Academic literature on the topic '(p)ppGpp Metabolism'

ABSTRACTBacteria grow in constantly changing environments that can suddenly become completely depleted of essential nutrients. The stringent response, a rewiring of the cellular metabolism mediated by the alarmone (p)ppGpp, plays a crucial role in adjusting bacterial growth to the severity of the nutritional stress. The ability of (p)ppGpp to trigger a slowdown of cell growth or induce bacterial dormancy has been widely investigated. However, little is known about the role of (p)ppGpp in promoting growth recovery after severe growth inhibition. In this study, we performed a time-resolved analysis of (p)ppGpp metabolism in Escherichia coli as it recovered from a sudden slowdown in growth. The results show that E. coli recovers by itself from the growth disruption provoked by the addition of serine hydroxamate, the serine analogue that we used to induce the stringent response. Growth inhibition was accompanied by a severe disturbance of metabolic activity and, more surprisingly, a transient overflow of valine and alanine. Our data also show that ppGpp is crucial for growth recovery since in the absence of ppGpp, E. coli’s growth recovery was slower. In contrast, an increased concentration of pppGpp was found to have no significant effect on growth recovery. Interestingly, the observed decrease in intracellular ppGpp levels in the recovery phase correlated with bacterial growth, and the main effect involved in the return to the basal level was identified by flux calculation as growth dilution. This report thus significantly expands our knowledge of (p)ppGpp metabolism in E. coli physiology.IMPORTANCE The capacity of microbes to resist and overcome environmental insults, known as resilience, allows them to survive in changing environments but also to resist antibiotic and biocide treatments and immune system responses. Although the role of the stringent response in bacterial resilience to nutritional stresses has been well studied, little is known about its importance in the ability of the bacteria to not just resist but also recover from these disturbances. To address this important question, we investigated growth disruption resilience in the model bacterium Escherichia coli and its dependence on the stringent response alarmone (p)ppGpp by quantifying ppGpp and pppGpp levels as growth was disrupted and then recovered. Our findings may thus contribute to understanding how ppGpp improves E. coli’s resilience to nutritional stress and other environmental insults.

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.

In nearly all bacterial species examined so far, amino acid starvation triggers the rapid accumulation of the nucleotide second messenger (p)ppGpp, the effector of the stringent response. While for years the enzymes involved in (p)ppGpp metabolism and the significance of (p)ppGpp accumulation to stress survival were considered well defined, a recent surge of interest in the field has uncovered an unanticipated level of diversity in how bacteria metabolize and utilize (p)ppGpp to rapidly synchronize a variety of biological processes important for growth and stress survival. In addition to the classic activation of the stringent response, it has become evident that (p)ppGpp exerts differential effects on cell physiology in an incremental manner rather than simply acting as a biphasic switch that controls growth or stasis. Of particular interest is the intimate relationship of (p)ppGpp with persister cell formation and virulence, which has spurred the pursuit of (p)ppGpp inhibitors as a means to control recalcitrant infections. Here, we present an overview of the enzymes responsible for (p)ppGpp metabolism, elaborate on the intricacies that link basal production of (p)ppGpp to bacterial homeostasis, and discuss the implications of targeting (p)ppGpp synthesis as a means to disrupt long-term bacterial survival strategies.

ABSTRACTThe alarmone (p)ppGpp plays pivotal roles in basic bacterial stress responses by increasing tolerance of various nutritional limitations and chemical insults, including antibiotics. Despite intensive studies since (p)ppGpp was discovered over 4 decades ago, (p)ppGpp binding proteins have not been systematically identified inEscherichia coli. We applied DRaCALA (differentialradialcapillaryaction ofligandassay) to identify (p)ppGpp-protein interactions. We discovered 12 new (p)ppGpp targets inE. colithat, based on their physiological functions, could be classified into four major groups, involved in (i) purine nucleotide homeostasis (YgdH), (ii) ribosome biogenesis and translation (RsgA, Era, HflX, and LepA), (iii) maturation of dehydrogenases (HypB), and (iv) metabolism of (p)ppGpp (MutT, NudG, TrmE, NadR, PhoA, and UshA). We present a comprehensive and comparative biochemical and physiological characterization of these novel (p)ppGpp targets together with a comparative analysis of relevant, known (p)ppGpp binding proteins. Via this, primary targets of (p)ppGpp inE. coliare identified. The GTP salvage biosynthesis pathway and ribosome biogenesis and translation are confirmed as targets of (p)ppGpp that are highly conserved betweenE. coliandFirmicutes. In addition, an alternative (p)ppGpp degradative pathway, involving NudG and MutT, was uncovered. This report thus significantly expands the known cohort of (p)ppGpp targets inE. coli.IMPORTANCEAntibiotic resistance and tolerance exhibited by pathogenic bacteria have resulted in a global public health crisis. Remarkably, almost all bacterial pathogens require the alarmone (p)ppGpp to be virulent. Thus, (p)ppGpp not only induces tolerance of nutritional limitations and chemical insults, including antibiotics, but is also often required for induction of virulence genes. However, understanding of the molecular targets of (p)ppGpp and the mechanisms by which (p)ppGpp influences bacterial physiology is incomplete. In this study, a systematic approach was used to uncover novel targets of (p)ppGpp inE. coli, the best-studied model bacterium. Comprehensive comparative studies of the targets revealed conserved target pathways of (p)ppGpp in both Gram-positive and -negative bacteria and novel targets of (p)ppGpp, including an alternative degradative pathway of (p)ppGpp. Thus, our discoveries may help in understanding of how (p)ppGpp increases the stress resilience and multidrug tolerance not only of the model organismE. colibut also of the pathogenic organisms in which these targets are conserved.

