RelA/SpoT Homolog Research Articles

A binuclear metallohydrolase model for RelA/SpoT-Homolog (RSH) hydrolases

When challenged by starvation, bacterial organisms synthesize guanosine penta- and tetraphosphate, collectively denoted as (p)ppGpp, as second messengers to reprogram metabolism towards slower growth and enhanced stress tolerance. When starvation is alleviated, the RelA-SpoT homolog (RSH) hydrolases down-regulate (p)ppGpp, cleaving the 3’-diphosphate to produce GTP or GDP. Metazoan RSH hydrolases (MESH) possess phosphatase activity responsible for converting cytoplasmic NADPH to NADH in mammalian cells. Inhibitor development for this family may therefore provide therapies to combat bacterial infection or metabolic dysregulation. Despite the availability of dozens of high-resolution structures, catalytic mechanisms of RSH hydrolases have remained poorly understood. All RSH hydrolases tightly bind a Mn2+ near its active center, which is believed sufficient for hydrolase activity. In contrast to this notion, we demonstrate, using the (p)ppGpp hydrolase SpoT from Acinetobacter baumannii, that a second divalent cation, presumably a Mg2+ under physiological conditions, is required for efficient catalysis. We also show that SpoT preferentially cleaves 3’-diphosphate over 3’-phosphate substrates, likely due to a key coordination between the β-phosphate and the second metal center. MESH replaces this β-phosphate with the side chain of an aspartate residue, thereby functioning as a phosphatase. We propose a binuclear metallohydrolase model where an invariant ED (Glu-Asp) diad, previously believed to activate the water nucleophile, instead coordinates to a Mg2+ center. The refined molecular and evolutionary blueprint of RSH hydrolases will provide a more reliable foundation for the development of small-molecule inhibitors of this important enzyme family.

Posttranslational regulation of photosynthetic activity via the TOR kinase in plants.

Chloroplasts are the powerhouse of the plant cell, and their activity must be matched to plant growth to avoid photooxidative damage. We have identified a posttranslational mechanism linking the eukaryotic target of rapamycin (TOR) kinase that promotes growth and the guanosine tetraphosphate (ppGpp) signaling pathway of prokaryotic origins that regulates chloroplast activity and photosynthesis in particular. We find that RelA SpoT homolog 3 (RSH3), a nuclear-encoded enzyme responsible for ppGpp biosynthesis, interacts directly with the TOR complex via a plant-specific amino-terminal region which is phosphorylated in a TOR-dependent manner. Down-regulating TOR activity causes a rapid increase in ppGpp synthesis in RSH3 overexpressors and reduces photosynthetic capacity in an RSH-dependent manner in wild-type plants. The TOR-RSH3 signaling axis therefore regulates the equilibrium between chloroplast activity and plant growth, setting a precedent for the regulation of organellar function by TOR.

The Mechanism of Inhibition of Mycobacterial (p)ppGpp Synthetases by a Synthetic Analog of Erogorgiaene.

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.

Direct detection of stringent alarmones (pp)pGpp using malachite green.

The alarmone (p)ppGpp serves as the signalling molecule for the bacterial universal stringent response and plays a crucial role in bacterial virulence, persistence, and stress adaptation. Consequently, there is a significant focus on developing new drugs that target and modulate the levels of (p)ppGpp as a potential strategy for controlling bacterial infections. However, despite the availability of various methods for detecting (p)ppGpp, a simple and straightforward detection method is needed. In this study, we demonstrated that malachite green, a well-established compound used for phosphate detection, can directly detect (p)ppGpp and its analogues esp., pGpp. By utilizing malachite green, we identified three new inhibitors of the hydrolase activity of SpoT, one of the two RelA-SpoT homolog (RSH) proteins responsible for making and hydrolyzing (p)ppGpp in Escherichia coli. These findings highlight the convenience and practicality of malachite green, which can be widely employed in high-throughput studies to investigate (pp)pGpp in vitro and discover novel regulators of RSH proteins.

Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola.

