherMycobacterium smegmatisstrains lacking the gene for either (p)ppGpp synthesis (ΔrelMsm) or c-di-GMP synthesis (ΔdcpA) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the ΔrelMsmand ΔdcpAstrains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls ofM. smegmatis. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids inM. smegmatis.
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Tauch, Andreas, Lutz Wehmeier, Susanne Götker, Alfred Pühler, and Jörn Kalinowski. "Relaxedrrnexpression and amino acid requirement of aCorynebacterium glutamicum relmutant defective in (p)ppGpp metabolism." FEMS Microbiology Letters 201, no. 1 (July 2001): 53–58. http://dx.doi.org/10.1111/j.1574-6968.2001.tb10732.x.
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Kim, Jeong Nam, Sang-Joon Ahn, and Robert A. Burne. "Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans." Applied and Environmental Microbiology 81, no. 15 (May 15, 2015): 5015–25. http://dx.doi.org/10.1128/aem.01160-15.
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ABSTRACTIn the dental caries pathogenStreptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackAmutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δptaor ΔptaΔackAmutant. The Δptaand ΔptaΔackAmutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackAstrain. Surprisingly, when exposed to oxidative stress, the ΔptaΔackAstrain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackAand ΔptaΔackAmutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackAand ΔptaΔackAmutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis ofS. mutans.
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VandenBerg, Kelsey E., Sarah Ahn, and Jonathan E. Visick. "(p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair." Applied and Environmental Microbiology 82, no. 17 (July 1, 2016): 5444–54. http://dx.doi.org/10.1128/aem.00623-16.
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ABSTRACTThel-isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival ofEscherichia coliunder stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identifiedpcmas a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δpcmmutant produced persisters for an extended period in stationary phase, and a ΔglpDmutation drastically increased persisters in a Δpcmbackground, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than thepcmmutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates inglpDmutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the ΔpcmΔglpDmutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δpcmmutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage.IMPORTANCEProtein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCM-directed repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population inEscherichia colibacteria increased, especially if the metabolic geneglpDwas also mutated. High levels of persisters inpcm glpDdouble mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage.
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Reiß, Swantje, Jan Pané-Farré, Stephan Fuchs, Patrice François, Manuel Liebeke, Jacques Schrenzel, Ulrike Lindequist, et al. "Global Analysis of the Staphylococcus aureus Response to Mupirocin." Antimicrobial Agents and Chemotherapy 56, no. 2 (November 21, 2011): 787–804. http://dx.doi.org/10.1128/aac.05363-11.
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ABSTRACTIn the present study, we analyzed the response ofS. aureusto mupirocin, the drug of choice for nasal decolonization. Mupirocin selectively inhibits the bacterial isoleucyl-tRNA synthetase (IleRS), leading to the accumulation of uncharged isoleucyl-tRNA and eventually the synthesis of (p)ppGpp. The alarmone (p)ppGpp induces the stringent response, an important global transcriptional and translational control mechanism that allows bacteria to adapt to nutritional deprivation. To identify proteins with an altered synthesis pattern in response to mupirocin treatment, we used the highly sensitive 2-dimensional gel electrophoresis technique in combination with mass spectrometry. The results were complemented by DNA microarray, Northern blot, and metabolome analyses. Whereas expression of genes involved in nucleotide biosynthesis, DNA metabolism, energy metabolism, and translation was significantly downregulated, expression of isoleucyl-tRNA synthetase, the branched-chain amino acid pathway, and genes with functions in oxidative-stress resistance (ahpCandkatA) and putative roles in stress protection (theyvyDhomologueSACOL0815andSACOL1759andSACOL2131) and transport processes was increased. A comparison of the regulated genes to known regulons suggests the involvement of the global regulators CodY and SigB in shaping the response ofS. aureusto mupirocin. Of particular interest was the induced transcription of genes encoding virulence-associated regulators (i.e.,arlRS,saeRS,sarA,sarR,sarS, andsigB), as well as genes directly involved in the virulence ofS. aureus(i.e.,fnbA,epiE,epiG, andseb).
