ppGpp Signaling Research Articles

Optimal resource allocation in micro-organisms under periodic nutrient fluctuations

Although microorganisms often live in dynamic environments, most studies, both experimental and theoretical, are carried out under static conditions. In this work, we investigate the issue of optimal resource allocation in bacteria growing in periodic environments. We consider a dynamic model describing the microbial metabolism under varying conditions, involving a control variable quantifying the protein precursors allocation. Our objective is to determine the optimal strategies maximizing the long-term growth of cells under a piecewise-constant periodic environment. Firstly, we perform a theoretical analysis of the resulting optimal control problem (OCP), based on the application the Pontryagin’s Maximum Principle (PMP). We determine that the structure of the optimal control must be bang–bang, with possibly some singular arcs corresponding to optimal equilibria of the system. If the control presents singular arcs, then these can only be reached and left through chattering arcs. We also use a direct optimization method, implemented in the BOCOP software, to solve the studied OCP. Our study reveals that the optimal solution over a large time horizon is related to the one over a single period of the varying environment with periodic constraints. Moreover, we observe that the maximal average growth rate attainable under periodic conditions can be higher than the one under a constant environment. We further extend our analysis to conduct a qualitative comparison between the predictions from our model and some recent biological experiments on E. coli. This analysis particularly highlights the mechanisms of action of the ppGpp signaling molecule, thus providing relevant explanations of the experimental observations. In conclusion, our study corroborates previous research indicating that this molecule plays a crucial role in the regulation of resource allocation of protein precursors in E. coli.

(p)ppGpp interacts with TrmE and regulates the PcoI/PcoR quorum-sensing system of Pseudomonas fluorescens 2P241

The efficient colonization of plant-beneficial Pseudomonas spp. is a prerequisite for their biocontrol capacity. Prior work revealed that the PcoI/PcoR quorum-sensing (QS) system plays a pivotal role in the root colonization of P. fluorescens 2P24. During the colonization, strain 2P24 has faced diverse impacts from plant-derived reactive oxygen species and other environmental stress. However, the molecular mechanism by which the PcoI/PcoR QS system is regulated under unfavored conditions remains unclear. Thus, in this study, the role of the (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT in the PcoI/PcoR QS system of P. fluorescens was investigated. Our data indicated that the deficiency of relA and spoT genes remarkably improved the expression of the pcoI gene, whereas the mutation of the spoT gene significantly repressed the expression of the pcoI gene. We further demonstrated that the regulation of the PcoI/PcoR QS system by (p)ppGpp was dependent on the function of the trmE gene, which encodes a tRNA modification GTPase. Furthermore, the mutation of relA, spoT, or both significantly influenced the motility, biofilm formation, oxidative stress, osmotic tolerance, and rhizosphere colonization. Collectively, our data indicated that the (p)ppGpp signaling pathway mediated by the relA gene and spoT gene was important to the function of the PcoI/PcoR QS system and had important implications for the understanding of the molecular mechanism of (p)ppGpp in epiphytic fitness via TrmE of P. fluorescens.

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.

New perspectives on the molecular mechanisms of stress signalling by the nucleotide guanosine tetraphosphate (ppGpp), an emerging regulator of photosynthesis in plants and algae.

The nucleotides guanosine tetraphosphate and guanosine pentaphosphate (together (p)ppGpp) are found in a wide range of prokaryotic and eukaryotic organisms where they are associated with stress signalling. In this review, we will discuss recent research highlighting the role of (p)ppGpp signalling as a conserved regulator of photosynthetic activity in the chloroplasts of plants and algae, and the latest discoveries that open up new perspectives on the emerging roles of (p)ppGpp in acclimation to environmental stress. We explore how rapid advances in the study of (p)ppGpp signalling in prokaryotes are now revealing large gaps in our understanding of the molecular mechanisms of signalling by (p)ppGpp and related nucleotides in plants and algae. Filling in these gaps is likely to lead to the discovery of conserved as well as new plant- and algal-specific (p)ppGpp signalling mechanisms that will offer new insights into the taming of the chloroplast and the regulation of stress tolerance.