The stringent response is a rapid response system that is ubiquitous in bacteria, allowing them to sense changes in the external environment and undergo extensive physiological transformations. However, the regulators (p)ppGpp and DksA have extensive and complex regulatory patterns. Our previous studies demonstrated that (p)ppGpp and DksA in Yersinia enterocolitica positively co-regulated motility, antibiotic resistance, and environmental tolerance but had opposite roles in biofilm formation. To reveal the cellular functions regulated by (p)ppGpp and DksA comprehensively, the gene expression profiles of wild-type, ΔrelA, ΔrelAΔspoT, and ΔdksAΔrelAΔspoT strains were compared using RNA-Seq. Results showed that (p)ppGpp and DksA repressed the expression of ribosomal synthesis genes and enhanced the expression of genes involved in intracellular energy and material metabolism, amino acid transport and synthesis, flagella formation, and the phosphate transfer system. Additionally, (p)ppGpp and DksA inhibited amino acid utilization (such as arginine and cystine) and chemotaxis in Y. enterocolitica. Overall, the results of this study unraveled the link between (p)ppGpp and DksA in the metabolic networks, amino acid utilization, and chemotaxis in Y. enterocolitica and enhanced the understanding of stringent responses in Enterobacteriaceae.

The (p)ppGpp-mediated stringent response is important for bacterial survival in nutrient limiting conditions. For maximal effect, (p)ppGpp interacts with the cofactor DksA, which stabilizes (p)ppGpp's interaction with RNA polymerase. We previously demonstrated that (p)ppGpp was required for the virulence ofHaemophilus ducreyiin humans. Here, we constructed anH. ducreyidksAmutant and showed it was also partially attenuated for pustule formation in human volunteers. To understand the roles of (p)ppGpp and DksA in gene regulation inH. ducreyi, we defined genes potentially altered by (p)ppGpp and DksA deficiency using transcriptome sequencing (RNA-seq). In bacteria collected at stationary phase, lack of (p)ppGpp and DksA altered expression of 28% and 17% ofH. ducreyiopen reading frames, respectively, including genes involved in transcription, translation, and metabolism. There was significant overlap in genes differentially expressed in the (p)ppGpp mutant relative to thedksAmutant. Loss of (p)ppGpp or DksA resulted in the dysregulation of several known virulence determinants. Deletion ofdksAdownregulatedlspBand rendered the organism less resistant to phagocytosis and increased its sensitivity to oxidative stress. Both mutants had reduced ability to attach to human foreskin fibroblasts; the defect correlated with reduced expression of the Flp adhesin proteins in the (p)ppGpp mutant but not in thedksAmutant, suggesting that DksA regulates the expression of an unknown cofactor(s) required for Flp-mediated adherence. We conclude that both (p)ppGpp and DksA serve as major regulators ofH. ducreyigene expression in stationary phase and have both overlapping and unique contributions to pathogenesis.

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.

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.

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.

