ppGpp Research Articles

A Physiological Basis for Nonheritable Antibiotic Resistance.

Antibiotics constitute one of the cornerstones of modern medicine. However, individuals may succumb to a bacterial infection if a pathogen survives exposure to antibiotics. The ability of bacteria to survive bactericidal antibiotics results from genetic changes in the preexisting bacterial genome, from the acquisition of genes from other organisms, and from nonheritable phenomena that give rise to antibiotic tolerance. Nonheritable antibiotic tolerance can be exhibited by a large fraction of the bacterial population or by a small subpopulation referred to as persisters. Nonheritable resistance to antibiotics has been ascribed to the activity of toxins that are part of toxin-antitoxin modules, to the universal energy currency ATP, and to the signaling molecule guanosine (penta) tetraphosphate. However, these molecules are dispensable for nonheritable resistance to antibiotics in many organisms. By contrast, nutrient limitation, treatment with bacteriostatic antibiotics, or expression of genes that slow bacterial growth invariably promote nonheritable resistance. We posit that antibiotic persistence results from conditions promoting feedback inhibition among core cellular processes, resulting phenotypically in a slowdown or halt in bacterial growth.

Acclimation of bacterial cell state for high-throughput enzyme engineering using a DmpR-dependent transcriptional activation system

Genetic circuit-based biosensors have emerged as an effective analytical tool in synthetic biology; these biosensors can be applied to high-throughput screening of new biocatalysts and metabolic pathways. Sigma 54 (σ54)-dependent transcription factor (TF) can be a valuable component of these biosensors owing to its intrinsic silent property compared to most of the housekeeping sigma 70 (σ70) TFs. Here, we show that these unique characteristics of σ54-dependent TFs can be used to control the host cell state to be more appropriate for high-throughput screening. The acclimation of cell state was achieved by using guanosine (penta)tetraphosphate ((p)ppGpp)-related genes (relA, spoT) and nutrient conditions, to link the σ54 TF-based reporter expression with the target enzyme activity. By controlling stringent programmed responses and optimizing assay conditions, catalytically improved tyrosine phenol lyase (TPL) enzymes were successfully obtained using a σ54-dependent DmpR as the TF component, demonstrating the practical feasibility of this biosensor. This combinatorial strategy of biosensors using σ factor-dependent TFs will allow for more effective high-throughput enzyme engineering with broad applicability.

The stringent response regulator (p) ppGpp mediates virulence gene expression and survival in Erwinia amylovora

BackgroundThe nucleotide second messengers, i.e., guanosine tetraphosphate and pentaphosphate [collectively referred to as (p) ppGpp], trigger the stringent response under nutrient starvation conditions and play an essential role in virulence in the fire blight pathogen Erwinia amylovora. Here, we present transcriptomic analyses to uncover the overall effect of (p) ppGpp-mediated stringent response in E. amylovora in the hrp-inducing minimal medium (HMM).ResultsIn this study, we investigated the transcriptomic changes of the (p) ppGpp0 mutant under the type III secretion system (T3SS)-inducing condition using RNA-seq. A total of 1314 differentially expressed genes (DEGs) was uncovered, representing more than one third (36.8%) of all genes in the E. amylovora genome. Compared to the wild-type, the (p) ppGpp0 mutant showed down-regulation of genes involved in peptide ATP-binding cassette (ABC) transporters and virulence-related processes, including type III secretion system (T3SS), biofilm, and motility. Interestingly, in contrast to previous reports, the (p) ppGpp0 mutant showed up-regulation of amino acid biosynthesis genes, suggesting that it might be due to that these amino acid biosynthesis genes are indirectly regulated by (p) ppGpp in E. amylovora or represent specific culturing condition used. Furthermore, the (p) ppGpp0 mutant exhibited up-regulation of genes involved in translation, SOS response, DNA replication, chromosome segregation, as well as biosynthesis of nucleotide, fatty acid and lipid.ConclusionThese findings suggested that in HMM environment, E. amylovora might use (p) ppGpp as a signal to activate virulence gene expression, and simultaneously mediate the balance between virulence and survival by negatively regulating DNA replication, translation, cell division, as well as biosynthesis of nucleotide, amino acid, fatty acid, and lipid. Therefore, (p) ppGpp could be a promising target for developing novel control measures to fight against this devastating disease of apples and pears.

