Статті в журналах: "Alarmone (p)ppGpp"

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Steinchen, Wieland, Jan S. Schuhmacher, Florian Altegoer, Christopher D. Fage, Vasundara Srinivasan, Uwe Linne, Mohamed A. Marahiel, and Gert Bange. "Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone." Proceedings of the National Academy of Sciences 112, no. 43 (October 12, 2015): 13348–53. http://dx.doi.org/10.1073/pnas.1505271112.

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Nucleotide-based second messengers serve in the response of living organisms to environmental changes. In bacteria and plant chloroplasts, guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively named “(p)ppGpp”] act as alarmones that globally reprogram cellular physiology during various stress conditions. Enzymes of the RelA/SpoT homology (RSH) family synthesize (p)ppGpp by transferring pyrophosphate from ATP to GDP or GTP. Little is known about the catalytic mechanism and regulation of alarmone synthesis. It also is unclear whether ppGpp and pppGpp execute different functions. Here, we unravel the mechanism and allosteric regulation of the highly cooperative alarmone synthetase small alarmone synthetase 1 (SAS1) fromBacillus subtilis. We determine that the catalytic pathway of (p)ppGpp synthesis involves a sequentially ordered substrate binding, activation of ATP in a strained conformation, and transfer of pyrophosphate through a nucleophilic substitution (SN2) reaction. We show that pppGpp—but not ppGpp—positively regulates SAS1 at an allosteric site. Although the physiological significance remains to be elucidated, we establish the structural and mechanistic basis for a biological activity in which ppGpp and pppGpp execute different functional roles.

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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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Beljantseva, Jelena, Pavel Kudrin, Liis Andresen, Victoria Shingler, Gemma C. Atkinson, Tanel Tenson, and Vasili Hauryliuk. "Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA." Proceedings of the National Academy of Sciences 114, no. 14 (March 20, 2017): 3726–31. http://dx.doi.org/10.1073/pnas.1617868114.

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The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ’s enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ’s enzymatic activity destabilizes the RelQ:RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5α. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.

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Ancona, Veronica, Jae Hoon Lee, Tiyakhon Chatnaparat, Jinrok Oh, Jong-In Hong, and Youfu Zhao. "The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora." Journal of Bacteriology 197, no. 8 (February 9, 2015): 1433–43. http://dx.doi.org/10.1128/jb.02551-14.

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ABSTRACTThe hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor inErwinia amylovora. Previous studies have demonstrated that the T3SS inE. amylovorais transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence ofE. amylovorawas investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp0) as well as adksAmutant ofE. amylovorawas completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence ofE. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp0anddksAmutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp0anddksAmutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size ofE. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activatedhrpAandhrpLpromoter activities inhrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles inE. amylovoravirulence and indicated thatE. amylovorautilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence.IMPORTANCEThe type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of anspoTnull mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional.

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Dasgupta, Shreya, Pallabi Basu, Ritesh Ranjan Pal, Satyabrata Bag, and Rupak K. Bhadra. "Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae." Microbiology 160, no. 9 (September 1, 2014): 1855–66. http://dx.doi.org/10.1099/mic.0.079319-0.

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In Vibrio cholerae, the causative agent of cholera, products of three genes, relA, spoT and relV, govern nutritional stress related stringent response (SR). SR in bacteria is critically regulated by two intracellular small molecules, guanosine 3′-diphosphate 5′-triphosphate (pppGpp) and guanosine 3′,5′-bis(diphosphate) (ppGpp), collectively called (p)ppGpp or alarmone. Evolution of relV is unique in V. cholerae because other Gram-negative bacteria carry only relA and spoT genes. Recent reports suggest that RelV is needed for pathogenesis. RelV carries a single (p)ppGpp synthetase or RelA-SpoT domain (SYNTH/RSD) and belongs to the small alarmone synthetase (SAS) family of proteins. Here, we report extensive functional characterizations of the relV gene by constructing several deletion and site-directed mutants followed by their controlled expression in (p)ppGpp0 cells of Escherichia coli or V. cholerae. Substitution analysis indicated that the amino acid residues K107, D129, R132, L150 and E188 of the RSD region of RelV are essential for its activity. While K107, D129 and E188 are highly conserved in RelA and SAS proteins, L150 appears to be conserved in the latter group of enzymes, and the R132 residue was found to be unique in RelV. Extensive progressive deletion analysis indicated that the amino acid residues at positions 59 and 248 of the RelV protein are the functional N- and C-terminal boundaries, respectively. Since the minimal functional length of RelV was found to be 189 aa, which includes the 94 aa long RSD region, it seems that the flanking residues of the RSD are also important for maintaining the (p)ppGpp synthetase activity.