The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH) proteins, play central roles in the stringent response. The pathogenic oral spirochete bacterium Treponema denticola lacks a long-RSH homologue but encodes putative small alarmone synthetase (Tde-SAS, TDE1711) and small alarmone hydrolase (Tde-SAH, TDE1690) proteins. Here, we characterize the respective in vitro and in vivo activities of Tde-SAS and Tde-SAH, which respectively belong to the previously uncharacterized RSH families DsRel and ActSpo2. The tetrameric 410-amino acid (aa) Tde-SAS protein preferentially synthesizes ppGpp over pppGpp and a third alarmone, pGpp. Unlike RelQ homologues, alarmones do not allosterically stimulate the synthetic activities of Tde-SAS. The ~180 aa C-terminal tetratricopeptide repeat (TPR) domain of Tde-SAS acts as a brake on the alarmone synthesis activities of the ~220-aa N-terminal catalytic domain. Tde-SAS also synthesizes "alarmone-like" nucleotides such as adenosine tetraphosphate (ppApp), albeit at considerably lower rates. The 210-aa Tde-SAH protein efficiently hydrolyzes all guanosine and adenosine-based alarmones in a Mn(II) ion-dependent manner. Using a growth assays with a ΔrelAΔspoT strain of Escherichia coli that is deficient in pppGpp/ppGpp synthesis, we demonstrate that Tde-SAS can synthesize alarmones in vivo to restore growth in minimal media. Taken together, our results add to our holistic understanding of alarmone metabolism across diverse bacterial species. IMPORTANCE The spirochete bacterium Treponema denticola is a common component of the oral microbiota. However, it may play important pathological roles in multispecies oral infectious diseases such as periodontitis: a severe and destructive form of gum disease, which is a major cause of tooth loss in adults. The operation of the stringent response, a highly conserved survival mechanism, is known to help many bacterial species cause persistent or virulent infections. By characterizing the biochemical functions of the proteins putatively responsible for the stringent response in T. denticola, we may gain molecular insight into how this bacterium can survive within harsh oral environments and promote infection. Our results also expand our general understanding of proteins that synthesize nucleotide-based intracellular signaling molecules in bacteria.

Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts through Specific RelA/SpoT Homologues.

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RelA-SpoT homologue (RSH) enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope-labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. Transcript analyses showed an upregulation of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. Arabidopsis mutants deficient in RSH2 and RSH3 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting the involvement of these synthases in PAMP-triggered innate immune responses to pathogens within the chloroplast.

The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives.

The treatment of many bacterial diseases remains a significant problem due to the increasing antibiotic resistance of their infectious agents. Among others, this is related to Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA) and Mycobacterium tuberculosis. In the present article, we report on antibacterial compounds with activity against both S. aureus and MRSA. A straightforward approach to 2-(1H-indol-3-yl)quinazolin-4(3H)-one and their analogues was developed. Their structural and functional relationships were also considered. The antimicrobial activity of the synthesized compounds against Mycobacterium tuberculosis H37Rv, S. aureus ATCC 25923, MRSA ATCC 43300, Candida albicans ATCC 10231, and their role in the inhibition of the biofilm formation of S. aureus were reported. 2-(5-Iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) showed a low minimum inhibitory concentration (MIC) of 0.98 μg/mL against MRSA. The synthesized compounds were assessed via molecular docking for their ability to bind long RSH (RelA/SpoT homolog) proteins using mycobacterial and streptococcal (p)ppGpp synthetase structures as models. The cytotoxic activity of some synthesized compounds was studied. Compounds 3c, f, g, k, r, and 3z displayed significant antiproliferative activities against all the cancer cell lines tested. Indolylquinazolinones 3b, 3e, and 3g showed a preferential suppression of the growth of rapidly dividing A549 cells compared to slower growing fibroblasts of non-tumor etiology.