A Modular RNA Domain That Confers Differential Ligand Specificity.

The modularity of protein domains is well-known, but the existence of independent domains that confer function in RNA is less established. Recently, a family of RNA aptamers termed ykkC was discovered; they bind at least four ligands of very different chemical composition, including guanidine, phosphoribosyl pyrophosphate (PRPP), and guanosine tetraphosphate (ppGpp) (graphical abstract). Structures of these aptamers revealed an architecture characterized by two coaxial helical stacks. The first helix appears to be a generic scaffold, while the second helix forms the most contacts to the ligands. To determine if these two regions within the aptamer are modular units for ligand recognition, we swapped the ligand-binding coaxial stacks of a guanidine aptamer and a PRPP aptamer. This operation, in combination with a single mutation in the scaffold domain, achieved full switching of ligand specificity. This finding suggests that the ligand-binding helix largely dictates the ligand specificity of ykkC RNAs and that the scaffold coaxial stack is generally compatible with various ykkC ligand-binding modules. This work presents an example of RNA domain modularity comparable to that of a ligand-binding protein, showcasing the versatility of RNA as an entity capable of molecular evolution through adaptation of existing motifs.

SpoT Induces Intracellular Salmonella Virulence Programs in the Phagosome.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as "ctd." Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA.IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.

The (p)ppGpp-mediated stringent response regulatory system globally inhibits primary metabolism and activates secondary metabolism in Pseudomonas protegens H78.

Pseudomonas protegens H78 produces multiple secondary metabolites, including antibiotics and iron carriers. The guanosine pentaphosphate or tetraphosphate ((p)ppGpp)-mediated stringent response is utilized by bacteria to survive during nutritional starvation and other stresses. RelA/SpoT homologues are responsible for the biosynthesis and degradation of the alarmone (p)ppGpp. Here, we investigated the global effect of relA/spoT dual deletion on the transcriptomic profiles, physiology, and metabolism of P. protegens H78 grown to mid- to late log phase. Transcriptomic profiling revealed that relA/spoT deletion globally upregulated the expression of genes involved in DNA replication, transcription, and translation; amino acid metabolism; carbohydrate and energy metabolism; ion transport and metabolism; and secretion systems. Bacterial growth was partially increased, while the cell survival rate was significantly reduced by relA/spoT deletion in H78. The utilization of some nutritional elements (C, P, S, and N) was downregulated due to relA/spoT deletion. In contrast, relA/spoT mutation globally inhibited the expression of secondary metabolic gene clusters (plt, phl, prn, ofa, fit, pch, pvd, and has). Correspondingly, antibiotic and iron carrier biosynthesis, iron utilization, and antibiotic resistance were significantly downregulated by the relA/spoT mutation. This work highlights that the (p)ppGpp-mediated stringent response regulatory system plays an important role in inhibiting primary metabolism and activating secondary metabolism in P. protegens.

RelZ-Mediated Stress Response in Mycobacterium smegmatis: pGpp Synthesis and Its Regulation.

Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate [collectively termed (p)ppGpp] to reprogram their cellular processes under stress. In mycobacteria, these alarmones govern a multitude of cellular phenotypes, such as cell division, biofilm formation, antibiotic tolerance, and long-term survival. Mycobacterium smegmatis possesses the bifunctional RelMsm as a (p)ppGpp synthetase and hydrolase. In addition, it contains a short alarmone synthetase MS_RHII-RSD (renamed RelZ), which contains an RNase H domain in tandem with the (p)ppGpp synthetase domain. The physiological functions of RelMsm have been well documented, but there is no clear picture about the cellular functions of RelZ in M. smegmatis RelZ has been implicated in R-loop induced stress response due to its unique domain architecture. In this study, we elucidate the differential substrate utilization pattern of RelZ compared to that of RelMsm We unveil the ability of RelZ to use GMP as a substrate to synthesize pGpp, thereby expanding the repertoire of second messengers known in mycobacteria. We have demonstrated that the pGpp synthesis activity of RelZ is negatively regulated by RNA and pppGpp. Furthermore, we investigated its role in biofilm formation and antibiotic tolerance. Our findings highlight the complex role played by the RelZ in cellular physiology of M. smegmatis and sheds light upon its functions distinct from those of RelMsmIMPORTANCE Bacteria utilize nucleotide messengers to survive the hostile environmental conditions and the onslaught of attacks within the host. The second messengers guanosine tetraphosphate and pentaphosphate [(p)ppGpp] have a profound impact on the long-term survival, biofilm formation, antibiotic tolerance, virulence, and pathogenesis of bacteria. Therefore, understanding the stress response mechanism regulated by (p)ppGpp is essential for discovering inhibitors of stress response and potential drug targets. Mycobacterium smegmatis contains two (p)ppGpp synthetases: RelMsm and RelZ. Our study unravels the novel regulatory mechanisms of RelZ activity and its role in mediating antibiotic tolerance. We further reveal its ability to synthesize novel second messenger pGpp, which may have regulatory roles in mycobacteria.

Physiology and Transcriptional Analysis of (p)ppGpp-Related Regulatory Effects in Corynebacterium glutamicum.

The alarmone species ppGpp and pppGpp are elementary components of bacterial physiology as they both coordinate the bacterial stress response and serve as fine-tuners of general metabolism during conditions of balanced growth. Since the regulation of (p)ppGpp metabolism and the effects of (p)ppGpp on cellular processes are highly complex and show massive differences between bacterial species, the underlying molecular mechanisms have so far only been insufficiently investigated for numerous microorganisms. In this study, (p)ppGpp physiology in the actinobacterial model organism Corynebacterium glutamicum was analyzed by phenotypic characterization and RNAseq-based transcriptome analysis. Total nutrient starvation was identified as the most effective method to induce alarmone production, whereas traditional induction methods such as the addition of serine hydroxamate (SHX) or mupirocin did not show a strong accumulation of (p)ppGpp. The predominant alarmone in C. glutamicum represents guanosine tetraphosphate, whose stress-associated production depends on the presence of the bifunctional RSH enzyme Rel. Interestingly, in addition to ppGpp, another substance yet not identified accumulated strongly under inducing conditions. A C. glutamicum triple mutant (Δrel,ΔrelS,ΔrelH) unable to produce alarmones [(p)ppGpp0 strain] exhibited unstable growth characteristics and interesting features such as an influence of illumination on its physiology, production of amino acids as well as differences in vitamin and carotenoid production. Differential transcriptome analysis using RNAseq provided numerous indications for the molecular basis of the observed phenotype. An evaluation of the (p)ppGpp-dependent transcriptional regulation under total nutrient starvation revealed a complex interplay with the involvement of ribosome-mediated transcriptional attenuation, the stress-responsive sigma factors σB and σH and transcription factors such as McbR, the master regulator of sulfur metabolism. In addition to the differential regulation of genes connected with various cell functions, the transcriptome analysis revealed conserved motifs within the promoter regions of (p)ppGpp-dependently and independently regulated genes. In particular, the representatives of translation-associated genes are both (p)ppGpp-dependent transcriptionally downregulated and show a highly conserved and so far unknown TTTTG motif in the −35 region, which is also present in other actinobacterial genera.

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.

Structure and activity of PPX/GppA homologs from Escherichiacoli and Helicobacterpylori.