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Bugrysheva, Julia V., Christopher J. Pappas, Darya A. Terekhova, Radha Iyer, Henry P. Godfrey, Ira Schwartz, and Felipe C. Cabello. "Characterization of the RelBbu Regulon in Borrelia burgdorferi Reveals Modulation of Glycerol Metabolism by (p)ppGpp." PLOS ONE 10, no. 2 (February 17, 2015): e0118063. http://dx.doi.org/10.1371/journal.pone.0118063.
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Wu, Lingyu, Zheng Wang, Yejun Guan, Xianqing Huang, Huimin Shi, Yujie Liu, and Xuehong Zhang. "The (p)ppGpp-mediated stringent response regulatory system globally inhibits primary metabolism and activates secondary metabolism in Pseudomonas protegens H78." Applied Microbiology and Biotechnology 104, no. 7 (February 3, 2020): 3061–79. http://dx.doi.org/10.1007/s00253-020-10421-5.
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Huang, Can, Wenqian Li, and Jingyu Chen. "Stringent Response Factor DksA Contributes to Fatty Acid Degradation Function to Influence Cell Membrane Stability and Polymyxin B Resistance of Yersinia enterocolitica." International Journal of Molecular Sciences 24, no. 15 (July 26, 2023): 11951. http://dx.doi.org/10.3390/ijms241511951.
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DksA is a proteobacterial regulator that binds directly to the secondary channel of RNA polymerase with (p)ppGpp and is responsible for various bacterial physiological activities. While (p)ppGpp is known to be involved in the regulation and response of fatty acid metabolism pathways in many foodborne pathogens, the role of DksA in this process has yet to be clarified. This study aimed to characterize the function of DksA on fatty acid metabolism and cell membrane structure in Yersinia enterocolitica. Therefore, comparison analysis of gene expression, growth conditions, and membrane permeabilization among the wide-type (WT), DksA-deficient mutant (YEND), and the complemented strain was carried out. It confirmed that deletion of DksA led to a more than four-fold decrease in the expression of fatty acid degradation genes, including fadADEIJ. Additionally, YEND exhibited a smaller growth gap compared to the WT strain at low temperatures, indicating that DksA is not required for the growth of Y. enterocolitica in cold environments. Given that polymyxin B is a cationic antimicrobial peptide that targets the cell membrane, the roles of DksA under polymyxin B exposure were also characterized. It was found that DksA positively regulates the integrity of the inner and outer membranes of Y. enterocolitica under polymyxin B, preventing the leakage of intracellular nucleic acids and proteins and ultimately reducing the sensitivity of Y. enterocolitica to polymyxin B. Taken together, this study provides insights into the functions of DksA and paves the way for novel fungicide development.
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Gupta, Kuldeepkumar Ramnaresh, Gunjan Arora, Abid Mattoo, and Andaleeb Sajid. "Stringent Response in Mycobacteria: From Biology to Therapeutic Potential." Pathogens 10, no. 11 (November 1, 2021): 1417. http://dx.doi.org/10.3390/pathogens10111417.
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Mycobacterium tuberculosis is a human pathogen that can thrive inside the host immune cells for several years and cause tuberculosis. This is due to the propensity of M. tuberculosis to synthesize a sturdy cell wall, shift metabolism and growth, secrete virulence factors to manipulate host immunity, and exhibit stringent response. These attributes help M. tuberculosis to manage the host response, and successfully establish and maintain an infection even under nutrient-deprived stress conditions for years. In this review, we will discuss the importance of mycobacterial stringent response under different stress conditions. The stringent response is mediated through small signaling molecules called alarmones “(pp)pGpp”. The synthesis and degradation of these alarmones in mycobacteria are mediated by Rel protein, which is both (p)ppGpp synthetase and hydrolase. Rel is important for all central dogma processes—DNA replication, transcription, and translation—in addition to regulating virulence, drug resistance, and biofilm formation. Rel also plays an important role in the latent infection of M. tuberculosis. Here, we have discussed the literature on alarmones and Rel proteins in mycobacteria and highlight that (p)ppGpp-analogs and Rel inhibitors could be designed and used as antimycobacterial compounds against M. tuberculosis and non-tuberculous mycobacterial infections.