Identification of a broadly conserved family of enzymes that hydrolyze (p)ppApp

Bacteria produce a variety of nucleotide second messengers to adapt to their surroundings. Although chemically similar, the nucleotides guanosine penta- and tetraphosphate [(p)ppGpp] and adenosine penta- and tetraphosphate [(p)ppApp] have distinct functions in bacteria. (p)ppGpp mediates survival under nutrient-limiting conditions and its intracellular levels are regulated by synthetases and hydrolases belonging to the RelA-SpoT homolog (RSH) family of enzymes. By contrast, (p)ppApp is not known to be involved in nutrient stress responses and is synthesized by RSH-resembling toxins that inhibit the growth of bacterial cells. However, it remains unclear whether there exists a family of hydrolases that specifically act on (p)ppApp to reverse its toxic effects. Here, we present the structure and biochemical characterization of adenosine 3'-pyrophosphohydrolase 1 (Aph1), the founding member of a monofunctional (p)ppApp hydrolase family of enzymes. Our work reveals that Aph1 adopts a histidine-aspartate (HD)-domain fold characteristic of phosphohydrolase metalloenzymes and its activity mitigates the growth inhibitory effects of (p)ppApp-synthesizing toxins. Using an informatic approach, we identify over 2,000 putative (p)ppApp hydrolases that are widely distributed across bacterial phyla and found in diverse genomic contexts, and we demonstrate that 12 representative members hydrolyze ppApp. In addition, our in silico analyses reveal a unique molecular signature that is specific to (p)ppApp hydrolases, and we show that mutation of two residues within this signature broadens the specificity of Aph1 to promiscuously hydrolyze (p)ppGpp invitro. Overall, our findings indicate that like (p)ppGpp hydrolases, (p)ppApp hydrolases are widespread in bacteria and may play important and underappreciated role(s) in bacterial physiology.

Mechanism of regulation and neutralization of tRNA-phosphorylating small RelA-SpoT homologue toxins

Mechanism of regulation and neutralization of tRNA-phosphorylating small RelA-SpoT homologue toxins

New Chemotypes for the Inhibition of (p)ppGpp Synthesis in the Quest for New Antimicrobial Compounds.

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.

Regulation of ppGpp Synthesis and Its Impact on Chloroplast Biogenesis during Early Leaf Development in Rice.

Guanosine tetraphosphate (ppGpp) is known as an alarmone that mediates bacterial stress responses. In plants, ppGpp is synthesized in chloroplasts from GTP and ATP and functions as a regulator of chloroplast gene expression to affect photosynthesis and plant growth. This observation indicates that ppGpp metabolism is closely related to chloroplast function, but the regulation of ppGpp and its role in chloroplast differentiation are not well understood. In rice, ppGpp directly inhibits plastidial guanylate kinase (GKpm), a key enzyme in GTP biosynthesis. GKpm is highly expressed during early leaf development in rice, and the GKpm-deficient mutant, virescent-2 (v2), develops chloroplast-deficient chlorotic leaves under low-temperature conditions. To examine the relationship between GTP synthesis and ppGpp homeostasis, we generated transgenic rice plants over-expressing RSH3, a protein known to act as a ppGpp synthase. When RSH3 was overexpressed in v2, the leaf chlorosis was more severe. Although the RSH3 overexpression in the wild type caused no visible effects, pulse amplitude modulation fluorometer measurements indicated that photosynthetic rates were reduced in this line. This finding implies that the regulation of ppGpp synthesis in rice is involved in the maintenance of the GTP pool required to regulate plastid gene expression during early chloroplast biogenesis. We further investigated changes in the expressions of RelA/SpoT Homolog (RSH) genes encoding ppGpp synthases and hydrolases during the same period. Comparing the expression of these genes with the cellular ppGpp content suggests that the basal ppGpp level is determined by the antagonistic action of multiple RSH enzymatic activities during early leaf development in rice.

Nonhydrolysable Analogues of (p)ppGpp and (p)ppApp Alarmone Nucleotides as Novel Molecular Tools

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.

Two P or Not Two P: Understanding Regulation by the Bacterial Second Messengers (p)ppGpp.

Under stressful growth conditions and nutrient starvation, bacteria adapt by synthesizing signaling molecules that profoundly reprogram cellular physiology. At the onset of this process, called the stringent response, members of the RelA/SpoT homolog (RSH) protein superfamily are activated by specific stress stimuli to produce several hyperphosphorylated forms of guanine nucleotides, commonly referred to as (p)ppGpp. Some bifunctional RSH enzymes also harbor domains that allow for degradation of (p)ppGpp by hydrolysis. (p)ppGpp synthesis or hydrolysis may further be executed by single-domain alarmone synthetases or hydrolases, respectively. The downstream effects of (p)ppGpp rely mainly on direct interaction with specific intracellular effectors, which are widely used throughout most cellular processes. The growing number of identified (p)ppGpp targets allows us to deduce both common features of and differences between gram-negative and gram-positive bacteria. In this review, we give an overview of (p)ppGpp metabolism with a focus on the functional and structural aspects of the enzymes involved and discuss recent findings on alarmone-regulated cellular effectors.

RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

Emerging and divergent roles of pyrophosphorylated nucleotides in bacterial physiology and pathogenesis.

Bacteria inhabit diverse environmental niches and consequently must modulate their metabolism to adapt to stress. The nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) (collectively referred to as (p)ppGpp) are essential for survival during nutrient starvation. (p)ppGpp is synthesized by the RelA-SpoT homologue (RSH) protein family and coordinates the control of cellular metabolism through its combined effect on over 50 proteins. While the role of (p)ppGpp has largely been associated with nutrient limitation, recent studies have shown that (p)ppGpp and related nucleotides have a previously underappreciated effect on different aspects of bacterial physiology, such as maintaining cellular homeostasis and regulating bacterial interactions with a host, other bacteria, or phages. (p)ppGpp produced by pathogenic bacteria facilitates the evasion of host defenses such as reactive nitrogen intermediates, acidic pH, and the complement system. Additionally, (p)ppGpp and pyrophosphorylated derivatives of canonical adenosine nucleotides called (p)ppApp are emerging as effectors of bacterial toxin proteins. Here, we review the RSH protein family with a focus on its unconventional roles during host infection and bacterial competition.

Homology Model of a Catalytically Competent Bifunctional Rel Protein.

Bacteria have developed different bet hedging strategies to survive hostile environments and stressful conditions with persistency being maybe the most elegant yet still poorly understood one. Persisters’ temporary tolerance to antibiotic treatment hints at their role not only in chronic and recurrent infections but also in the insurgence of resistant strains. Therefore, hampering persisters formation might represent an innovative strategy in the quest for new effective antimicrobial compounds. Among the molecular mechanisms postulated for the persister phenotypic switch, we decided to focus our attention on the stringent response and, in particular, on the upstream triggering step that is the accumulation of guanosine tetra- and pentaphosphate, collectivity called (p)ppGpp. Intracellular levels of (p)ppGpp are regulated by a superfamily of enzymes called RSH (RelA/SpoT homologue) that are able to promote its synthesis via pyrophosphate transfer from an ATP molecule to the 3’ position of either GDP or GTP. These enzymes are classified based on the structural domain(s) present (only synthetase, only hydrolase, or both). Here we present our work on RelSeq (from S. equisimilis), still the only bifunctional Rel protein for which a GDP-bound “synthetase-ON” structure is available. Analysis of the synthetase site, occupied only by GDP, revealed a partially active state, where the supposed ATP binding region is not conformationally apt to accommodate it. In order to achieve a protein model that gets closer to a fully active state, we generated a chimera structure of RelSeq by homology modeling, starting from the crystal structure of the catalytically competent state of RelP, a smaller, single-domain, Rel protein from S. aureus. Molecular dynamics simulations allowed verifying the stability of the generated chimera model. Virtual screening and ligand design studies are underway.

Dual role of a (p)ppGpp- and (p)ppApp-degrading enzyme in biofilm formation and interbacterial antagonism.

The guanosine nucleotide-based second messengers ppGpp and pppGpp (collectively: (p)ppGpp) enable adaptation of microorganisms to environmental changes and stress conditions. In contrast, the closely related adenosine nucleotides (p)ppApp are involved in type VI secretion system (T6SS)-mediated killing during bacterial competition. Long RelA-SpoT Homolog (RSH) enzymes regulate synthesis and degradation of (p)ppGpp (and potentially also (p)ppApp) through their synthetase and hydrolase domains, respectively. Small alarmone hydrolases (SAH) that consist of only a hydrolase domain are found in a variety of bacterial species, including the opportunistic human pathogen Pseudomonas aeruginosa. Here, we present the structure and mechanism of P. aeruginosa SAH showing that the enzyme promiscuously hydrolyses (p)ppGpp and (p)ppApp in a strictly manganese-dependent manner. While being dispensable for P. aeruginosa growth or swimming, swarming, and twitching motilities, its enzymatic activity is required for biofilm formation. Moreover, (p)ppApp-degradation by SAH provides protection against the T6SS (p)ppApp synthetase effector Tas1, suggesting that SAH enzymes can also serve as defense proteins during interbacterial competition.

