Статті в журналах: "(p)ppGpp Synthetases"

1

Ababneh, Qutaiba O., and Jennifer K. Herman. "RelA Inhibits Bacillus subtilis Motility and Chaining." Journal of Bacteriology 197, no. 1 (October 20, 2014): 128–37. http://dx.doi.org/10.1128/jb.02063-14.

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The nucleotide second messengers pppGpp and ppGpp [(p)ppGpp] are responsible for the global downregulation of transcription, translation, DNA replication, and growth rate that occurs during the stringent response. More recent studies suggest that (p)ppGpp is also an important effector in many nonstringent processes, including virulence, persister cell formation, and biofilm production. InBacillus subtilis, (p)ppGpp production is primarily determined by the net activity of RelA, a bifunctional (p)ppGpp synthetase/hydrolase, and two monofunctional (p)ppGpp synthetases, YwaC and YjbM. We observe that inB. subtilis, arelAmutant grows exclusively as unchained, motile cells, phenotypes regulated by the alternative sigma factor SigD. Our data indicate that therelAmutant is trapped in a SigD “on” state during exponential growth, implicating RelA and (p)ppGpp levels in the regulation of cell chaining and motility inB. subtilis. Our results also suggest that minor variations in basal (p)ppGpp levels can significantly skew developmental decision-making outcomes.

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2

Tozawa, Yuzuru, Akira Nozawa, Takuya Kanno, Takakuni Narisawa, Shinji Masuda, Koji Kasai, and Hideaki Nanamiya. "Calcium-activated (p)ppGpp Synthetase in Chloroplasts of Land Plants." Journal of Biological Chemistry 282, no. 49 (October 15, 2007): 35536–45. http://dx.doi.org/10.1074/jbc.m703820200.

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The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5′-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH2 terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca2+ and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles.

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3

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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4

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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5

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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6

Natori, Yousuke, Kazumi Tagami, Kana Murakami, Sawako Yoshida, Osamu Tanigawa, Yoonsuh Moh, Kenta Masuda, et al. "Transcription Activity of Individual rrn Operons in Bacillus subtilis Mutants Deficient in (p)ppGpp Synthetase Genes, relA, yjbM, and ywaC." Journal of Bacteriology 191, no. 14 (May 15, 2009): 4555–61. http://dx.doi.org/10.1128/jb.00263-09.

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ABSTRACT In Bacillus subtilis a null mutation of the relA gene, whose gene product is involved in the synthesis and/or hydrolysis of (p)ppGpp, causes a growth defect that can be suppressed by mutation(s) of yjbM and/or ywaC coding for small (p)ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, either yjbM or ywaC, by mapping and sequencing their mutations, suggesting that there are no (p)ppGpp synthetases other than RelA, YjbM, and YwaC in B. subtilis. In order to understand better the relation between RelA and rRNA synthesis, we studied in the relA mutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B) individually after integration of a promoter- and terminatorless cat gene. We identified the transcriptional start sites of each rrn operon (a G) and found that transcription of all rrn operons from their P1 promoters was drastically reduced in the relA mutant while this was almost completely restored in the relA yjbM ywaC triple mutant. Taken together with previous results showing that the intracellular GTP concentration was reduced in the relA mutant while it was restored in the triple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.

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7

Zhu, Jiawen, Tengfei Zhang, Zhipeng Su, Lu Li, Dong Wang, Ran Xiao, Muye Teng, Meifang Tan, and Rui Zhou. "(p)ppGpp synthetases regulate the pathogenesis of zoonotic Streptococcus suis." Microbiological Research 191 (October 2016): 1–11. http://dx.doi.org/10.1016/j.micres.2016.05.007.

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8

Wang, Xin, Yiluo Cheng, Wenting Zhang, Qin Lu, Guoyuan Wen, Qingping Luo, Huabin Shao, Zishu Pan, and Tengfei Zhang. "(p)ppGpp synthetases are required for the pathogenicity of Salmonella Pullorum in chickens." Microbiological Research 245 (April 2021): 126685. http://dx.doi.org/10.1016/j.micres.2020.126685.