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Yan, Xue, Aurélie Budin-Verneuil, Yanick Auffray, and Vianney Pichereau. "Proteome phenotyping of ΔrelA mutants in Enterococcus faecalis V583." Canadian Journal of Microbiology 60, no. 8 (August 2014): 525–31. http://dx.doi.org/10.1139/cjm-2014-0254.
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The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583. A 2-dimensional electrophoresis proteomic analysis of 2 relA mutants, i.e., ΔrelA carrying a complete deletion of the relA gene, and ΔrelAsp that is deleted from only its 3′ extremity, showed that 31 proteins were deregulated in 1 or both of these mutants. Mass spectrometry identification of these proteins showed that 10 are related to translation, including 5 ribosomal proteins, 3 proteins involved in translation elongation, and 2 proteins in tRNA synthesis; 14 proteins are involved in diverse metabolisms and biosynthesis (8 in sugar and energy metabolisms, 2 in fatty acid biosynthesis, 2 in amino acid biosynthesis, and 2 in nucleotide metabolism). Five proteins were relevant to the adaptation to different environmental stresses, i.e., SodA and a Dps family protein, 2 cold-shock domain proteins, and Ef1744, which is a general stress protein that plays an important role in the response to ethanol stress. The potential role of these proteins in the development of stress phenotypes associated with these mutations is discussed.
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Gomez-Escribano, Juan P., Juan F. Martín, A. Hesketh, M. J. Bibb, and P. Liras. "Streptomyces clavuligerus relA-null mutants overproduce clavulanic acid and cephamycin C: negative regulation of secondary metabolism by (p)ppGpp." Microbiology 154, no. 3 (March 1, 2008): 744–55. http://dx.doi.org/10.1099/mic.0.2007/011890-0.
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Hogg, Tanis, Undine Mechold, Horst Malke, Mike Cashel, and Rolf Hilgenfeld. "Conformational Antagonism between Opposing Active Sites in a Bifunctional RelA/SpoT Homolog Modulates (p)ppGpp Metabolism during the Stringent Response." Cell 117, no. 1 (April 2004): 57–68. http://dx.doi.org/10.1016/s0092-8674(04)00260-0.
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Lu, Hang, and Yili Huang. "Transcriptome Analysis of Novosphingobium pentaromativorans US6-1 Reveals the Rsh Regulon and Potential Molecular Mechanisms of N-acyl-l-homoserine Lactone Accumulation." International Journal of Molecular Sciences 19, no. 9 (September 5, 2018): 2631. http://dx.doi.org/10.3390/ijms19092631.
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In most bacteria, a bifunctional Rsh responsible for (p)ppGpp metabolism is the key player in stringent response. To date, no transcriptome-wide study has been conducted to investigate the Rsh regulon, and the molecular mechanism of how Rsh affects the accumulation of N-acyl-l-homoserine lactone (AHL) remains unknown in sphingomonads. In this study, we identified an rshUS6–1 gene by sequence analysis in Novosphingobium pentaromativorans US6-1, a member of the sphingomonads. RNA-seq was used to determine transcription profiles of the wild type and the ppGpp-deficient rshUS6–1 deletion mutant (∆rsh). There were 1540 genes in the RshUS6–1 regulon, including those involved in common traits of sphingomonads such as exopolysaccharide biosynthesis. Furthermore, both RNA-seq and quantitative real-time polymerase chain reaction (qRT-PCR) showed essential genes for AHL production (novI and novR) were positively regulated by RshUS6–1 during the exponential growth phase. A degradation experiment indicated the reason for the AHL absence in ∆rsh was unrelated to the AHL degradation. According to RNA-seq, we proposed σE, DksA, Lon protease and RNA degradation enzymes might be involved in the RshUS6–1-dependent expression of novI and novR. Here, we report the first transcriptome-wide analysis of the Rsh regulon in sphingomonads and investigate the potential mechanisms regulating AHL accumulation, which is an important step towards understanding the regulatory system of stringent response in sphingomonads.
