Acetate is a beneficial nutrient for E. coli at low glycolytic flux.

Acetate, a major by-product of glycolytic metabolism in Escherichia coli and many other microorganisms, has long been considered a toxic waste compound that inhibits microbial growth. This counterproductive auto-inhibition represents a major problem in biotechnology and has puzzled the scientific community for decades. Recent studies have however revealed that acetate is also a co-substrate of glycolytic nutrients and a global regulator of E. coli metabolism and physiology. Here, we used a systems biology strategy to investigate the mutual regulation of glycolytic and acetate metabolism in E. coli. Computational and experimental analyses demonstrate that decreasing the glycolytic flux enhances co-utilization of acetate with glucose. Acetate metabolism thus compensates for the reduction in glycolytic flux and eventually buffers carbon uptake so that acetate, rather than being toxic, actually enhances E. coli growth under these conditions. We validated this mechanism using three orthogonal strategies: chemical inhibition of glucose uptake, glycolytic mutant strains, and alternative substrates with a natively low glycolytic flux. In summary, acetate makes E. coli more robust to glycolytic perturbations and is a valuable nutrient, with a beneficial effect on microbial growth.

Chemical and Metabolic Controls on Dihydroxyacetone Metabolism Lead to Suboptimal Growth of Escherichia coli.

In this work, we shed light on the metabolism of dihydroxyacetone (DHA), a versatile, ubiquitous, and important intermediate for various chemicals in industry, by analyzing its metabolism at the system level in Escherichia coli Using constraint-based modeling, we show that the growth of E. coli on DHA is suboptimal and identify the potential causes. Nuclear magnetic resonance analysis shows that DHA is degraded nonenzymatically into substrates known to be unfavorable to high growth rates. Transcriptomic analysis reveals that DHA promotes genes involved in biofilm formation, which may reduce the bacterial growth rate. Functional analysis of the genes involved in DHA metabolism proves that under the aerobic conditions used in this study, DHA is mainly assimilated via the dihydroxyacetone kinase pathway. In addition, these results show that the alternative routes of DHA assimilation (i.e., the glycerol and fructose-6-phosphate aldolase pathways) are not fully activated under our conditions because of anaerobically mediated hierarchical control. These pathways are therefore certainly unable to sustain fluxes as high as the ones predicted in silico for optimal aerobic growth on DHA. Overexpressing some of the genes in these pathways releases these constraints and restores the predicted optimal growth on DHA.IMPORTANCE DHA is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species, from microorganisms to humans, and can be used by Escherichia coli as a growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli We show that under aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical, and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value-added chemicals.

The post-transcriptional regulatory system CSR controls the balance of metabolic pools in upper glycolysis of Escherichia coli.

Metabolic control in Escherichia coli is a complex process involving multilevel regulatory systems but the involvement of post-transcriptional regulation is uncertain. The post-transcriptional factor CsrA is stated as being the only regulator essential for the use of glycolytic substrates. A dozen enzymes in the central carbon metabolism (CCM) have been reported as potentially controlled by CsrA, but its impact on the CCM functioning has not been demonstrated. Here, a multiscale analysis was performed in a wild-type strain and its isogenic mutant attenuated for CsrA (including growth parameters, gene expression levels, metabolite pools, abundance of enzymes and fluxes). Data integration and regulation analysis showed a coordinated control of the expression of glycolytic enzymes. This also revealed the imbalance of metabolite pools in the csrA mutant upper glycolysis, before the phosphofructokinase PfkA step. This imbalance is associated with a glucose-phosphate stress. Restoring PfkA activity in the csrA mutant strain suppressed this stress and increased the mutant growth rate on glucose. Thus, the carbon storage regulator system is essential for the effective functioning of the upper glycolysis mainly through its control of PfkA. This work demonstrates the pivotal role of post-transcriptional regulation to shape the carbon metabolism.

Differential quantitative proteome analysis of Escherichia coli grown on acetate versus glucose.

Relative protein abundances of Escherichia coli MG1655 growing exponentially on minimal medium with acetate or glucose as the sole carbon source were investigated in a quantitative shotgun proteome analysis with TMT6-plex isobaric tags. Peptides were separated by high resolution high/low pH 2D-LC, using an optimized fraction pooling scheme followed by mass spectrometric analysis. Quantitative data were acquired for 2099 proteins covering 49% of the predicted E. coli proteins, showing system-wide effects of growth conditions. In total, 507 proteins showed a fold change of at least 1.5 and 205 proteins changed by more than twofold. Significant differences in abundance were observed for most of the proteins in the central carbon metabolism and in proteins relevant for amino acid and protein synthesis, processing of environmental information and scavenging of a variety of alternate carbon sources. Periplasmic-binding proteins were also more abundant on acetate, especially proteins involved in scavenging extracellular resources such as sugars. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD003863).

Acetate Exposure Determines the Diauxic Behavior of Escherichia coli during the Glucose-Acetate Transition.