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9

Wang, Xin, Yiluo Cheng, Wenting Zhang, Qin Lu, Guoyuan Wen, Qingping Luo, Huabin Shao, Zishu Pan, and Tengfei Zhang. "(p)ppGpp synthetases are required for the pathogenicity of Salmonella Pullorum in chickens." Microbiological Research 245 (April 2021): 126685. http://dx.doi.org/10.1016/j.micres.2020.126685.

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10

Zhu, Jiawen, Tengfei Zhang, Zhipeng Su, Liping Feng, Hanyang Liu, Zhenying Xu, Yongsheng Wu, Ting Gao, Huabin Shao, and Rui Zhou. "Co-regulation of CodY and (p)ppGpp synthetases on morphology and pathogenesis of Streptococcus suis." Microbiological Research 223-225 (June 2019): 88–98. http://dx.doi.org/10.1016/j.micres.2019.04.001.

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11

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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12

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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13

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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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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16

Okada, Yumiko, Sou-ichi Makino, Toru Tobe, Nobuhiko Okada, and Shouji Yamazaki. "Cloning of rel from Listeria monocytogenes as an Osmotolerance Involvement Gene." Applied and Environmental Microbiology 68, no. 4 (April 2002): 1541–47. http://dx.doi.org/10.1128/aem.68.4.1541-1547.2002.

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ABSTRACT Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.

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17

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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Van der Henst, Mathilde, Elodie Carlier, and Xavier De Bolle. "Intracellular Growth and Cell Cycle Progression are Dependent on (p)ppGpp Synthetase/Hydrolase in Brucella abortus." Pathogens 9, no. 7 (July 14, 2020): 571. http://dx.doi.org/10.3390/pathogens9070571.

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Brucella abortus is a pathogenic bacterium able to proliferate inside host cells. During the first steps of its trafficking, it is able to block the progression of its cell cycle, remaining at the G1 stage for several hours, before it reaches its replication niche. We hypothesized that starvation mediated by guanosine tetra- or penta-phosphate, (p)ppGpp, could be involved in the cell cycle arrest. Rsh is the (p)ppGpp synthetase/hydrolase. A B. abortus ∆rsh mutant is unable to grow in minimal medium, it is unable to survive in stationary phase in rich medium and it is unable to proliferate inside RAW 264.7 macrophages. A strain producing the heterologous constitutive (p)ppGpp hydrolase Mesh1b is also unable to proliferate inside these macrophages. Altogether, these data suggest that (p)ppGpp is necessary to allow B. abortus to adapt to its intracellular growth conditions. The deletion of dksA, proposed to mediate a part of the effect of (p)ppGpp on transcription, does not affect B. abortus growth in culture or inside macrophages. Expression of a gene coding for a constitutively active (p)ppGpp synthetase slows down growth in rich medium and inside macrophages. Using an mCherry–ParB fusion able to bind to the replication origin of the main chromosome of B. abortus, we observed that expression of the constitutive (p)ppGpp synthetase gene generates an accumulation of bacteria at the G1 phase. We thus propose that (p)ppGpp accumulation could be one of the factors contributing to the G1 arrest observed for B. abortus in RAW 264.7 macrophages.

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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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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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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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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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23

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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24

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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Holley, Concerta, Dharanesh Gangaiah, Wei Li, Kate R. Fortney, Diane M. Janowicz, Sheila Ellinger, Beth Zwickl, Barry P. Katz, and Stanley M. Spinola. "A (p)ppGpp-Null Mutant of Haemophilus ducreyi Is Partially Attenuated in Humans Due to Multiple Conflicting Phenotypes." Infection and Immunity 82, no. 8 (June 9, 2014): 3492–502. http://dx.doi.org/10.1128/iai.01994-14.