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Sidorov, Roman Yu, and Alexander G. Tkachenko. "DMNP, a Synthetic Analog of Erogorgiaene, Inhibits the ppGpp Synthetase Activity of the Small Alarmone Synthetase RelZ." BIO Web of Conferences 57 (2023): 08002. http://dx.doi.org/10.1051/bioconf/20235708002.
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Suppression of the stringent response is a promising strategy for the treatment of persistent bacterial infections. A novel class of compounds having a mechanism of action based on alarmone synthetase inhibition and suppressing the synthesis of (p)ppGpp alarmones in bacteria may provide a more effective treatment for latent infections and resolve problems associated with bacterial persistence. Conventional antibiotics primarily act on actively growing bacteria, but they are inactive against persister cells with a slowed metabolism. Alarmone synthetase inhibitors have antipersister properties that may enhance conventional antibiotics’ antibacterial action. Two groups of RSH proteins are responsible for the synthesis of alarmones: long RelA/SpoT homologs and small alarmone synthetases. Many species of bacteria possess both types of enzymes. Despite the fact that a number of inhibitors of bifunctional long synthetases/hydrolases have been described to date, their properties with respect to monofunctional small alarmone synthetases have been studied poorly. This study investigated the effect of the alarmone synthetase inhibitor DMNP on the purified RelZ small alarmone synthetase protein from Mycolicibacterium smegmatis.
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Reverón, Inés, Laura Plaza-Vinuesa, Laura Santamaría, Juan Carlos Oliveros, Blanca de las Rivas, Rosario Muñoz, and Félix López de Felipe. "Transcriptomic Evidence of Molecular Mechanisms Underlying the Response of Lactobacillus plantarum WCFS1 to Hydroxytyrosol." Antioxidants 9, no. 5 (May 20, 2020): 442. http://dx.doi.org/10.3390/antiox9050442.
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This study was aimed to gain new insights into the molecular mechanisms used by Lactobacillus plantarum WCFS1 to respond to hydroxytyrosol (HXT), one of the main and health-relevant plant phenolics present in olive oil. To this goal, whole genome transcriptomic profiling was used to better understand the contribution of differential gene expression in the adaptation to HXT by this microorganism. The transcriptomic profile reveals an HXT-triggered antioxidant response involving genes from the ROS (reactive oxygen species) resistome of L. plantarum, genes coding for H2S-producing enzymes and genes involved in the response to thiol-specific oxidative stress. The expression of a set of genes involved in cell wall biogenesis was also upregulated, indicating that this subcellular compartment was a target of HXT. The expression of several MFS (major facilitator superfamily) efflux systems and ABC-transporters was differentially affected by HXT, probably to control its transport across the membrane. L. plantarum transcriptionally reprogrammed nitrogen metabolism and involved the stringent response (SR) to adapt to HXT, as indicated by the reduced expression of genes involved in cell proliferation or related to the metabolism of (p)ppGpp, the molecule that triggers the SR. Our data have identified, at genome scale, the antimicrobial mechanisms of HXT action as well as molecular mechanisms that potentially enable L. plantarum to cope with the effects of this phenolic compound.
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Gangaiah, Dharanesh, Xinjun Zhang, Beth Baker, Kate R. Fortney, Hongyu Gao, Concerta L. Holley, Robert S. Munson, Yunlong Liu, and Stanley M. Spinola. "Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers." Infection and Immunity 84, no. 5 (February 29, 2016): 1514–25. http://dx.doi.org/10.1128/iai.00048-16.