Guanosine tetraphosphate (ppGpp) accumulation inhibits chloroplast gene expression and promotes super grana formation in the moss Physcomitrium (Physcomitrella) patens.

The nucleotides guanosine tetraphosphate and pentaphosphate (or (p)ppGpp) are implicated in the regulation of chloroplast function in plants. (p)ppGpp signalling is best understood in the model vascular plant Arabidopsis thaliana in which it acts to regulate plastid gene expression to influence photosynthesis, plant development and immunity. However, little information is known about the conservation or diversity of (p)ppGpp signalling in other land plants. We studied the function of ppGpp in the moss Physcomitrium (previously Physcomitrella) patens using an inducible system for triggering ppGpp accumulation. We used this approach to investigate the effects of ppGpp on chloroplast function, photosynthesis and growth. We demonstrate that ppGpp accumulation causes a dramatic drop in photosynthetic capacity by inhibiting chloroplast gene expression. This was accompanied by the unexpected reorganisation of the thylakoid system into super grana. Surprisingly, these changes did not affect gametophore growth, suggesting that bryophytes and vascular plants may have different tolerances to defects in photosynthesis. Our findings point to the existence of both highly conserved and more specific targets of (p)ppGpp signalling in the land plants that may reflect different growth strategies.

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.

Diversity of mechanisms to control bacterial GTP homeostasis by the mutually exclusive binding of adenine and guanine nucleotides to IMP dehydrogenase.

IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate‐limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine‐tuned by post‐translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide‐controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.

Diverse molecular mechanisms of transcription regulation by the bacterial alarmone ppGpp.

Bacteria must rapidly detect and respond to stressful environmental conditions. Guanosine tetraphosphate (ppGpp) is a universal stress signal that, in most bacteria, drives the reprograming of transcription at a global level. However, recent studies have revealed that the molecular mechanisms utilized by ppGpp to rewire bacterial transcriptomes are unexpectedly diverse. In Proteobacteria, ppGpp regulates the expression of hundreds of genes by directly binding to two sites on RNA polymerase (RNAP), one in combination with the transcription factor, DksA. Conversely, ppGpp indirectly regulates transcription in Firmicutes by controlling GTP levels. In this case, ppGpp inhibits enzymes that salvage and synthesize GTP, which indirectly represses transcription from rRNA and other promoters that use GTP for initiation. More recently, two different mechanisms of transcription regulation involving the direct binding of transcription factors by ppGpp have been described. First, in Francisella tularensis, ppGpp was shown to modulate the formation of a tripartite transcription factor complex that binds RNAP and activates virulence genes. Second, in Firmicutes, ppGpp allosterically regulates the transcription repressor, PurR, which controls purine biosynthesis genes. The diversity in bacterial ppGpp signaling revealed in these studies suggests the likelihood that additional paradigms in ppGpp‐mediated transcription regulation await discovery.

Many birds with one stone: targeting the (p)ppGpp signaling pathway of bacteria to improve antimicrobial therapy.

Winning the war against resistant bacteria will require a change of paradigm in antibiotic discovery. A promising new direction is the targeting of non-essential pathways required for successful infection, such as quorum-sensing, virulence, and biofilm formation. Similarly important will be strategies to prevent or revert antibiotic resistance. Here, we argue that the (p)ppGpp signaling pathway should be a prime target of this effort, since its inactivation could potentially achieve all these goals simultaneously. The hyperphosphorylated guanine nucleotide (p)ppGpp is an ancient and universal second messenger of bacteria that has pleotropic effects on the physiology of these organisms. Initially described as a stress signal-an alarmone-it is now clear that (p)ppGpp plays a more general and fundamental role in bacterial adaptation, by integrating multiple internal and environmental signals to establish the optimal balance between growth and maintenance functions at any given time. Given such centrality, perturbation of the (p)ppGpp pathway will affect bacteria in multiple ways, from the ability to adjust metabolism to the available nutrients to the capacity to differentiate into developmental forms adapted to colonize different niches. Here, we provide an overview of the (p)ppGpp pathway, how it affects bacterial growth, survival and virulence, and its connection with antibiotic tolerance and persistence. We will emphasize the dysfunctions of cells living without (p)ppGpp and finalize by reviewing the efforts and prospects of developing inhibitors of this pathway, and how these could be employed to improve current antibiotic therapy.