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ABSTRACT(p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species.Haemophilus ducreyi, the causative agent of chancroid, has homologs of bothrelAandspoT, which primarily synthesize and hydrolyze (p)ppGpp inEscherichia coli. We constructedrelAandrelA spoTdeletion mutants to assess the contribution of (p)ppGpp toH. ducreyipathogenesis. Both therelAsingle mutant and therelA spoTdouble mutant failed to synthesize (p)ppGpp, suggesting thatrelAis the primary synthetase of (p)ppGpp inH. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented intrans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription oflspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. InE. coli, overexpression of DksA compensates for the loss of (p)ppGpp; theH. ducreyidouble mutant expressed higher transcript levels ofdksAthan the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.

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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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27

Germain, Elsa, Mohammad Roghanian, Kenn Gerdes, and Etienne Maisonneuve. "Stochastic induction of persister cells by HipA through (p)ppGpp-mediated activation of mRNA endonucleases." Proceedings of the National Academy of Sciences 112, no. 16 (April 6, 2015): 5171–76. http://dx.doi.org/10.1073/pnas.1423536112.

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The model organismEscherichia colicodes for at least 11 type II toxin–antitoxin (TA) modules, all implicated in bacterial persistence (multidrug tolerance). Ten of these encode messenger RNA endonucleases (mRNases) inhibiting translation by catalytic degradation of mRNA, and the 11th module,hipBA, encodes HipA (high persister protein A) kinase, which inhibits glutamyl tRNA synthetase (GltX). In turn, inhibition of GltX inhibits translation and induces the stringent response and persistence. Previously, we presented strong support for a model proposing (p)ppGpp (guanosine tetra and penta-phosphate) as the master regulator of persistence. Stochastic variation of [(p)ppGpp] in single cells induced TA-encoded mRNases via a pathway involving polyphosphate and Lon protease. Polyphosphate activated Lon to degrade all known type II antitoxins ofE. coli. In turn, the activated mRNases induced persistence and multidrug tolerance. However, even though it was known that activation of HipA stimulated (p)ppGpp synthesis, our model did not explain howhipBAinduced persistence. Here we show that, in support of and consistent with our initial model, HipA-induced persistence depends not only on (p)ppGpp but also on the 10 mRNase-encoding TA modules, Lon protease, and polyphosphate. Importantly, observations with single cells convincingly show that the high level of (p)ppGpp caused by activation of HipA does not induce persistence in the absence of TA-encoded mRNases. Thus, slow growth per se does not induce persistence in the absence of TA-encoded toxins, placing these genes as central effectors of bacterial persistence.

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Manuel, Jerrylynn, Chrystal Berry, Carrie Selin, W. G. Dilantha Fernando, and Teresa R. de Kievit. "Repression of the Antifungal Activity of Pseudomonas sp. Strain DF41 by the Stringent Response." Applied and Environmental Microbiology 77, no. 16 (June 24, 2011): 5635–42. http://dx.doi.org/10.1128/aem.02875-10.

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ABSTRACTThe stringent response (SR) enables bacteria to adapt to nutrient limitation through production of the nucleotides guanosine tetraphosphate and guanosine pentaphosphate, collectively known as (p)ppGpp. Two enzymes are responsible for the intracellular pools of (p)ppGpp: RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. We investigated how the SR affects the ability of the biological control agentPseudomonassp. strain DF41 to inhibit the fungal pathogenSclerotinia sclerotiorum(Lib.) de Bary. Strain DF41relAandrelA spoTmutants were generated and found to exhibit increased antifungal activity. Strain DF41 produces a lipopeptide (LP) molecule that is essential forSclerotiniabiocontrol. LP production and protease activity were both elevated in therelAandrelA spoTmutants. Addition ofrelAbut notspoTintransrestored the mutant phenotype to that of the parent. Next, we investigated whether an association exists between the SR and known regulators of biocontrol, including the Gac system and RpoS. AgacSmutant of strain DF41 produced less (p)ppGpp and exhibited a 1.7-fold decrease inrelAexpression compared to the wild type, suggesting thatrelAforms part of the Gac regulon. We discovered thatrpoStranscription was reduced significantly in the SR mutants. Furthermore,rpoSprovided intransrestored protease activity to wild-type levels but did not attenuate antifungal activity. Finally,relAexpression was decreased in the mutants, indicating that the SR is required for maximum expression ofrelA.