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Haemophilus ducreyicauses the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans,H. ducreyiresides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. Munson, Jr., E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014,http://dx.doi.org/10.1128/mBio.01081-13) suggested thatH. ducreyiencounters growth conditions in human lesions resembling those found in stationary phase. However, howH. ducreyitranscriptionally responds to stress during human infection is unknown. Here, we determined theH. ducreyitranscriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that thein vivotranscriptome is distinct from those ofin vitrogrowth. Compared to the inoculum (mid-log-phase bacteria),H. ducreyiharvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis.H. ducreyiupregulated few genes (hgbA,flp-tad, andlspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressedin vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that thein vivotranscriptome is distinct from those ofin vitrogrowth and that adaptation to nutrient stress and anaerobiosis is crucial forH. ducreyisurvival in humans.
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Grucela, Paulina Katarzyna, Tobias Fuhrer, Uwe Sauer, Yanjie Chao, and Yong Everett Zhang. "Ribose 5-phosphate: the key metabolite bridging the metabolisms of nucleotides and amino acids during stringent response in Escherichia coli?" Microbial Cell 10, no. 7 (July 3, 2023): 141–44. http://dx.doi.org/10.15698/mic2023.07.799.
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The bacterial stringent response and its effector alarmone guanosine penta- or tetra – phosphates (p)ppGpp are vital for bacterial tolerance and survival of various stresses in environments (including antibiotics) and host cells (virulence). (p)ppGpp does so by binding to its numerous target proteins and reprograming bacterial transcriptome to tune down the synthesis of nucleotides and rRNA/tRNA, and up-regulate amino acid biosynthesis genes. Recent identification of more novel (p)ppGpp direct binding proteins in Escherichia coli and their deep studies have unveiled unprecedented details of how (p)ppGpp coordinates the nucleotide and amino acid metabolic pathways upon stringent response; however, the mechanistic link between nucleotide and amino acid metabolisms remains still incompletely understood. Here we propose the metabolite ribose 5’-phosphate as the key link between nucleotide and amino acid metabolisms and a working model integrating both the transcriptional and metabolic effects of (p)ppGpp on E. coli physiological adaptation during the stringent response.
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Vogeleer, Philippe, and Fabien Létisse. "Dynamic Metabolic Response to (p)ppGpp Accumulation in Pseudomonas putida." Frontiers in Microbiology 13 (April 14, 2022). http://dx.doi.org/10.3389/fmicb.2022.872749.
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The stringent response is a ubiquitous bacterial reaction triggered by nutrient deprivation and mediated by the intracellular concentrations of ppGpp and pppGpp. These alarmones, jointly referred to as (p)ppGpp, control gene transcription, mRNA translation and protein activity to adjust the metabolism and growth rate to environmental changes. While the ability of (p)ppGpp to mediate cell growth slowdown and metabolism adaptation has been demonstrated in Escherichia coli, it’s role in Pseudomonas putida remains unclear. The aims of this study were therefore to determine which forms of (p)ppGpp are synthetized in response to severe growth inhibition in P. putida, and to decipher the mechanisms of (p)ppGpp-mediated metabolic regulation in this bacterium. We exposed exponentially growing cells of P. putida to serine hydroxamate (SHX), a serine analog known to trigger the stringent response, and tracked the dynamics of intra- and extracellular metabolites using untargeted quantitative MS and NMR-based metabolomics, respectively. We found that SHX promotes ppGpp and pppGpp accumulation few minutes after exposure and arrests bacterial growth. Meanwhile, central carbon metabolites increase in concentration while purine pathway intermediates drop sharply. Importantly, in a ΔrelA mutant and a ppGpp0 strain in which (p)ppGpp synthesis genes were deleted, SHX exposure inhibited cell growth but led to an accumulation of purine pathway metabolites instead of a decrease, suggesting that as observed in other bacteria, (p)ppGpp downregulates the purine pathway in P. putida. Extracellular accumulations of pyruvate and acetate were observed as a specific metabolic consequence of the stringent response. Overall, our results show that (p)ppGpp rapidly remodels the central carbon metabolism and the de novo purine biosynthesis pathway in P. putida. These data represent a hypothesis-generating resource for future studies on the stringent response.