Rapid adaptation to environmental changes is crucial for bacterial survival. Almost all bacteria possess a conserved stringent response system to prompt transcriptional and metabolic responses toward stress. The adaptive process relies on alarmones, guanosine pentaphosphate (pppGpp), and tetraphosphate (ppGpp), to regulate global gene expression. The ppGpp is more potent than pppGpp in the regulatory activity, and pppGpp phosphohydrolase (GppA) plays a key role in (p)ppGpp homeostasis. Sharing a similar domain structure, GppA is indistinguishable from exopolyphosphatase (PPX), which mediates the metabolism of cellular inorganic polyphosphate. Here, our phylogenetic analysis of PPX/GppA homologs in bacteria shows a wide distribution with several distinct subfamilies, and our structural and functional analysis of Escherichiacoli GppA and Helicobacterpylori PPX/GppA reveals unique properties of each homolog. These results explain how each homolog possesses its distinct functionality.

Identification of novel scaffolds targeting Mycobacterium tuberculosis.

Drug resistance in Mycobacterium tuberculosis is relentlessly progressing while only a handful of novel drug candidates are developed. Here we describe a GFP-based high-throughput screening of 386,496 diverse compounds to identify putative tuberculosis drug candidates. In an exploratory analysis of the model organism M. bovis BCG and M. smegmatis and the subsequent screening of the main library, we identified 6354 compounds with anti-mycobacterial activity. These hit compounds were predominantly selective for mycobacteria while dozens had activity in the low μM range. We tested toxicity against the human monocyte/macrophage cell line THP-1 and elaborated activity against M. tuberculosis growing in liquid broth, under unique conditions such as non-replicating persistence or inhibition of M. tuberculosis residing in macrophages. Finally, spontaneous compound-resistant M. tuberculosis mutants were selected and subsequently analyzed by whole genome sequencing. In addition to compounds targeting the well-described proteins Pks13 and MmpL3, we identified two novel scaffolds targeting the bifunctional guanosine pentaphosphate synthetase/ polyribonucleotide nucleotidyltransferase GpsI, or interacting with the aminopeptidase PepB, a probable pro-drug activator. KEY MESSAGES: A newly identified scaffold targets the bifunctional enzyme GpsI. The aminopeptidase PepB is interacting with a second novel scaffold. Phenotypic screenings regularly identify novel compounds targeting Pks13 and MmpL3.

Stringent response regulators (p)ppGpp and DksA positively regulate virulence and host adaptation of Xanthomonas citri.

SummaryThe bacterial stringent response is a response to nutrition deprivation and other stress conditions. In Gram‐negative bacteria, this process is mediated by the small signal molecules guanosine pentaphosphate pppGpp and guanosine tetraphosphate ppGpp (collectively referred to as (p)ppGpp), and the RNA polymerase‐binding transcription factor DksA. The (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT are responsible for cellular (p)ppGpp levels. Here, we investigated the roles of DksA and (p)ppGpp in the virulence traits of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker. ΔdksA and (p)ppGpp‐deficient ΔspoTΔrelA strains caused reduced virulence and compromised growth in host plants, indicating that DksA and (p)ppGpp are required for full virulence of Xcc. To characterize the effect of stringent response regulators on gene expression, RNA‐seq was conducted using ΔdksA and ΔspoTΔrelA mutant strains grown in hrp‐inducing XVM2 medium. Transcriptome analyses showed that DksA and (p)ppGpp repressed the expression of genes encoding tRNAs, ribosome proteins, iron acquisition and flagellum assembly, and enhanced the expression of genes for histidine metabolism, type 3 secretion system (T3SS), type 2 secretion system (T2SS) and TonB‐dependent transporters. Phenotypically, the ΔdksA and ΔspoTΔrelA strains displayed altered motility, enhanced siderophore production and were unable to cause the hypersensitive response on non‐host plants. In conclusion, stringent response regulators DksA and (p)ppGpp play an important role in virulence, nutrition uptake and host adaptation of Xcc.