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Merrikh, Houra, Alexander E. Ferrazzoli, and Susan T. Lovett. "Growth Phase and (p)ppGpp Control of IraD, a Regulator of RpoS Stability, in Escherichia coli." Journal of Bacteriology 191, no. 24 (October 9, 2009): 7436–46. http://dx.doi.org/10.1128/jb.00412-09.

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ABSTRACT The antiadaptor protein IraD inhibits the proteolysis of the alternative sigma factor, RpoS, which promotes the synthesis of >100 genes during the general stress response and during stationary phase. Our previous results showed that IraD determines RpoS steady-state levels during exponential growth and mediates its stabilization after DNA damage. In this study, we show by promoter fusions that iraD was upregulated during the transition from exponential growth to stationary phase. The levels of RpoS likewise rose during this transition in a partially IraD-dependent manner. The expression of iraD was under the control of ppGpp. The expression of iraD required RelA and SpoT (p)ppGpp synthetase activities and was dramatically induced by a “stringent” allele of RNA polymerase, culminating in elevated levels of RpoS. Surprisingly, DksA, normally required for transcriptional effects of the stringent response, repressed iraD expression, suggesting that DksA can exert regulatory effects independent of and opposing those of (p)ppGpp. Northern blot analysis and 5′ rapid amplification of cDNA ends revealed two transcripts for iraD in wild-type strains; the smaller was regulated positively by RelA during growth; the larger transcript was induced specifically upon transition to stationary phase and was RelA SpoT dependent. A reporter fusion to the distal promoter indicated that it accounts for growth-phase regulation and DNA damage inducibility. DNA damage inducibility occurred in strains unable to synthesize (p)ppGpp, indicating an additional mode of regulation. Our results suggest that the induction of RpoS during transition to stationary phase and by (p)ppGpp occurs at least partially through IraD.

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Zhou, Yan Ning, William G. Coleman, Zhaoxu Yang, Yi Yang, Nathaniel Hodgson, Fuxiang Chen, and Ding Jun Jin. "Regulation of Cell Growth during Serum Starvation and Bacterial Survival in Macrophages by the Bifunctional Enzyme SpoT in Helicobacter pylori." Journal of Bacteriology 190, no. 24 (October 3, 2008): 8025–32. http://dx.doi.org/10.1128/jb.01134-08.

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ABSTRACT In Helicobacter pylori the stringent response is mediated solely by spoT. The spoT gene is known to encode (p)ppGpp synthetase activity and is required for H. pylori survival in the stationary phase. However, neither the hydrolase activity of the H. pylori SpoT protein nor the role of SpoT in the regulation of growth during serum starvation and intracellular survival of H. pylori in macrophages has been determined. In this study, we examined the effects of SpoT on these factors. Our results showed that the H. pylori spoT gene encodes a bifunctional enzyme with both a hydrolase activity and the previously described (p)ppGpp synthetase activity, as determined by introducing the gene into Escherichia coli relA and spoT defective strains. Also, we found that SpoT mediates a serum starvation response, which not only restricts the growth but also maintains the helical morphology of H. pylori. Strikingly, a spoT null mutant was able to grow to a higher density in serum-free medium than the wild-type strain, mimicking the “relaxed” growth phenotype of an E. coli relA mutant during amino acid starvation. Finally, SpoT was found to be important for intracellular survival in macrophages during phagocytosis. The unique role of (p)ppGpp in cell growth during serum starvation, in the stress response, and in the persistence of H. pylori is discussed.