Plastidial (p)ppGpp Synthesis by the Ca2+-Dependent RelA-SpoT Homolog Regulates the Adaptation of Chloroplast Gene Expression to Darkness in Arabidopsis.

In bacteria, the hyper-phosphorylated nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), functions as a secondary messenger under stringent conditions. ppGpp levels are controlled by two distinct enzymes, namely RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3 and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2 and RSH3 were undertaken, which showed that the ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here, to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase in ppGpp was observed for 30 min in the wild type (WT) after the light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyses were performed with the rsh2-rsh3 double and rsh1-rsh2-rsh3 triple mutants and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase in ppGpp in the WT and rsh2 rsh3 accompanied decrements in the mRNA levels of some plastidial genes transcribed by the plastid-encoded plastid RNA polymerase. These results indicate that the transient increase in ppGpp at night is due to CRSH-dependent ppGpp synthesis and that the ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2 and RSH3 to accustom plastidial gene expression to darkness.

Pleiotropic Effects of Bacterial Small Alarmone Synthetases: Underscoring the Dual-Domain Small Alarmone Synthetases in Mycobacterium smegmatis.

The nucleotide alarmone (p)ppGpp, signaling the stringent response, is known for more than 5 decades. The cellular turnover of the alarmone is regulated by RelA/SpoT homolog (RSH) superfamily of enzymes. There are long RSHs (RelA, SpoT, and Rel) and short RSHs [small alarmone synthetases (SAS) and small alarmone hydrolases (SAH)]. Long RSHs are multidomain proteins with (p)ppGpp synthesis, hydrolysis, and regulatory functions. Short RSHs are single-domain proteins with a single (p)ppGpp synthesis/hydrolysis function with few exceptions having two domains. Mycobacterial RelZ is a dual-domain SAS with RNase HII and the (p)ppGpp synthetase activity. SAS is known to impact multiple cellular functions independently and in accordance with the long RSH. Few SAS in bacteria including RelZ synthesize pGpp, the third small alarmone, along with the conventional (p)ppGpp. SAS can act as an RNA-binding protein for the negative allosteric inhibition of (p)ppGpp synthesis. Here, we initially recap the important features and molecular functions of different SAS that are previously characterized to understand the obligation for the “alarmone pool” produced by the long and short RSHs. Then, we focus on the RelZ, especially the combined functions of RNase HII and (p)ppGpp synthesis from a single polypeptide to connect with the recent findings of SAS as an RNA-binding protein. Finally, we conclude with the possibilities of using single-stranded RNA (ssRNA) as an additional therapeutic strategy to combat the persistent infections by inhibiting the redundant (p)ppGpp synthetases.

Structural Basis for Regulation of the Opposing (p)ppGpp Synthetase and Hydrolase within the Stringent Response Orchestrator Rel

The stringent response enables metabolic adaptation of bacteria under stress conditions and is governed by RelA/SpoT Homolog (RSH)-type enzymes. Long RSH-type enzymes encompass an N-terminal domain (NTD) harboring the second messenger nucleotide (p)ppGpp hydrolase and synthetase activity and a stress-perceiving and regulatory C-terminal domain (CTD). CTD-mediated binding of Rel to stalled ribosomes boosts (p)ppGpp synthesis. However, how the opposing activities of the NTD are controlled in the absence of stress was poorly understood. Here, we demonstrate on the RSH-type protein Rel that the critical regulative elements reside within the TGS (ThrRS, GTPase, and SpoT) subdomain of the CTD, which associates to and represses the synthetase to concomitantly allow for activation of the hydrolase. Furthermore, we show that Rel forms homodimers, which appear to control the interaction with deacylated-tRNA, but not the enzymatic activity of Rel. Collectively, our study provides a detailed molecular view into the mechanism of stringent response repression in the absence of stress.