Disruption of transcription-translation coordination in Escherichia coli leads to premature transcriptional termination.

Tight coordination between transcription and translation is crucial to maintaining the integrity of gene expression in bacteria, yet how bacteria manage to coordinate these two processes remains unclear. Possible direct physical coupling between the RNA polymerase and ribosome has been thoroughly investigated in recent years. Here, we quantitatively characterize the transcriptional kinetics of Escherichia coli under different growth conditions. Transcriptional and translational elongation remain coordinated under various nutrient conditions, as previously reported. However, transcriptional elongation was not affected under antibiotics that slowed down translational elongation. This result was also found by introducing nonsense mutation that completely dissociated transcription from translation. Our data thus provide direct evidence that translation is not required to maintain the speed of transcriptional elongation. In cases where transcription and translation are dissociated, our study provides quantitative characterization of the resulting process of premature transcriptional termination (PTT). PTT-mediated polarity caused by translation-targeting antibiotics substantially affected the coordinated expression of genes in several long operons, contributing to the key physiological effects of these antibiotics. Our results also suggest a model in which the coordination between transcriptional and translational elongation under normal growth conditions is implemented by guanosine tetraphosphate.

Cell-length heterogeneity: a population-level solution to growth/virulence trade-offs in the plant pathogen Dickeya dadantii.

Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen. Infected plants lose tissue integrity and functional immunity as a result, exposing the nutrient rich, decayed tissues to the environment. One challenge for the necrotrophs to successfully cause secondary infection (infection spread from an initially infected plant to the nearby uninfected plants) is to effectively utilize nutrients released from hosts towards building up a large population before other saprophytes come. In this study, we observed that the necrotrophic pathogen Dickeya dadantii exhibited heterogeneity in bacterial cell length in an isogenic population during infection of potato tuber. While some cells were regular rod-shape (<10μm), the rest elongated into filamentous cells (>10μm). Short cells tended to occur at the interface of healthy and diseased tissues, during the early stage of infection when active attacking and killing is occurring, while filamentous cells tended to form at a later stage of infection. Short cells expressed all necessary virulence factors and motility, whereas filamentous cells did not engage in virulence, were non-mobile and more sensitive to environmental stress. However, compared to the short cells, the filamentous cells displayed upregulated metabolic genes and increased growth, which may benefit the pathogens to build up a large population necessary for the secondary infection. The segregation of the two subpopulations was dependent on differential production of the alarmone guanosine tetraphosphate (ppGpp). When exposed to fresh tuber tissues or freestanding water, filamentous cells quickly transformed to short virulent cells. The pathogen adaptation of cell length heterogeneity identified in this study presents a model for how some necrotrophs balance virulence and vegetative growth to maximize fitness during infection.

Slow growth determines nonheritable antibiotic resistance in Salmonella enterica.

Bacteria can withstand killing by bactericidal antibiotics through phenotypic changes mediated by their preexisting genetic repertoire. These changes can be exhibited transiently by a large fraction of the bacterial population, giving rise to tolerance, or displayed by a small subpopulation, giving rise to persistence. Apart from undermining the use of antibiotics, tolerant and persistent bacteria foster the emergence of antibiotic-resistant mutants. Persister formation has been attributed to alterations in the abundance of particular proteins, metabolites, and signaling molecules, including toxin-antitoxin modules, adenosine triphosphate, and guanosine (penta) tetraphosphate, respectively. Here, we report that persistent bacteria form as a result of slow growth alone, despite opposite changes in the abundance of such proteins, metabolites, and signaling molecules. Our findings argue that transitory disturbances to core activities, which are often linked to cell growth, promote a persister state regardless of the underlying physiological process responsible for the change in growth.

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC.

The stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation in Escherichia coli remains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factor dnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication, oriC Active transcription normally introduces negative supercoils into oriC to help promote replication initiation, so the accumulation of ppGpp reduces initiation potential at oriC by reducing transcription. We find that maintaining transcription near oriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein gene seqA also relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils into oriCIMPORTANCE To survive bouts of starvation, cells must inhibit DNA replication. In bacteria, starvation triggers production of a signaling molecule called ppGpp (guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While ppGpp has been known to block replication initiation in Escherichia coli for decades, the mechanism responsible was unknown. Early work suggested that ppGpp drives a decrease in levels of the replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that ppGpp leads to a change in DNA topology that prevents initiation. ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.

Abstract 2667: Genetic removal of metazoan SpoT homolog I (MESH1) inhibits proliferation through the repression of HIPPO effector TAZ

Abstract The capacity of tumor cells to survive various stresses is an essential property that endows them invulnerability. One of the bacterial key adaptive strategies against nutrient deprivation is stringent response. In bacteria, nutrient deprivation leads to an elevation of a signaling alarmone, guanosine tetraphosphate ((p)ppGpp) to repress the protein synthesis and proliferation activity in order to maintain cell viability. The accumulation of (p)ppGpp is partially caused by the repression of its hydrolase, SpoT. However, this strategy still remains elusive in humans, but the human ortholog of SpoT-MESH1 has been discovered. MESH1 also has the hydrolase activity on (p)ppGpp in vitro, but human cells lack the required alarmone (p)ppGpp and its synthetase, which restricts the discovery of stringent response in humans. Unexpectedly, our recent data suggests that MESH1 depletion allows cell survival under cystine deprivation, an extremely oxidative stress, yet with a slow proliferation rate. These correspond to bacterial stringent response phenotypes. Moreover, our gene expression profiling shows that MESH1 silencing triggers a global transcriptional reprogramming with a similarity to bacterial transcriptional responses of stringent response. Altogether, these evidences inspire us to speculate on the existence of human stringent response with similar cell protective functions, albeit in the absence of (p)ppGpp. In this study, I aim to identify the molecular pathways and regulatory mechanisms that MESH1 adopts to explain the MESH1-related stringent response phenotypes in human cancer cell lines. One interesting phenomenon we observed from our genome-wide RNA screen is that MESH1 silencing significantly represses TAZ RNA level. TAZ serves as a key co-activator involved in the HIPPO pathway that has been well studied to regulate cell proliferation, cell stemness, and etc, which are perfectly relevant to the MESH1-related stringent response phenotypes. Therefore, I will specifically determine the functional roles of TAZ repression upon MESH1 removal in the expression of stringent response features by genetic tools. Furthermore, I will characterize the transcriptional control of TAZ and other gene signatures of human stringent response. We observed that MESH1 depletion significantly represses the acetyl-CoA:CoA ratio and further inhibits the histone acetylation activity that presumably turns off/on certain gene transcription. Based on these facts, I will determine the importance of histone modification on the transcriptional reprogramming upon MESH1 silencing. Successful completion of this study will equip us with more thorough understandings of stress response mechanisms in humans so that we can improve the treatment for stress-related diseases, such as cancer, by providing a novel therapeutic target, MESH1. Note: This abstract was not presented at the meeting. Citation Format: Tianai Sun, Chien-Kuang Ding, Jen-Tsan A. Chi. Genetic removal of metazoan SpoT homolog I (MESH1) inhibits proliferation through the repression of HIPPO effector TAZ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2667.

Ppn2 endopolyphosphatase overexpressed in Saccharomyces cerevisiae: Comparison with Ppn1, Ppx1, and Ddp1 polyphosphatases