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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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Kashevarova, N. M., A. V. Akhova, E. A. Khaova, and A. G. Tkachenko. "Role of alarmone (p)ppGpp in the regulation of indole formation depending on glucose content in <i> Escherichia coli </i>." Acta Biomedica Scientifica 7, no. 3 (July 5, 2022): 162–68. http://dx.doi.org/10.29413/abs.2022-7.3.17.

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Signaling molecules such as indole (product of tryptophan catabolism) and (p)ppGpp (stringent response regulator) are involved in regulation of physiological processes in bacterial cells aimed to adapt to antibiotics and stresses. However, question of existence of relationship between the stringent response and indole signaling requires more detailed investigation.The aim. To study effect of stringent response regulator (p)ppGpp on indole production in Escherichia coli depending on glucose content.Materials and methods. In this work, we studied the dynamics of indole accumulation in batch cultures of parent E. coli BW25141 ((p)ppGpp+ strain) and deletion mutant BW25141∆relA∆spoT ((p)ppGpp0 strain) in glucose-mineral tryptophan-free M9 medium, as well as with 2 mM tryptophan addition. In order to study effect of starvation stress on bacterial cell ability to synthesize indole, we used a model of growth limitation by carbon substrate at two glucose concentrations, 0.1 % and 0.4 %.Results. We have shown here that (p)ppGpp absence in E. coli cells reduces their ability to produce indole in the tryptophan-free medium and significantly slows down the rate of its accumulation in the tryptophan-containing one. Low glucose concentration (0.1 %) leads to decrease in indole production by (p)ppGpp+ cells in the tryptophan-free medium. The presence of indole synthesis precursor, tryptophan, in growth medium, on the contrary, increases the production of indole at lower glucose concentration in both (p)ppGpp+ and (p)ppGpp0 strains demonstrating direct dependence of delay time for onset of indole formation on glucose content, which is more pronounced in the culture of deletion mutant unable of synthesizing (p) ppGpp. The data obtained can be interpreted as result of complex regulatory effect of catabolic repression and the stringent response caused by alarmone (p)ppGpp action on expression level of tnaCAB operon responsible for indole biosynthesis.

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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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Abranches, Jacqueline, Alaina R. Martinez, Jessica K. Kajfasz, Violeta Chávez, Danielle A. Garsin, and José A. Lemos. "The Molecular Alarmone (p)ppGpp Mediates Stress Responses, Vancomycin Tolerance, and Virulence in Enterococcus faecalis." Journal of Bacteriology 191, no. 7 (January 23, 2009): 2248–56. http://dx.doi.org/10.1128/jb.01726-08.

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ABSTRACT The stringent response is a global bacterial response to stress that is mediated by accumulation of the alarmone (p)ppGpp. In this study, treatment with mupirocin was shown to induce high levels of (p)ppGpp production in Enterococcus faecalis, indicating that this nosocomial pathogen can mount a classic stringent response. In addition, (p)ppGpp was found to accumulate in cells subjected to heat shock, alkaline shock, and inhibitory concentrations of vancomycin. Sequence analysis of the E. faecalis genome indicated that (p)ppGpp synthesis is catalyzed by the bifunctional synthetase/hydrolase RelA and the RelQ small synthase. The (p)ppGpp profiles of ΔrelA, ΔrelQ, and ΔrelAQ strains revealed that RelA is the major enzyme responsible for the accumulation of (p)ppGpp during antibiotic or physical stresses, while RelQ appears to be responsible for maintaining basal levels of alarmone during homeostatic growth. Compared to its parent, the ΔrelA strain was more susceptible to several stress conditions, whereas complete elimination of (p)ppGpp in a ΔrelAQ double mutant restored many of the stress-sensitive phenotypes of ΔrelA. Interestingly, growth curves and time-kill studies indicated that tolerance to vancomycin is enhanced in the ΔrelA strain but diminished in the ΔrelQ and ΔrelAQ strains. Finally, virulence of the ΔrelAQ strain but not of the ΔrelA or ΔrelQ strain was significantly attenuated in the Caenorhabditis elegans model. Taken together, these results indicate that (p)ppGpp pools modulate environmental stress responses, vancomycin tolerance, and virulence in this important nosocomial pathogen.

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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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Mojr, Viktor, Mohammad Roghanian, Hedvig Tamman, Duy Dinh Do Pham, Magdalena Petrová, Radek Pohl, Hiraku Takada, et al. "Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools." ACS Chemical Biology 16, no. 9 (September 3, 2021): 1680–91. http://dx.doi.org/10.1021/acschembio.1c00398.

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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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Rhee, Hyun-Woo, Chang-Ro Lee, Seung-Hyon Cho, Mi-Ryung Song, Michael Cashel, Hyon E. Choy, Young-Jae Seok, and Jong-In Hong. "Selective Fluorescent Chemosensor for the Bacterial Alarmone (p)ppGpp." Journal of the American Chemical Society 130, no. 3 (January 2008): 784–85. http://dx.doi.org/10.1021/ja0759139.