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Ossa, Faisury, Michelle E. Diodati, Nora B. Caberoy, Krista M. Giglio, Mick Edmonds, Mitchell Singer, and Anthony G. Garza. "The Myxococcus xanthus Nla4 Protein Is Important for Expression of Stringent Response-Associated Genes, ppGpp Accumulation, and Fruiting Body Development." Journal of Bacteriology 189, no. 23 (September 28, 2007): 8474–83. http://dx.doi.org/10.1128/jb.00894-07.

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ABSTRACT Changes in gene expression are important for the landmark morphological events that occur during Myxococcus xanthus fruiting body development. Enhancer binding proteins (EBPs), which are transcriptional activators, play prominent roles in the coordinated expression of developmental genes. A mutation in the EBP gene nla4 affects the timing of fruiting body formation, the morphology of mature fruiting bodies, and the efficiency of sporulation. In this study, we showed that the nla4 mutant accumulates relatively low levels of the stringent nucleotide ppGpp. We also found that the nla4 mutant is defective for early developmental events and for vegetative growth, phenotypes that are consistent with a deficiency in ppGpp accumulation. Further studies revealed that nla4 cells produce relatively low levels of GTP, a precursor of RelA-dependent synthesis of (p)ppGpp. In addition, the normal expression patterns of all stringent response-associated genes tested, including the M. xanthus ppGpp synthetase gene relA, are altered in nla4 mutant cells. These findings indicate that Nla4 is part of regulatory pathway that is important for mounting a stringent response and for initiating fruiting body development.

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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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Avarbock, Andrew, David Avarbock, Jiah-Shin Teh, Michael Buckstein, Zhi-mei Wang, and Harvey Rubin. "Functional Regulation of the Opposing (p)ppGpp Synthetase/Hydrolase Activities of RelMtbfromMycobacteriumtuberculosis†." Biochemistry 44, no. 29 (July 2005): 9913–23. http://dx.doi.org/10.1021/bi0505316.

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Tagami, Kazumi, Hideaki Nanamiya, Yuka Kazo, Marie Maehashi, Shota Suzuki, Eri Namba, Masahiro Hoshiya, et al. "Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp 0 mutant of Bacillus subtilis triggers YvyD‐dependent dimerization of ribosome." MicrobiologyOpen 1, no. 2 (April 3, 2012): 115–34. http://dx.doi.org/10.1002/mbo3.16.

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Nanamiya, Hideaki, Koji Kasai, Akira Nozawa, Choong-Soo Yun, Takakuni Narisawa, Kana Murakami, Yousuke Natori, Fujio Kawamura, and Yuzuru Tozawa. "Identification and functional analysis of novel (p)ppGpp synthetase genes in Bacillus subtilis." Molecular Microbiology 67, no. 2 (December 7, 2007): 291–304. http://dx.doi.org/10.1111/j.1365-2958.2007.06018.x.

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Murdeshwar, M. S., and D. Chatterji. "MS_RHII-RSD, a Dual-Function RNase HII-(p)ppGpp Synthetase from Mycobacterium smegmatis." Journal of Bacteriology 194, no. 15 (May 25, 2012): 4003–14. http://dx.doi.org/10.1128/jb.00258-12.

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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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Zhang, Shuyu, та W. G. Haldenwang. "RelA Is a Component of the Nutritional Stress Activation Pathway of the Bacillus subtilis Transcription Factor σB". Journal of Bacteriology 185, № 19 (1 жовтня 2003): 5714–21. http://dx.doi.org/10.1128/jb.185.19.5714-5721.2003.