Saccharomyces cerevisiae has high level of inorganic polyphosphate and a multicomponent system of its metabolism, including polyphosphatases Ppx1, Ppn1, Ddp1, and Ppn2.The aim of the study was to construct the yeast strain overexpressing Ppn2 and to compare the properties of Ppn2, Ppx1, Ppn1, and Ddp1 purified from overexpressing strains of S. cerevisiae. We overexpressed Ppn2 in S. cerevisiae under a strong constitutive promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase-encoding gene and suggested biochemical criteria for distinguishing among yeast polyphosphatases, which is important for their identification and understanding of their functions. Ppn2, Ppn1, and Ddp1 had endopolyphosphatase activities, whereas Ppx1 did not. Ppx1 and Ppn1 exhibited high and Ddp1 and Ppn2 low exopolyphosphatase activity: 240, 500, 0.05 and 0.1 U/mg protein, respectively. The enzymes had distinct patterns of exopolyphosphatase activities stimulation by divalent metal ions. Ppn2, Ppn1 and Ddp1 displayed endopolyphosphatase activity in the presence of 1 mM Mg2+. The endopolyphosphatase activities of Ppn2 and Ppn1 were induced by 0.01 mM of Co2+ or Zn2+, whereas that of Ddp1 required 0.1 mM of these cations. The endopolyphosphatase activity of Ppn1 was inhibited by 0.01 mg mL−1 of heparin, while endopolphosphatase activity of Ppn2 was weakly sensitive to 0.25 mg mL−1 of heparin. The Ppx1 and Ppn1 activity with guanosine tetraphosphate was nearly 80% of activity with long-chain polyphosphates. The Ppn1 hydrolyzed dATP, while Ppx1 did not. The differences in the mode of polyphosphate hydrolysis, substrate specificity, metal ion dependence and cell localization suggest distinct roles of these enzymes in yeast.

Intricate Crosstalk Between Lipopolysaccharide, Phospholipid and Fatty Acid Metabolism in Escherichia coli Modulates Proteolysis of LpxC.

Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In Escherichia coli, the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification.

The Stringent Response And Its Involvement In The Reactions Of Bacterial Cells To Stress

Abstract The stringent response is a form of bacterial response to adverse environmental conditions. Its effectors are guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp], which are synthetized by RelA, SpoT and their homologs (RSH). RelA, a (p)ppGpp synthase, is activated when there is a shortage of amino acids, whereas SpoT, which has the ability to synthetize and hydrolyze (p)ppGpp, responds to fatty acids, iron and carbon limits. Accumulation of (p)ppGpp causes an inhibition of translation, replication, a decrease in the transcription of many genes, e.g. rRNA, tRNA, encoding ribosomal proteins, and an increase in the transcription of genes whose proteins are important in bacterial stress response. The stringent response alarmones are crucial for bacterial resistance to oxidative stress and antibiotics. They also regulate the production of specific molecules, the so-called quorum sensing autoinducers, which help bacteria communicate the density of their own population, which enables them to adjust their metabolism to the prevailing conditions, to form a biofilm – a community of microorganisms attached to a certain surface, ensuring them appropriate conditions to survive in an unfavourable environment, and to colonize new niches. (p)ppGpp has a positive impact on biofilm formation not only via the regulation of quorum sensing, but also by stimulating the synthesis of potential elements of the biofilm. It also appears that the stringent response alarmones decrease the ability of Agrobacterium tumefaciens bacteria to transform plants and thus their potential to cause disease. (p)ppGpp enables the bacteria to perform swarming motility, a movement that increases their resistance to adverse environmental factors. 1. Introduction. 2. RelA, SpoT and RSH proteins – enzymes that metabolize the alarmones of the stringent response. 2.1. The regulation of transcription via stringent response alarmones in Gram-negative bacteria. 2.2. The regulation of transcription via (p)ppGpp in Gram-positive bacteria. 2.3. The influence of stringent response alarmones on translation and replication. 3. The role of the stringent response in the regulation of other physiological processes. 3.1. The role of the stringent response in the production of siderophores and antibiotics. 4. Bacterial cell resistance to stress and the stringent response. 4.1. The participation of the stringent response in quorum sensing regulation. 4.2. The regulation of exopolysacharide production and biofilm formation dependent on the stringent response. 4.3. The role of the stringent response in the regulation of bacterial swarming motility. 5. Summary