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Bærentsen, René. "Bacterial nucleosidase is regulated by the alarmone (p)ppGpp." Acta Crystallographica Section A Foundations and Advances 75, a2 (August 18, 2019): e151-e151. http://dx.doi.org/10.1107/s2053273319094051.

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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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Lee, Jae-Woo, Young-Ha Park, and Yeong-Jae Seok. "Rsd balances (p)ppGpp level by stimulating the hydrolase activity of SpoT during carbon source downshift inEscherichia coli." Proceedings of the National Academy of Sciences 115, no. 29 (June 18, 2018): E6845—E6854. http://dx.doi.org/10.1073/pnas.1722514115.

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

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Giramma, Christina N., McKenna B. DeFoer, and Jue D. Wang. "The Alarmone (p)ppGpp Regulates Primer Extension by Bacterial Primase." Journal of Molecular Biology 433, no. 19 (September 2021): 167189. http://dx.doi.org/10.1016/j.jmb.2021.167189.

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Kanjee, Usheer, Koji Ogata, and Walid A. Houry. "Direct binding targets of the stringent response alarmone (p)ppGpp." Molecular Microbiology 85, no. 6 (August 2, 2012): 1029–43. http://dx.doi.org/10.1111/j.1365-2958.2012.08177.x.

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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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Wolz, Christiane, Tobias Geiger, and Christiane Goerke. "The synthesis and function of the alarmone (p)ppGpp in firmicutes." International Journal of Medical Microbiology 300, no. 2-3 (February 2010): 142–47. http://dx.doi.org/10.1016/j.ijmm.2009.08.017.

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Mouery, Kyle, Bethany A. Rader, Erin C. Gaynor, and Karen Guillemin. "The Stringent Response Is Required for Helicobacter pylori Survival of Stationary Phase, Exposure to Acid, and Aerobic Shock." Journal of Bacteriology 188, no. 15 (August 1, 2006): 5494–500. http://dx.doi.org/10.1128/jb.00366-06.

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ABSTRACT The gastric pathogen Helicobacter pylori must adapt to fluctuating conditions in the harsh environment of the human stomach with the use of a minimal number of transcriptional regulators. We investigated whether H. pylori utilizes the stringent response, involving signaling through the alarmone (p)ppGpp, as a survival strategy during environmental stresses. We show that the H. pylori homologue of the bifunctional (p)ppGpp synthetase and hydrolase SpoT is responsible for all cellular (p)ppGpp production in response to starvation conditions. Furthermore, the H. pylori spoT gene complements the growth defect of Escherichia coli mutants lacking (p)ppGpp. An H. pylori spoT deletion mutant is impaired for stationary-phase survival and undergoes a premature transformation to a coccoid morphology. In addition, the spoT deletion mutant is unable to survive specific environmental stresses, including aerobic shock and acid exposure, which are likely to be encountered by this bacterium during infection and transmission.

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Jiang, Mengxi, Susan M. Sullivan, Patrice K. Wout, and Janine R. Maddock. "G-Protein Control of the Ribosome-Associated Stress Response Protein SpoT." Journal of Bacteriology 189, no. 17 (July 6, 2007): 6140–47. http://dx.doi.org/10.1128/jb.00315-07.

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ABSTRACT The bacterial response to stress is controlled by two proteins, RelA and SpoT. RelA generates the alarmone (p)ppGpp under amino acid starvation, whereas SpoT is responsible for (p)ppGpp hydrolysis and for synthesis of (p)ppGpp under a variety of cellular stress conditions. It is widely accepted that RelA is associated with translating ribosomes. The cellular location of SpoT, however, has been controversial. SpoT physically interacts with the ribosome-associated GTPase CgtA, and we show here that, under an optimized salt condition, SpoT is also associated with a pre-50S particle. Analysis of spoT and cgtA mutants and strains overexpressing CgtA suggests that the ribosome associations of SpoT and CgtA are mutually independent. The steady-state level of (p)ppGpp is increased in a cgtA mutant, but the accumulation of (p)ppGpp during amino acid starvation is not affected, providing strong evidence that CgtA regulates the (p)ppGpp level during exponential growth but not during the stringent response. We show that CgtA is not associated with pre-50S particles during amino acid starvation, indicating that under these conditions in which (p)ppGpp accumulates, CgtA is not bound either to the pre-50S particle or to SpoT. We propose that, in addition to its role as a 50S assembly factor, CgtA promotes SpoT (p)ppGpp degradation activity on the ribosome and that the loss of CgtA from the ribosome is necessary for maximal (p)ppGpp accumulation under stress conditions. Intriguingly, we found that in the absence of spoT and relA, cgtA is still an essential gene in Escherichia coli.