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Anderson, Brent W., Danny K. Fung, and Jue D. Wang. "Regulatory Themes and Variations by the Stress-Signaling Nucleotide Alarmones (p)ppGpp in Bacteria." Annual Review of Genetics 55, no. 1 (August 20, 2021). http://dx.doi.org/10.1146/annurev-genet-021821-025827.
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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. Expected final online publication date for the Annual Review of Genetics, Volume 55 is November 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Colomer-Winter, C., A. L. Flores-Mireles, S. Kundra, S. J. Hultgren, and J. A. Lemos. "(p)ppGpp and CodY Promote Enterococcus faecalis Virulence in a Murine Model of Catheter-Associated Urinary Tract Infection." mSphere 4, no. 4 (July 24, 2019). http://dx.doi.org/10.1128/msphere.00392-19.
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ABSTRACT 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.
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Prusa, Jerome, Dennis X. Zhu, and Christina L. Stallings. "The stringent response and Mycobacterium tuberculosis pathogenesis." Pathogens and Disease 76, no. 5 (June 11, 2018). http://dx.doi.org/10.1093/femspd/fty054.
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Abstract During infection, the host restrains Mycobacterium tuberculosis (Mtb) from proliferating by imposing an arsenal of stresses. Despite this onslaught of attacks, Mtb is able to persist for the lifetime of the host, indicating that this pathogen has substantial molecular mechanisms to resist host-inflicted damage. The stringent response is a conserved global stress response in bacteria that involves the production of the hyperphosphorylated guanine nucleotides ppGpp and pppGpp (collectively called (p)ppGpp). (p)ppGpp then regulates a number of cellular processes to adjust the physiology of the bacteria to promote survival in different environments. Survival in the presence of host-generated stresses is an essential quality of successful pathogens, and the stringent response is critical for the intracellular survival of a number of pathogenic bacteria. In addition, the stringent response has been linked to virulence gene expression, persistence, latency and drug tolerance. In Mtb, (p)ppGpp synthesis is required for survival in low nutrient conditions, long term culture and during chronic infection in animal models, all indicative of a strict requirement for (p)ppGpp during exposure to stresses associated with infection. In this review we discuss (p)ppGpp metabolism and how this functions as a critical regulator of Mtb virulence.
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Bange, Gert, Ditlev E. Brodersen, Anastasia Liuzzi, and Wieland Steinchen. "Two P or Not Two P: Understanding Regulation by the Bacterial Second Messengers (p)ppGpp." Annual Review of Microbiology 75, no. 1 (August 3, 2021). http://dx.doi.org/10.1146/annurev-micro-042621-122343.
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Under stressful growth conditions and nutrient starvation, bacteria adapt by synthesizing signaling molecules that profoundly reprogram cellular physiology. At the onset of this process, called the stringent response, members of the RelA/SpoT homolog (RSH) protein superfamily are activated by specific stress stimuli to produce several hyperphosphorylated forms of guanine nucleotides, commonly referred to as (p)ppGpp. Some bifunctional RSH enzymes also harbor domains that allow for degradation of (p)ppGpp by hydrolysis. (p)ppGpp synthesis or hydrolysis may further be executed by single-domain alarmone synthetases or hydrolases, respectively. The downstream effects of (p)ppGpp rely mainly on direct interaction with specific intracellular effectors, which are widely used throughout most cellular processes. The growing number of identified (p)ppGpp targets allows us to deduce both common features of and differences between gram-negative and gram-positive bacteria. In this review, we give an overview of (p)ppGpp metabolism with a focus on the functional and structural aspects of the enzymes involved and discuss recent findings on alarmone-regulated cellular effectors. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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Gaca, Anthony O., Jessica K. Kajfasz, James H. Miller, Kuanqing Liu, Jue D. Wang, Jacqueline Abranches, and José A. Lemos. "Basal Levels of (p)ppGpp in Enterococcus faecalis: the Magic beyond the Stringent Response." mBio 4, no. 5 (September 24, 2013). http://dx.doi.org/10.1128/mbio.00646-13.