PpGpp signaling plays a critical role in virulence of Acinetobacter baumannii

ABSTRACT Acinetobacter baumannii, a major nosocomial pathogen, survives in diverse hospital environments, and its multidrug resistance is a major concern. The ppGpp-dependent stringent response mediates the reprogramming of genes with diverse functions in several bacteria. We investigated whether ppGpp is involved in A. baumannii’s pathogenesis by examining biofilm formation, surface motility, adhesion, invasion, and mouse infection studies. Transcriptome analysis of early stationary phase cultures revealed 498 differentially-expressed genes (≥ 2-fold change) in a ppGpp-deficient A. baumannii strain; 220 and 278 genes were up and downregulated, respectively. Csu operon expression, important in pilus biosynthesis during early biofilm formation, was significantly reduced in the ppGpp-deficient strain. Our findings suggest that ppGpp signaling influences A. baumannii biofilm formation, surface motility, adherence, and virulence. We showed the association between ppGpp and pathogenicity in A. baumannii for the first time; ppGpp may be a novel antivirulence target in A. baumannii.

PpGpp influences protein protection, growth and photosynthesis in Phaeodactylum tricornutum.

Summary Chloroplasts retain elements of a bacterial stress response pathway that is mediated by the signalling nucleotides guanosine penta‐ and tetraphosphate ((p)ppGpp). In the model flowering plant Arabidopsis, ppGpp acts as a potent regulator of plastid gene expression and influences photosynthesis, plant growth and development. However, little is known about ppGpp metabolism or its evolution in other photosynthetic eukaryotes.Here, we studied the function of ppGpp in the diatom Phaeodactylum tricornutum using transgenic lines containing an inducible system for ppGpp accumulation. We used these lines to investigate the effects of ppGpp on growth, photosynthesis, lipid metabolism and protein expression.We demonstrate that ppGpp accumulation reduces photosynthetic capacity and promotes a quiescent‐like state with reduced proliferation and ageing. Strikingly, using nontargeted proteomics, we discovered that ppGpp accumulation also leads to the coordinated upregulation of a protein protection response in multiple cellular compartments.Our findings highlight the importance of ppGpp as a fundamental regulator of chloroplast function across different domains of life, and lead to new questions about the molecular mechanisms and roles of (p)ppGpp signalling in photosynthetic eukaryotes.

Survival of the Fittest: The Relationship of (p)ppGpp With Bacterial Virulence.

The signaling nucleotide (p)ppGpp has been the subject of intense research in the past two decades. Initially discovered as the effector molecule of the stringent response, a bacterial stress response that reprograms cell physiology during amino acid starvation, follow-up studies indicated that many effects of (p)ppGpp on cell physiology occur at levels that are lower than those needed to fully activate the stringent response, and that the repertoire of enzymes involved in (p)ppGpp metabolism is more diverse than initially thought. Of particular interest, (p)ppGpp regulation has been consistently linked to bacterial persistence and virulence, such that the scientific pursuit to discover molecules that interfere with (p)ppGpp signaling as a way to develop new antimicrobials has grown substantially in recent years. Here, we highlight contemporary studies that have further supported the intimate relationship of (p)ppGpp with bacterial virulence and studies that provided new insights into the different mechanisms by which (p)ppGpp modulates bacterial virulence.