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Manav, Melek Cemre, Jelena Beljantseva, Martin S. Bojer, Tanel Tenson, Hanne Ingmer, Vasili Hauryliuk, and Ditlev E. Brodersen. "Structural basis for (p)ppGpp synthesis by theStaphylococcus aureussmall alarmone synthetase RelP." Journal of Biological Chemistry 293, no. 9 (January 11, 2018): 3254–64. http://dx.doi.org/10.1074/jbc.ra117.001374.

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24

Bager, Ragnhild, Mohammad Roghanian, Kenn Gerdes, and David J. Clarke. "Alarmone (p)ppGpp regulates the transition from pathogenicity to mutualism inPhotorhabdus luminescens." Molecular Microbiology 100, no. 4 (March 11, 2016): 735–47. http://dx.doi.org/10.1111/mmi.13345.

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25

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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Laurie, Andrew D., Lisandro M. D. Bernardo, Chun Chau Sze, Eleonore Skärfstad, Agnieszka Szalewska-Palasz, Thomas Nyström та Victoria Shingler. "The Role of the Alarmone (p)ppGpp in ςNCompetition for Core RNA Polymerase". Journal of Biological Chemistry 278, № 3 (5 листопада 2002): 1494–503. http://dx.doi.org/10.1074/jbc.m209268200.

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Bachman, Michael A., and Michele S. Swanson. "Genetic Evidence that Legionella pneumophila RpoS Modulates Expression of the Transmission Phenotype in Both the Exponential Phase and the Stationary Phase." Infection and Immunity 72, no. 5 (May 2004): 2468–76. http://dx.doi.org/10.1128/iai.72.5.2468-2476.2004.

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ABSTRACT The opportunistic pathogen Legionella pneumophila alternates between two states: replication within phagocytes and transmission between host amoebae or macrophages. In broth cultures that model this life cycle, during the replication period, CsrA inhibits expression of transmission traits. When nutrients become limiting, the alarmone (p)ppGpp accumulates and the sigma factors RpoS and FliA and the positive activators LetA/S and LetE promote differentiation to the transmissible form. Here we show that when cells enter the postexponential growth phase, RpoS increases expression of the transmission genes fliA, flaA, and mip, factors L. pneumophila needs to establish a new replication niche. In contrast, in exponential (E)-phase cells whose (p)ppGpp levels are low, rpoS inhibits expression of transmission traits, on the basis of three separate observations. First, rpoS RNA levels peak in the E phase, suggestive of a role for RpoS during replication. Second, in multiple copies, rpoS decreases the amounts of csrA, letE, fliA, and flaA transcripts and inhibits the transmission traits of motility, infectivity, and cytotoxicity. Third, rpoS blocks expression of cytotoxicity and motility by E-phase bacteria that have been induced to express the LetA activator ectopically. The data are discussed in the context of a model in which the alarmone (p)ppGpp enables RpoS to outcompete other sigma factors for binding to RNA polymerase to promote transcription of transmission genes, while LetA/S acts in parallel to relieve CsrA posttranscriptional repression of the transmission regulon. By coupling transcriptional and posttranscriptional control pathways, intracellular L. pneumophila could respond to stress by rapidly differentiating to a transmissible form.

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Lemos, José A., Marcelle M. Nascimento, Vanessa K. Lin, Jacqueline Abranches, and Robert A. Burne. "Global Regulation by (p)ppGpp and CodY in Streptococcus mutans." Journal of Bacteriology 190, no. 15 (June 6, 2008): 5291–99. http://dx.doi.org/10.1128/jb.00288-08.

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ABSTRACT The RelA, RelP, and RelQ enzymes are responsible for the production of the alarmone (p)ppGpp in Streptococcus mutans. A strain lacking all three synthetases (ΔrelAPQ) does not grow in minimal medium lacking the branched-chain amino acids (BCAA) leucine or valine but grows well if isoleucine is also omitted. Here, we investigated whether there was a correlation between growth in the absence of leucine and valine with (p)ppGpp pools and the activation of CodY. By using a combination of single, double, and triple mutants lacking the (p)ppGpp synthetase enzymes, we demonstrated that the ability to grow in the absence of leucine or valine required basal levels of (p)ppGpp production by RelP and RelQ. The introduction of a codY mutation into the ΔrelAPQ strain fully restored growth in medium lacking leucine or valine, revealing that the growth-defective phenotype of ΔrelAPQ was directly linked to CodY. Lowering GTP levels through the addition of decoyinine did not alleviate CodY repression or affect the expression of genes involved in BCAA biosynthesis, suggesting that S. mutans CodY is not activated by GTP. The results of phenotypic studies revealed that the codY mutant had a reduced capacity to form biofilms and that its growth was more sensitive to low pH, showing a role for CodY in two key virulence properties of S. mutans. Microarray results revealed the extent of the CodY regulon. Notably, the identification of putative CodY-binding boxes upstream of genes that were downregulated in the codY mutant indicates that CodY may also function as a transcriptional activator in S. mutans.