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ABSTRACTThe stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. InEnterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for “Rel SpoT homologue”; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization ofE. faecalisstrains lackingrsh,relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays ofE. faecalisstrains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the ∆rsh∆relQ[(p)ppGpp0] strain, suggesting that a lack of basal (p)ppGpp places the cell in a “transcriptionally relaxed” state. Alterations in the fermentation profile and increased production of H2O2in the (p)ppGpp0strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen inBacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions.IMPORTANCEDrug-resistant bacterial infections continue to pose a significant public health threat by limiting therapeutic options available to care providers. The stringent response (SR), mediated by the accumulation of two modified guanine nucleotides collectively known as (p)ppGpp, is a highly conserved stress response that broadly remodels bacterial physiology to a survival state. Given the strong correlation of the SR with the ability of bacteria to survive antibiotic treatment and the direct association of (p)ppGpp production with bacterial infectivity, understanding how bacteria produce and utilize (p)ppGpp may reveal potential targets for the development of new antimicrobial therapies. Using the multidrug-resistant pathogenEnterococcus faecalisas a model, we show that small alterations to (p)ppGpp levels, well below concentrations needed to trigger the SR, severely affected bacterial metabolism and antibiotic survival. Our findings highlight the often-underappreciated contribution of basal (p)ppGpp levels to metabolic balance and stress tolerance in bacteria.
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Avilan, Luisana, Carine Puppo, Adrien Villain, Emanuelle Bouveret, Benoit Menand, Ben Field, and Brigitte Gontero. "RSH enzyme diversity for (p)ppGpp metabolism in Phaeodactylum tricornutum and other diatoms." Scientific Reports 9, no. 1 (November 27, 2019). http://dx.doi.org/10.1038/s41598-019-54207-w.
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AbstractThe nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.
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Kundra, Shivani, Cristina Colomer-Winter, and José A. Lemos. "Survival of the Fittest: The Relationship of (p)ppGpp With Bacterial Virulence." Frontiers in Microbiology 11 (December 3, 2020). http://dx.doi.org/10.3389/fmicb.2020.601417.
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The signaling nucleotide (p)ppGpp has been the subject of intense research in the past two decades. Initially discovered as the effector molecule of the stringent response, a bacterial stress response that reprograms cell physiology during amino acid starvation, follow-up studies indicated that many effects of (p)ppGpp on cell physiology occur at levels that are lower than those needed to fully activate the stringent response, and that the repertoire of enzymes involved in (p)ppGpp metabolism is more diverse than initially thought. Of particular interest, (p)ppGpp regulation has been consistently linked to bacterial persistence and virulence, such that the scientific pursuit to discover molecules that interfere with (p)ppGpp signaling as a way to develop new antimicrobials has grown substantially in recent years. Here, we highlight contemporary studies that have further supported the intimate relationship of (p)ppGpp with bacterial virulence and studies that provided new insights into the different mechanisms by which (p)ppGpp modulates bacterial virulence.
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Léger, Loïc, Deborah Byrne, Paul Guiraud, Elsa Germain, and Etienne Maisonneuve. "NirD curtails the stringent response by inhibiting RelA activity in Escherichia coli." eLife 10 (July 29, 2021). http://dx.doi.org/10.7554/elife.64092.