Sublethal Paraquat Confers Multidrug Tolerance in Pseudomonas aeruginosa by Inducing Superoxide Dismutase Activity and Lowering Envelope Permeability.

Stressors and environmental cues shape the physiological state of bacteria, and thus how they subsequently respond to antibiotic toxicity. To understand how superoxide stress can modulate survival to bactericidal antibiotics, we examined the effect of intracellular superoxide generators, paraquat and menadione, on stationary-phase antibiotic tolerance of the opportunistic pathogen, Pseudomonas aeruginosa. We tested how pre-challenge with sublethal paraquat and menadione alters the tolerance to ofloxacin and meropenem in wild-type P. aeruginosa and mutants lacking superoxide dismutase (SOD) activity (sodAB), the paraquat responsive regulator soxR, (p)ppGpp signaling (relA spoT mutant), or the alternative sigma factor rpoS. We confirmed that loss of SOD activity impairs ofloxacin and meropenem tolerance in stationary phase cells, and found that sublethal superoxide generators induce drug tolerance by stimulating SOD activity. This response is rapid, requires de novo protein synthesis, and is RpoS-dependent but does not require (p)ppGpp signaling nor SoxR. We further showed that pre-challenge with sublethal paraquat induces a SOD-dependent reduction in cell-envelope permeability and ofloxacin penetration. Our results highlight a novel mechanism of hormetic protection by superoxide generators, which may have important implications for stress-induced antibiotic tolerance in P. aeruginosa cells.

Synthesis of ppGpp impacts type IX secretion and biofilm matrix formation in Porphyromonas gingivalis

In order to persist, bacteria need to adjust their physiological state in response to external and internal cues. External stimuli are often referred to as stressors. The stringent response, mediated by the alarmone (p)ppGpp, is central to the stress response in many bacteria; yet, there is limited knowledge regarding the role of (p)ppGpp signaling in bacteria belonging to the phylum Bacteroidetes. Like its counterparts in the gut (e.g., Bacteroides thetaiotaomicron and Bacteroides fragilis), Porphyromonas gingivalis persists in close association with its human host. Given the potential for numerous perturbations in the oral cavity, and the fact that P. gingivalis can enter and replicate within host cells, we hypothesized that (p)ppGpp is a key signaling molecule for stress adaptation and persistence. Here, we show that accumulation of ppGpp in P. gingivalis is governed by two homologous enzymes, designated Rel, and RshB, and that ppGpp signaling affects growth rate, survival, biofilm formation, production of outer membrane vesicles, and expression of genes encoding type IX secretion structural and cargo proteins. Overall, our findings provide a potential mechanism by which biofilm formation and virulence of P. gingivalis are integrated via ppGpp signaling, a regulatory mechanism central to bacterial survival in dynamic environments.

Stress responses linked to antimicrobial resistance in Acinetobacter species

Since the last 20years, bacteria of the genus Acinetobacter have been the leading cause of hospital-acquired infections. In addition to the ability of Acinetobacter species to acquire rapid antibiotic resistance, limited knowledge on the mechanisms of multidrug resistance to antibiotics limits the treatment options for such infections. Here, we present a review of cellular processes, including oxidative stress defense, energy metabolism, ppGpp signaling, toxin-antitoxin system, and quorum sensing network in Acinetobacter species and their roles in antimicrobial resistance. Although inhibition of stress responses is an attractive approach to the development of effective antimicrobial therapeutic agents, it is crucial to understand the mechanisms that cause antibiotic resistance in Acinetobacter species, as they are not as well studied as those in other pathogenic bacteria. RelA/SpoT has been shown to be involved in ppGpp synthesis in all 50 genomes of 35 Acinetobacter species. However, toxin-antitoxin (TA) systems are present in less than 30% of the 50 genomes (28/30% of SplT/A; 14/14% of HigB/A; 4/6% of HicA/B), except the RelE/B system (30/78%). These data suggested that ppGpp signaling is conserved in Acinetobacter species, but TA systems are not. This review describes our current knowledge on stress responses with respect to antibiotic resistance or tolerance in pathogenic and non-pathogenic Acinetobacter species.