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Kasai, Koji, Tomoyasu Nishizawa, Kosaku Takahashi, Takeshi Hosaka, Hiroyuki Aoki, and Kozo Ochi. "Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium, Thermus thermophilus." Journal of Bacteriology 188, no. 20 (October 1, 2006): 7111–22. http://dx.doi.org/10.1128/jb.00574-06.

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ABSTRACT Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel +; wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNASer aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

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Sze, Chun Chau, and Victoria Shingler. "The alarmone (p)ppGpp mediates physiological-responsive control at the sigma54-dependent Po promoter." Molecular Microbiology 31, no. 4 (February 1999): 1217–28. http://dx.doi.org/10.1046/j.1365-2958.1999.01264.x.

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Gerdes, Kenn. "Hypothesis: type I toxin–antitoxin genes enter the persistence field—a feedback mechanism explaining membrane homoeostasis." Philosophical Transactions of the Royal Society B: Biological Sciences 371, no. 1707 (November 5, 2016): 20160189. http://dx.doi.org/10.1098/rstb.2016.0189.

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Bacteria form persisters, cells that are tolerant to multiple antibiotics and other types of environmental stress. Persister formation can be induced either stochastically in single cells of a growing bacterial ensemble, or by environmental stresses, such as nutrient starvation, in a subpopulation of cells. In many cases, the molecular mechanisms underlying persistence are still unknown. However, there is growing evidence that, in enterobacteria, both stochastically and environmentally induced persistence are controlled by the second messenger (p)ppGpp. For example, the ‘alarmone’ (p)ppGpp activates Lon, which, in turn, activates type II toxin–antitoxin (TA) modules to thereby induce persistence. Recently, it has been shown that a type I TA module, hokB / sokB , also can induce persistence. In this case, the underlying mechanism depends on the universally conserved GTPase Obg and, surprisingly, also (p)ppGpp. In the presence of (p)ppGpp, Obg stimulates hokB transcription and induces persistence. HokB toxin expression is under both negative and positive control: SokB antisense RNA inhibits hokB mRNA translation, while (p)ppGpp and Obg together stimulate hokB transcription. HokB is a small toxic membrane protein that, when produced in modest amounts, leads to membrane depolarization, cell stasis and persistence. By contrast, overexpression of HokB disrupts the membrane potential and kills the cell. These observations raise the question of how expression of HokB is regulated. Here, I propose a homoeostatic control mechanism that couples HokB expression to the membrane-bound RNase E that degrades and inactivates SokB antisense RNA. This article is part of the themed issue ‘The new bacteriology’.

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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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Carmona, Manuel, Maria J. Rodríguez, Óscar Martínez-Costa та Víctor de Lorenzo. "In Vivo and In Vitro Effects of (p)ppGpp on the ς54 Promoter Pu of the TOL Plasmid ofPseudomonas putida". Journal of Bacteriology 182, № 17 (1 вересня 2000): 4711–18. http://dx.doi.org/10.1128/jb.182.17.4711-4718.2000.

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ABSTRACT The connection between the physiological control of the ς54-dependent Pu promoter of the TOL plasmid pWW0 of Pseudomonas putida and the stringent response mediated by the alarmone (p)ppGpp has been examined in vivo an in vitro. To this end, the key regulatory elements of the system were faithfully reproduced in an Escherichia coli strain and assayed as lacZ fusions in various genetic backgrounds lacking (p)ppGpp or overexpressing relA. Neither the responsiveness of Pu to 3-methyl benzylalcohol mediated by its cognate activator XylR nor the down-regulation of the promoter by rapid growth were affected in relA/spoT strains to an extent which could account for the known physiological control that governs this promoter. Overexpression of the relA gene [predicted to increase intracellullar (p)ppGpp levels] did, however, cause a significant gain in Pu activity. Since such a gain might be the result of indirect effects, we resorted to an in vitro transcription system to assay directly the effect of ppGpp on the transcriptional machinery. Although we did observe a significant increase in Pu performance through a range of ς54-RNAP concentrations, such an increase never exceeded twofold. The difference between these results and the behavior of the related Po promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic signals recruited for the physiological control of ς54-systems.

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Moris, Martine, Kristien Braeken, Eric Schoeters, Christel Verreth, Serge Beullens, Jos Vanderleyden, and Jan Michiels. "Effective Symbiosis between Rhizobium etli and Phaseolus vulgaris Requires the Alarmone ppGpp." Journal of Bacteriology 187, no. 15 (August 1, 2005): 5460–69. http://dx.doi.org/10.1128/jb.187.15.5460-5469.2005.

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ABSTRACT The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the σN-dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific σN copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.