Adaptation to a rapidly fluctuating environment is the key to the survival of an organism. Bacteria sense and respond to stress by an overall reprogramming of the cellular processes to shut down the energy-consuming processes and switch to pathways that ensure the survival under stress. One of the strategies utilized by bacteria is to mount ‘stringent response’ which is mediated by the second messengers (p)ppGpp. (p)ppGpp governs a multitude of phenotypes in Mycobacteria and for a long time the bifunctional Rel was believed to be its only (p)ppGpp synthetase. A serendipitous detection of (p)ppGpp in a Mycobacterium smegmatis strain devoid of Rel led to the discovery of a second (p)ppGpp synthetase thereby broadening the horizon of stringent response in Mycobacteria (Murdeshwar and Chatterji, 2012). This unique protein contained a RNaseH domain along with the (p)ppGpp synthesis domain suggesting a role distinct from that of Rel. Subsequent characterisation of the protein revealed that neither domain is active in isolation raising a question about the link between these activities. Due to the crucial role played by (p)ppGpp, it becomes essential to analyse the (p)ppGpp null phenotype. Several bacterial species like Bacillus subtilis have short alarm one synthetases in addition to Rel and they have been proposed to be activated under particular stress conditions underlining the need to delineate the role of these (p)ppGpp synthetases (Nanamiya et al., 2008). Our study proposes a role for MS_RHII-RSD in vivo and deals with the phenotypic characterisation of the Δrel Δms_rhII-rsd strain. Chapter 1 reviews the available literature in the field of stringent response and provides the rationale behind this study. The discovery of (p)ppGpp and the plethora of functions regulated by it is explained along with a description of the key players in the (p)ppGpp metabolism. The chapter stresses upon the need to investigate the significance of a second (p)ppGpp synthetase in Mycobacteria and the scope of the current study. Chapter 2 deals with the elucidation of the in vivo significance of MS_RHII-RSD in M. smegmatis and proposes a role for the protein in R-loop removal during stress which requires both RNaseH activity and (p)ppGpp synthesis. The in vitro R-loop hydrolysis assays along with evidence for R-loop removal in M. smegmatis have been discussed along with the strategy used for the generation of the Δms_rhII-rsd strain. Chapter 3 explores the interdependence between the RNaseH and (p)ppGpp domains in MS_RHII-RSD in an attempt to unravel the necessity of the RNase H activity in a (p)ppGpp synthetase. The generation of active-site mutants of RNaseH and RSD along with their functional and biophysical characterisation has been described in detail. Oligomerisation studies with MS_RHII-RSD revealed the importance of a hexameric form for the protein. Chapter 4 further elaborates upon the link between the RNaseH activity and the (p)ppGpp synthesis activity and reveals a possible regulation of (p)ppGpp synthesis activity by RNA. Furthermore, the differing substrate specificities between Rel and MS_RHII-RSD are discussed. A possibility of the presence of pGpp due to MS_RHII-RSD in Mycobacteria has been outlined. Chapter 5 describes the attempts at generating a (p)ppGpp-deficient strain of M. smegmatis and reveals the surprising presence of yet another (p)ppGpp synthetase. The generation and characterisation of the Δrel Δms_rhII-rsd strain was performed and the physiological role of MS_RHII-RSD in biofilm formation and antibiotic tolerance has been highlighted. Chapter 6 summarizes the results of the study and points out the future directions for the work. Appendix 1 gives a comprehensive list of strains and plasmids used in this study. Appendix 2 provides a list of growth differences in antibiotics between the wild type and knockout strains of M. smegmatis obtained by Phenotype microarray. Appendix 3 is a commentary on the Pup-proteasome regulation in Mycobacteria.

RNA polymerase (RNAP) is the key enzyme in transcription and it is a multi-subunit enzyme made up of alpha, beta, beta prime and omega subunit in the stoichiometry of α2ββω. Except for the smallest subunit ω (rpoZ), all the subunits are essential for the cell survival. No clear phenotype was observed for the ΔrpoZ strain of E. coli for many years. Recently we isolated several dominant negtaive mutants of ω. These ω mutants were found to be structured as compared to the native ω which is unstructured. Mutant RNAP with the structured ω was found to be defective at the initiation step of transcription. This study showed the structural importance of ω subunit. Also, ω is linked to stringent response and its role is associated with key players of the stringent response i.e. ppGpp and protein DksA. ppGpp and DksA have been extensively studied with respect to the role played by them in cell survival under the stress. DksA and ppGpp show a more pronounced effect in vivo as compared to that of in vitro. ω has been found to be involved in binding of sigma factors and ppGpp to RNAP and its role has been evaluated in the present study in a more detailed manner. Our studies revealed both the structural and functional role of ω. The functional role of ω in stress response and its role in the distribution of RNAP across the E. coli genome has been studied. The importance of the unstructured ω in maintaining the catalytic activity of RNAP has been analysed. Also, the importance of flexible ω in ppGpp and σ factors binding to RNAP has been deciphered. Chapter 1 gives a brief introduction about the functional modulation of RNA polymerase. Transcription modulators which interact with RNA polymerase to orchestrate the transcription of genes are discussed. Chapter 2 presents our findings on the functional and structural role of ω subunit in the interaction of ppGpp to RNAP and its physiological importance in E. coli. Chapter 3 documents the assembly of the wild type ω and its dominant negative variant, ω6 with reconstituted RNAP (core1: α2ββ′). Subsequently, the interaction of σ-factors with reconstituted RNAP (core2: α2ββ′ω; mutated core2: α2ββ′ω6) has been described. Chapter 4 provides a broader perspective of the role played by ω in transcriptional machinery by looking into the gene selection pattern of ω-less RNA polymerase. Growth phenotype of ω deleted strain with various carbon substrates and its tolerance to different environmental stress like osmotic stress, pH and antibiotic, using phenotype microarray has been examined. Chapter 5 summarises the work that has been documented in this thesis. Appendix- Chapter 6 describes the co-immunoprecipitation studies which were done to analyse the binding profile of RNA polymerase in ΔrpoZ and ΔdksA strains. Phenotypic microarray and promoter activity assay were done to analyse the correlation of these factors in vivo. Appendix- Chapter 7 describes the differential role played by ω subunit in Gram-positive and Gram-negative bacteria.