RSH enzyme diversity for (p)ppGpp metabolism in Phaeodactylum tricornutum and other diatoms

The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.

(p)ppGpp: the magic governor of bacterial growth economy.

A fundamental question in microbiology is how bacterial cells manage to coordinate gene expression with cell growth during adapting to various environmental conditions. Although the cellular responses to changing environments have been extensively studied using transcriptomic and proteomic approaches, it remains poorly understood regarding the molecular strategy enabling bacteria to manipulate the global gene expression patterns. The alarmone (p)ppGpp is a key secondary messenger involved in regulating various biochemical and physiological processes of bacterial cells. However, despite of the extensive studies of (p)ppGpp signaling in stringent response during the past 50years, the connection between (p)ppGpp and exponential growth remains poorly understood. Our recent work demonstrates that (p)ppGpp is strongly involved in regulating cell growth of Escherichia coli through balancing the cellular investment on metabolic proteins and ribosomes, highlighting itself as a magic governor of bacterial global resource allocation. In this mini-review, we briefly summarize some historical perspectives and current progress of the relation between (p)ppGpp and bacterial exponential growth. Two important future directions are also highlighted: the first direction is to elucidate the cellular signal that triggers (p)ppGpp accumulation during poor growth conditions; the second direction is to investigate the relation between (p)ppGpp and exponential growth for bacterial species other than E. coli.

Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa

Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in Pseudomonas aeruginosa We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase P. aeruginosa Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the ΔrelA spoT mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase P. aeruginosa and suggest that inhibition of SOD activity may potentiate current antibiotics.

Mechanisms of Bacterial Tolerance and Persistence in the Gastrointestinal and Respiratory Environments.

Pathogens that infect the gastrointestinal and respiratory tracts are subjected to intense pressure due to the environmental conditions of the surroundings. This pressure has led to the development of mechanisms of bacterial tolerance or persistence which enable microorganisms to survive in these locations. In this review, we analyze the general stress response (RpoS mediated), reactive oxygen species (ROS) tolerance, energy metabolism, drug efflux pumps, SOS response, quorum sensing (QS) bacterial communication, (p)ppGpp signaling, and toxin-antitoxin (TA) systems of pathogens, such as Escherichia coli, Salmonella spp., Vibrio spp., Helicobacter spp., Campylobacter jejuni, Enterococcus spp., Shigella spp., Yersinia spp., and Clostridium difficile, all of which inhabit the gastrointestinal tract. The following respiratory tract pathogens are also considered: Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cenocepacia, and Mycobacterium tuberculosis Knowledge of the molecular mechanisms regulating the bacterial tolerance and persistence phenotypes is essential in the fight against multiresistant pathogens, as it will enable the identification of new targets for developing innovative anti-infective treatments.

Green magic: regulation of the chloroplast stress response by (p)ppGpp in plants and algae.

The hyperphosphorylated nucleotides guanosine pentaphosphate and tetraphosphate [together referred to as (p)ppGpp, or 'magic spot'] orchestrate a signalling cascade in bacteria that controls growth under optimal conditions and in response to environmental stress. (p)ppGpp is also found in the chloroplasts of plants and algae where it has also been shown to accumulate in response to abiotic stress. Recent studies suggest that (p)ppGpp is a potent inhibitor of chloroplast gene expression in vivo, and is a significant regulator of chloroplast function that can influence both the growth and the development of plants. However, little is currently known about how (p)ppGpp is wired into eukaryotic signalling pathways, or how it may act to enhance fitness when plants or algae are exposed to environmental stress. This review discusses our current understanding of (p)ppGpp metabolism and its extent in plants and algae, and how (p)ppGpp signalling may be an important factor that is capable of influencing growth and stress acclimation in this major group of organisms.