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Coppa, Crescenzo, Luca Sorrentino, Monica Civera, Marco Minneci, Francesca Vasile, and Sara Sattin. "New Chemotypes for the Inhibition of (p)ppGpp Synthesis in the Quest for New Antimicrobial Compounds." Molecules 27, no. 10 (May 12, 2022): 3097. http://dx.doi.org/10.3390/molecules27103097.

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Antimicrobial resistance (AMR) poses a serious threat to our society from both the medical and economic point of view, while the antibiotic discovery pipeline has been dwindling over the last decades. Targeting non-essential bacterial pathways, such as those leading to antibiotic persistence, a bacterial bet-hedging strategy, will lead to new molecular entities displaying low selective pressure, thereby reducing the insurgence of AMR. Here, we describe a way to target (p)ppGpp (guanosine tetra- or penta-phosphate) signaling, a non-essential pathway involved in the formation of persisters, with a structure-based approach. A superfamily of enzymes called RSH (RelA/SpoT Homolog) regulates the intracellular levels of this alarmone. We virtually screened several fragment libraries against the (p)ppGpp synthetase domain of our RSH chosen model RelSeq, selected three main chemotypes, and measured their interaction with RelSeq by thermal shift assay and STD-NMR. Most of the tested fragments are selective for the synthetase domain, allowing us to select the aminobenzoic acid scaffold as a hit for lead development.

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Jha, Varsha, Nishant A. Dafale, and Hemant J. Purohit. "Regulatory rewiring through global gene regulations by PhoB and alarmone (p)ppGpp under various stress conditions." Microbiological Research 227 (October 2019): 126309. http://dx.doi.org/10.1016/j.micres.2019.126309.

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Bowden, Steven D., Alison Eyres, Jade C. S. Chung, Rita E. Monson, Arthur Thompson, George P. C. Salmond, David R. Spring, and Martin Welch. "Virulence inPectobacterium atrosepticumis regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp." Molecular Microbiology 90, no. 3 (September 30, 2013): 457–71. http://dx.doi.org/10.1111/mmi.12369.

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Zhang, Hai-Bao, Chao Wang, and Lian-Hui Zhang. "The quormone degradation system of Agrobacterium tumefaciens is regulated by starvation signal and stress alarmone (p)ppGpp." Molecular Microbiology 52, no. 5 (April 28, 2004): 1389–401. http://dx.doi.org/10.1111/j.1365-2958.2004.04061.x.

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Chatnaparat, Tiyakhon, Zhong Li, Schuyler S. Korban, and Youfu Zhao. "The bacterial alarmone (p)ppGpp is required for virulence and controls cell size and survival ofPseudomonas syringaeon plants." Environmental Microbiology 17, no. 11 (March 4, 2015): 4253–70. http://dx.doi.org/10.1111/1462-2920.12744.

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40

Boyle, William K., Crystal L. Richards, Daniel P. Dulebohn, Amanda K. Zalud, Jeff A. Shaw, Sándor Lovas, Frank C. Gherardini, and Travis J. Bourret. "DksA-dependent regulation of RpoS contributes to Borrelia burgdorferi tick-borne transmission and mammalian infectivity." PLOS Pathogens 17, no. 2 (February 18, 2021): e1009072. http://dx.doi.org/10.1371/journal.ppat.1009072.

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Throughout its enzootic cycle, the Lyme disease spirochete Borreliella (Borrelia) burgdorferi, senses and responds to changes in its environment using a small repertoire of transcription factors that coordinate the expression of genes required for infection of Ixodes ticks and various mammalian hosts. Among these transcription factors, the DnaK suppressor protein (DksA) plays a pivotal role in regulating gene expression in B. burgdorferi during periods of nutrient limitation and is required for mammalian infectivity. In many pathogenic bacteria, the gene regulatory activity of DksA, along with the alarmone guanosine penta- and tetra-phosphate ((p)ppGpp), coordinate the stringent response to various environmental stresses, including nutrient limitation. In this study, we sought to characterize the role of DksA in regulating the transcriptional activity of RNA polymerase and its role in the regulation of RpoS-dependent gene expression required for B. burgdorferi infectivity. Using in vitro transcription assays, we observed recombinant DksA inhibits RpoD-dependent transcription by B. burgdorferi RNA polymerase independent of ppGpp. Additionally, we determined the pH-inducible expression of RpoS-dependent genes relies on DksA, but this relationship is independent of (p)ppGpp produced by Relbbu. Subsequent transcriptomic and western blot assays indicate DksA regulates the expression of BBD18, a protein previously implicated in the post-transcriptional regulation of RpoS. Moreover, we observed DksA was required for infection of mice following intraperitoneal inoculation or for transmission of B. burgdorferi by Ixodes scapularis nymphs. Together, these data suggest DksA plays a central role in coordinating transcriptional responses in B. burgdorferi required for infectivity through DksA’s interactions with RNA polymerase and post-transcriptional control of RpoS.