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ABSTRACT The general stress regulon of Bacillus subtilis is induced by the activation of the σB transcription factor. Activation of σB occurs when one of two phosphatases (RsbU and RsbP), each responding to a unique type of stress, actuates a positive regulator of σB by dephosphorylation. Nutritional stress triggers the RsbP phosphatase. The mechanism by which RsbP becomes active is unknown; however, its activation coincides with culture conditions that are likely to reduce the cell's levels of high-energy nucleotides. We now present evidence that RelA, a (p)ppGpp synthetase and the key enzyme of the stringent response, plays a role in nutritional stress activation of σB. An insertion mutation that disrupts relA blocks the activation of σB in response to PO4 or glucose limitation and inhibits the drop in ATP/GTP levels that normally accompanies σB induction under these conditions. In contrast, the activation of σB by physical stress (e.g., ethanol treatment) is not affected by the loss of RelA. RelA's role in σB activation appears to be distinct from its participation in the stringent response. Amino acid analogs which induce the stringent response and RelA-dependent (p)ppGpp synthesis do not trigger σB activity. In addition, neither a missense mutation in relA (relA240GE) nor a null mutation in rplK (rplK54), either of which is sufficient to inhibit the stringent response and RelA-dependent (p)ppGpp synthesis, fails to block σB activation by PO4 or glucose limitation.

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Yan, X., C. Zhao, A. Budin-Verneuil, A. Hartke, A. Rince, M. S. Gilmore, Y. Auffray, and V. Pichereau. "The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583." Microbiology 155, no. 10 (July 16, 2009): 3226–37. http://dx.doi.org/10.1099/mic.0.026146-0.

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Bag, Satyabrata, Bhabatosh Das, Shreya Dasgupta, and Rupak K. Bhadra. "Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis." Archives of Microbiology 196, no. 8 (May 24, 2014): 575–88. http://dx.doi.org/10.1007/s00203-014-0996-9.

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Gropp, Michal, Yael Strausz, Miriam Gross, and Gad Glaser. "Regulation of Escherichia coli RelA Requires Oligomerization of the C-Terminal Domain." Journal of Bacteriology 183, no. 2 (January 15, 2001): 570–79. http://dx.doi.org/10.1128/jb.183.2.570-579.2001.

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ABSTRACT The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD inrelA + cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

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Krishnan, Sushma, Anushya Petchiappan, Albel Singh, Apoorva Bhatt, and Dipankar Chatterji. "R-loop induced stress response by second (p)ppGpp synthetase inMycobacterium smegmatis: functional and domain interdependence." Molecular Microbiology 102, no. 1 (July 19, 2016): 168–82. http://dx.doi.org/10.1111/mmi.13453.

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Chakraburtty, Rekha, Janet White, Eriko Takano, and Mervyn Bibb. "Cloning, characterization and disruption of a (p)ppGpp synthetase gene (relA) of Streptomyces coelicolor A3(2)." Molecular Microbiology 19, no. 2 (January 1996): 357–68. http://dx.doi.org/10.1046/j.1365-2958.1996.390919.x.

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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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45

Yang, Xiaoming, and Edward E. Ishiguro. "Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli." Journal of Bacteriology 183, no. 22 (November 15, 2001): 6532–37. http://dx.doi.org/10.1128/jb.183.22.6532-6537.2001.

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ABSTRACT Amino acid-deprived rplK (previously known asrelC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative ofrplK encoding L11 lacking the N-terminal 36 amino acids (designated ′L11) was constructed. Bacteria overexpressing ′L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed ′L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.

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Das, Bhabatosh, Ritesh Ranjan Pal, Satyabrata Bag, and Rupak K. Bhadra. "Stringent response inVibrio cholerae: genetic analysis ofspoTgene function and identification of a novel (p)ppGpp synthetase gene." Molecular Microbiology 72, no. 2 (April 2009): 380–98. http://dx.doi.org/10.1111/j.1365-2958.2009.06653.x.

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47

Pausch, Patrick, Maha Abdelshahid, Wieland Steinchen, Heinrich Schäfer, Fabio Lino Gratani, Sven-Andreas Freibert, Christiane Wolz, Kürşad Turgay, Daniel N. Wilson, and Gert Bange. "Structural Basis for Regulation of the Opposing (p)ppGpp Synthetase and Hydrolase within the Stringent Response Orchestrator Rel." Cell Reports 32, no. 11 (September 2020): 108157. http://dx.doi.org/10.1016/j.celrep.2020.108157.