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Bacteria regulate their metabolism to adapt and survive adverse conditions, in particular to stressful downshifts in nutrient availability. These shifts trigger the so-called stringent response, coordinated by the signaling molecules guanosine tetra and pentaphosphate collectively referred to as (p)ppGpp. In Escherichia coli, accumulation of theses alarmones depends on the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthetase/hydrolase SpoT. A tight regulation of these intracellular activities is therefore crucial to rapidly adjust the (p)ppGpp levels in response to environmental stresses but also to avoid toxic consequences of (p)ppGpp over-accumulation. In this study, we show that the small protein NirD restrains RelA-dependent accumulation of (p)ppGpp and can inhibit the stringent response in E. coli. Mechanistically, our in vivo and in vitro studies reveal that NirD directly binds the catalytic domains of RelA to balance (p)ppGpp accumulation. Finally, we show that NirD can control RelA activity by directly inhibiting the rate of (p)ppGpp synthesis.
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Meyer, Laura, Elsa Germain, and Etienne Maisonneuve. "Regulation of ytfK by cAMP-CRP Contributes to SpoT-Dependent Accumulation of (p)ppGpp in Response to Carbon Starvation YtfK Responds to Glucose Exhaustion." Frontiers in Microbiology 12 (November 4, 2021). http://dx.doi.org/10.3389/fmicb.2021.775164.
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Guanosine penta- or tetraphosphate (known as (p)ppGpp) serves as second messenger to respond to nutrient downshift and other environmental stresses, a phenomenon called stringent response. Accumulation of (p)ppGpp promotes the coordinated inhibition of macromolecule synthesis, as well as the activation of stress response pathways to cope and adapt to harmful conditions. In Escherichia coli, the (p)ppGpp level is tightly regulated by two enzymes, the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified the small protein YtfK as a key regulator of SpoT-mediated activation of stringent response in E. coli. Here, we further characterized the regulation of ytfK. We observed that ytfK is subjected to catabolite repression and is positively regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Importantly, YtfK contributes to SpoT-dependent accumulation of (p)ppGpp and cell survival in response to glucose starvation. Therefore, regulation of ytfK by the cAMP-CRP appears important to adjust (p)ppGpp level and coordinate cellular metabolism in response to glucose availability.
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Steinchen, Wieland, Victor Zegarra, and Gert Bange. "(p)ppGpp: Magic Modulators of Bacterial Physiology and Metabolism." Frontiers in Microbiology 11 (September 7, 2020). http://dx.doi.org/10.3389/fmicb.2020.02072.
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Das, Bhabatosh, and Rupak K. Bhadra. "(p)ppGpp Metabolism and Antimicrobial Resistance in Bacterial Pathogens." Frontiers in Microbiology 11 (October 9, 2020). http://dx.doi.org/10.3389/fmicb.2020.563944.
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Kudrin, Pavel, Vallo Varik, Sofia Raquel Alves Oliveira, Jelena Beljantseva, Teresa Del Peso Santos, Ievgen Dzhygyr, Dominik Rejman, Felipe Cava, Tanel Tenson, and Vasili Hauryliuk. "Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to β-Lactams." Antimicrobial Agents and Chemotherapy 61, no. 4 (January 23, 2017). http://dx.doi.org/10.1128/aac.02173-16.
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ABSTRACTThe nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, theEscherichia coli(p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purifiedE. colicomponents, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.
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Juengert, Janina R., Marina Borisova, Christoph Mayer, Christiane Wolz, Christopher J. Brigham, Anthony J. Sinskey, and Dieter Jendrossek. "Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16." Applied and Environmental Microbiology 83, no. 13 (April 28, 2017). http://dx.doi.org/10.1128/aem.00755-17.
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ABSTRACT In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033–8044, 2012, https://doi.org/10.1128/AEM.01693-12 ) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutropha. IMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha. This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a high-performance liquid chromatography–mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha. This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.