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Mwangi, Michael M., Choonkeun Kim, Marilyn Chung, Jennifer Tsai, Govindan Vijayadamodar, Michelle Benitez, Thomas P. Jarvie, Lei Du та Alexander Tomasz. "Whole-Genome Sequencing Reveals a Link Between β-Lactam Resistance and Synthetases of the Alarmone (p)ppGpp inStaphylococcus aureus". Microbial Drug Resistance 19, № 3 (червень 2013): 153–59. http://dx.doi.org/10.1089/mdr.2013.0053.

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Schakermann, M., S. Langklotz, and F. Narberhaus. "FtsH-Mediated Coordination of Lipopolysaccharide Biosynthesis in Escherichia coli Correlates with the Growth Rate and the Alarmone (p)ppGpp." Journal of Bacteriology 195, no. 9 (February 15, 2013): 1912–19. http://dx.doi.org/10.1128/jb.02134-12.

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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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Honoki, Rina, Sumire Ono, Akira Oikawa, Kazuki Saito, and Shinji Masuda. "Significance of accumulation of the alarmone (p)ppGpp in chloroplasts for controlling photosynthesis and metabolite balance during nitrogen starvation in Arabidopsis." Photosynthesis Research 135, no. 1-3 (May 23, 2017): 299–308. http://dx.doi.org/10.1007/s11120-017-0402-y.

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45

Gao, Xiaohui, Jinki Yeom, and Eduardo A. Groisman. "The expanded specificity and physiological role of a widespread N-degron recognin." Proceedings of the National Academy of Sciences 116, no. 37 (August 26, 2019): 18629–37. http://dx.doi.org/10.1073/pnas.1821060116.

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All cells use proteases to maintain protein homeostasis. The proteolytic systems known as the N-degron pathways recognize signals at the N terminus of proteins and bring about the degradation of these proteins. The ClpS protein enforces the N-degron pathway in bacteria and bacteria-derived organelles by targeting proteins harboring leucine, phenylalanine, tryptophan, or tyrosine at the N terminus for degradation by the protease ClpAP. We now report that ClpS binds, and ClpSAP degrades, proteins still harboring the N-terminal methionine. We determine that ClpS recognizes a type of degron in intact proteins based on the identity of the fourth amino acid from the N terminus, showing a strong preference for large hydrophobic amino acids. We uncover natural ClpS substrates in the bacteriumSalmonella enterica, including SpoT, the essential synthase/hydrolase of the alarmone (p)ppGpp. Our findings expand both the specificity and physiological role of the widespread N-degron recognin ClpS.

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Delaby, Marie, Gaël Panis, and Patrick H. Viollier. "Bacterial cell cycle and growth phase switch by the essential transcriptional regulator CtrA." Nucleic Acids Research 47, no. 20 (October 10, 2019): 10628–44. http://dx.doi.org/10.1093/nar/gkz846.

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Abstract Many bacteria acquire dissemination and virulence traits in G1-phase. CtrA, an essential and conserved cell cycle transcriptional regulator identified in the dimorphic alpha-proteobacterium Caulobacter crescentus, first activates promoters in late S-phase and then mysteriously switches to different target promoters in G1-phase. We uncovered a highly conserved determinant in the DNA-binding domain (DBD) of CtrA uncoupling this promoter switch. We also show that it reprograms CtrA occupancy in stationary cells inducing a (p)ppGpp alarmone signal perceived by the RNA polymerase beta subunit. A simple side chain modification in a critical residue within the core DBD imposes opposing developmental phenotypes and transcriptional activities of CtrA and a proximal residue can direct CtrA towards activation of the dispersal (G1-phase) program. Hence, we propose that this conserved determinant in the CtrA primary structure dictates promoter reprogramming during the growth transition in other alpha-proteobacteria that differentiate from replicative cells into dispersal cells.

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Durfee, Tim, Anne-Marie Hansen, Huijun Zhi, Frederick R. Blattner, and Ding Jun Jin. "Transcription Profiling of the Stringent Response in Escherichia coli." Journal of Bacteriology 190, no. 3 (November 26, 2007): 1084–96. http://dx.doi.org/10.1128/jb.01092-07.

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ABSTRACT The bacterial stringent response serves as a paradigm for understanding global regulatory processes. It can be triggered by nutrient downshifts or starvation and is characterized by a rapid RelA-dependent increase in the alarmone (p)ppGpp. One hallmark of the response is the switch from maximum-growth-promoting to biosynthesis-related gene expression. However, the global transcription patterns accompanying the stringent response in Escherichia coli have not been analyzed comprehensively. Here, we present a time series of gene expression profiles for two serine hydroxymate-treated cultures: (i) MG1655, a wild-type E. coli K-12 strain, and (ii) an isogenic relAΔ251 derivative defective in the stringent response. The stringent response in MG1655 develops in a hierarchical manner, ultimately involving almost 500 differentially expressed genes, while the relAΔ251 mutant response is both delayed and limited in scope. We show that in addition to the down-regulation of stable RNA-encoding genes, flagellar and chemotaxis gene expression is also under stringent control. Reduced transcription of these systems, as well as metabolic and transporter-encoding genes, constitutes much of the down-regulated expression pattern. Conversely, a significantly larger number of genes are up-regulated. Under the conditions used, induction of amino acid biosynthetic genes is limited to the leader sequences of attenuator-regulated operons. Instead, up-regulated genes with known functions, including both regulators (e.g., rpoE, rpoH, and rpoS) and effectors, are largely involved in stress responses. However, one-half of the up-regulated genes have unknown functions. How these results are correlated with the various effects of (p)ppGpp (in particular, RNA polymerase redistribution) is discussed.