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Yang, Xiaoming, and Edward E. Ishiguro. "Dimerization of the RelA protein of Escherichia coli." Biochemistry and Cell Biology 79, no. 6 (December 1, 2001): 729–36. http://dx.doi.org/10.1139/o01-144.

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The RelA protein of Escherichia coli is a ribosome-associated (p)ppGpp synthetase that is activated by amino acid deprivation. It was recently reported that the activity of RelA is regulated by oligomerization mediated by the C-terminal domain of RelA. The oligomerization of RelA is further characterized in this study. The C-terminal domain consisting of amino acids 455744, designated 'RelA, formed homooligomers as well as heterooligomers with RelA as demonstrated by copurification of RelA and 'RelA and by an affinity blotting assay. Glutaraldehyde-induced cross-linking indicated that the oligomer was a dimer. The functional analysis of 'RelA was based on a combination of yeast two-hybrid analysis, the determination of the effects of overexpression of 'RelA derivatives on the stringent response, and the cellular localization of the overexpressed 'RelA derivatives. These studies indicated that two regions, designated 'RelA-1 (amino acids 455538) and 'RelA-2 (amino acids 550682), were involved in dimerization. The involvement of one of these two regions, RelA-2, is consistent with a previous site-directed mutagenesis study. In addition to dimerization, 'RelA-2 apparently contained the main ribosome-binding domain of RelA. The third region, 'RelA-3 (amino acids 682744), was not involved in either dimerization or ribosome binding. The overexpression of 'RelA-1 and 'RelA-2, but not 'RelA-3, inhibited the stringent response. These results support the previously proposed model which suggests a role for oligomerization in the regulation of (p)ppGpp synthetase.Key words: RelA, Escherichia coli, stringent response.

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Lemos, José A. C., Thomas A. Brown, and Robert A. Burne. "Effects of RelA on Key Virulence Properties of Planktonic and Biofilm Populations of Streptococcus mutans." Infection and Immunity 72, no. 3 (March 2004): 1431–40. http://dx.doi.org/10.1128/iai.72.3.1431-1440.2004.

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ABSTRACT Streptococcus mutans is a biofilm-forming bacterium that is adapted to tolerate rapid and dramatic fluctuations in nutrient availability, carbohydrate source, and pH in its natural environment, the human oral cavity. Dissecting the pathways used to form stable biofilms and to tolerate environmental stress is central to understanding the virulence of this organism. Here, we investigated the role of the S. mutans relA gene, which codes for a guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase/hydrolase, in biofilm formation and acid tolerance. Two mutants in which relA was insertionally inactivated or replaced by an antibiotic resistance determinant were constructed. Under normal growth and stress conditions, the mutants grew slower than the wild-type strain, although the final yields were similar. The mutants, which were still able to accumulate (p)ppGpp after the induction of a stringent response, showed significant reductions in biofilm formation on microtiter plates or hydroxylapatite disks. There was no difference in the sensitivities to acid killing of the parent and relA strains grown in planktonic cultures. However, when cells were grown in biofilms, the mutants became more acid resistant and could lower the pH through glycolysis faster and to a greater extent than the wild-type strain. Differences in acid resistance were not correlated with increases in F-ATPase activity, although bacterial sugar:phosphotransferase activity was elevated in the mutants. Expression of the luxS gene was increased as much as fivefold in the relA mutants, suggesting a link between AI-2 quorum sensing and the stringent response.

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Gratani, Fabio Lino, Petra Horvatek, Tobias Geiger, Marina Borisova, Christoph Mayer, Iwan Grin, Samuel Wagner, et al. "Regulation of the opposing (p)ppGpp synthetase and hydrolase activities in a bifunctional RelA/SpoT homologue from Staphylococcus aureus." PLOS Genetics 14, no. 7 (July 9, 2018): e1007514. http://dx.doi.org/10.1371/journal.pgen.1007514.

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