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Fritsch, Verena Nadin, Vu Van Loi, Tobias Busche, Quach Ngoc Tung, Roland Lill, Petra Horvatek, Christiane Wolz, Jörn Kalinowski, and Haike Antelmann. "The alarmone (p)ppGpp confers tolerance to oxidative stress during the stationary phase by maintenance of redox and iron homeostasis in Staphylococcus aureus." Free Radical Biology and Medicine 161 (December 2020): 351–64. http://dx.doi.org/10.1016/j.freeradbiomed.2020.10.322.

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Warner, Digby F., and Valerie Mizrahi. "Tuberculosis Chemotherapy: the Influence of Bacillary Stress and Damage Response Pathways on Drug Efficacy." Clinical Microbiology Reviews 19, no. 3 (July 2006): 558–70. http://dx.doi.org/10.1128/cmr.00060-05.

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SUMMARY The global tuberculosis (TB) control effort is focused on interrupting transmission of the causative agent, Mycobacterium tuberculosis, through chemotherapeutic intervention in active infectious disease. The insufficiency of this approach is manifest in the inexorable annual increase in TB infection and mortality rates and the emergence of multidrug-resistant isolates. Critically, the limited efficacy of the current frontline anti-TB drug combination suggests that heterogeneity of host and bacillary physiologies might impair drug activity. This review explores the possibility that strategies enabling adaptation of M. tuberculosis to hostile in vivo conditions might contribute to the subversion of anti-TB chemotherapy. In particular, evidence that infecting bacilli are exposed to environmental and host immune-mediated DNA-damaging insults suggests a role for error-prone DNA repair synthesis in the generation of chromosomally encoded antibiotic resistance mutations. The failure of frontline anti-TB drugs to sterilize a population of susceptible bacilli is independent of genetic resistance, however, and instead implies the operation of alternative tolerance mechanisms. Specifically, it is proposed that the emergence of persister subpopulations might depend on the switch to an altered metabolic state mediated by the stringent response alarmone, (p)ppGpp, possibly involving some or all of the many toxin-antitoxin modules identified in the M. tuberculosis genome.

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Min, Kyung Bae, and Sang Sun Yoon. "Transcriptome analysis reveals that the RNA polymerase–binding protein DksA1 has pleiotropic functions in Pseudomonas aeruginosa." Journal of Biological Chemistry 295, no. 12 (February 11, 2020): 3851–64. http://dx.doi.org/10.1074/jbc.ra119.011692.

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The stringent response (SR) is a highly conserved stress response in bacteria. It is composed of two factors, (i) a nucleotide alarmone, guanosine tetra- and pentaphosphate ((p)ppGpp), and (ii) an RNA polymerase-binding protein, DksA, that regulates various phenotypes, including bacterial virulence. The clinically significant opportunistic bacterial pathogen Pseudomonas aeruginosa possesses two genes, dksA1 and dksA2, that encode DksA proteins. It remains elusive, however, which of these two genes plays a more important role in SR regulation. In this work, we compared genome-wide, RNA-Seq–based transcriptome profiles of ΔdksA1, ΔdksA2, and ΔdksA1ΔdksA2 mutants to globally assess the effects of these gene deletions on transcript levels coupled with phenotypic analyses. The ΔdksA1 mutant exhibited substantial defects in a wide range of phenotypes, including quorum sensing (QS), anaerobiosis, and motility, whereas the ΔdksA2 mutant exhibited no significant phenotypic changes, suggesting that the dksA2 gene may not have an essential function in P. aeruginosa under the conditions used here. Of note, the ΔdksA1 mutants displayed substantially increased transcription of genes involved in polyamine biosynthesis, and we also detected increased polyamine levels in these mutants. Because SAM is a shared precursor for the production of both QS autoinducers and polyamines, these findings suggest that DksA1 deficiency skews the flow of SAM toward polyamine production rather than to QS signaling. Together, our results indicate that DksA1, but not DksA2, controls many important phenotypes in P. aeruginosa. We conclude that DksA1 may represent a potential target whose inhibition may help manage recalcitrant P. aeruginosa infections.

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