UNLABELLED: Growth of Escherichia coli on glucose in batch culture is accompanied by the excretion of acetate, which is consumed by the cells when glucose is exhausted. This glucose-acetate transition is classically described as a diauxie (two successive growth stages). Here, we investigated the physiological and metabolic properties of cells after glucose exhaustion through the analysis of growth parameters and gene expression. We found that E. coli cells grown on glucose in batch culture produce acetate and consume it after glucose exhaustion but do not grow on acetate. Acetate is catabolized, but key anabolic genes--such as the genes encoding enzymes of the glyoxylate shunt--are not upregulated, hence preventing growth. Both the induction of the latter anabolic genes and growth were observed only after prolonged exposure to low concentrations of acetate and could be accelerated by high acetate concentrations. We postulate that such decoupling between acetate catabolism and acetate anabolism might be an advantage for the survival of E. coli in the ever-changing environment of the intestine. IMPORTANCE: The glucose-acetate transition is a valuable experimental model for comprehensive investigations of metabolic adaptation and a current paradigm for developing modeling approaches in systems microbiology. Yet, the work reported in our paper demonstrates that the metabolic behavior of Escherichia coli during the glucose-acetate transition is much more complex than what has been reported so far. A decoupling between acetate catabolism and acetate anabolism was observed after glucose exhaustion, which has not been reported previously. This phenomenon could represent a strategy for optimal utilization of carbon resources during colonization and persistence of E. coli in the gut and is also of significant interest for biotechnological applications.

Toxic effect and inability of L-homoserine to be a nitrogen source for growth of Escherichia coli resolved by a combination of in vivo evolution engineering and omics analyses.

L-homoserine is a pivotal intermediate in the carbon and nitrogen metabolism of E. coli. However, this non-canonical amino acid cannot be used as a nitrogen source for growth. Furthermore, growth of this bacterium in a synthetic media is potently inhibited by L-homoserine. To understand this dual effect, an adapted laboratory evolution (ALE) was applied, which allowed the isolation of a strain able to grow with L-homoserine as the nitrogen source and was, at the same time, desensitized to growth inhibition by this amino acid. Sequencing of this evolved strain identified only four genomic modifications, including a 49 bp truncation starting from the stop codon of thrL. This mutation resulted in a modified thrL locus carrying a thrL* allele encoding a polypeptide 9 amino acids longer than the thrL encoded leader peptide. Remarkably, the replacement of thrL with thrL* in the original strain MG1655 alleviated L-homoserine inhibition to the same extent as strain 4E, but did not allow growth with this amino acid as a nitrogen source. The loss of L-homoserine toxic effect could be explained by the rapid conversion of L-homoserine into threonine via the thrL*-dependent transcriptional activation of the threonine operon thrABC. On the other hand, the growth of E. coli on a mineral medium with L-homoserine required an activation of the threonine degradation pathway II and glycine cleavage system, resulting in the release of ammonium ions that were likely recaptured by NAD(P)-dependent glutamate dehydrogenase. To infer about the direct molecular targets of L-homoserine toxicity, a transcriptomic analysis of wild-type MG1655 in the presence of 10 mM L-homoserine was performed, which notably identified a potent repression of locomotion-motility-chemotaxis process and of branched-chain amino acids synthesis. Since the magnitude of these effects was lower in a ΔthrL mutant, concomitant with a twofold lower sensitivity of this mutant to L-homoserine, it could be argued that growth inhibition by L-homoserine is due to the repression of these biological processes. In addition, L-homoserine induced a strong upregulation of genes in the sulfate reductive assimilation pathway, including those encoding its transport. How this non-canonical amino acid triggers these transcriptomic changes is discussed.

Control and regulation of acetate overflow in Escherichia coli.

Overflow metabolism refers to the production of seemingly wasteful by-products by cells during growth on glucose even when oxygen is abundant. Two theories have been proposed to explain acetate overflow in Escherichia coli - global control of the central metabolism and local control of the acetate pathway - but neither accounts for all observations. Here, we develop a kinetic model of E. coli metabolism that quantitatively accounts for observed behaviours and successfully predicts the response of E. coli to new perturbations. We reconcile these theories and clarify the origin, control, and regulation of the acetate flux. We also find that, in turns, acetate regulates glucose metabolism by coordinating the expression of glycolytic and TCA genes. Acetate should not be considered a wasteful end-product since it is also a co-substrate and a global regulator of glucose metabolism in E. coli. This has broad implications for our understanding of overflow metabolism.

Exploring the Glucose Fluxotype of the E. coli y-ome Using High-Resolution Fluxomics.

We have developed a robust workflow to measure high-resolution fluxotypes (metabolic flux phenotypes) for large strain libraries under fully controlled growth conditions. This was achieved by optimizing and automating the whole high-throughput fluxomics process and integrating all relevant software tools. This workflow allowed us to obtain highly detailed maps of carbon fluxes in the central carbon metabolism in a fully automated manner. It was applied to investigate the glucose fluxotypes of 180 Escherichia coli strains deleted for y-genes. Since the products of these y-genes potentially play a role in a variety of metabolic processes, the experiments were designed to be agnostic as to their potential metabolic impact. The obtained data highlight the robustness of E. coli's central metabolism to y-gene deletion. For two y-genes, deletion resulted in significant changes in carbon and energy fluxes, demonstrating the involvement of the corresponding y-gene products in metabolic function or regulation. This work also introduces novel metrics to measure the actual scope and quality of high-throughput fluxomics investigations.

A tripartite carbohydrate-binding module to functionalize cellulose nanocrystals.

The development of protein and microorganism engineering have led to rising expectations of biotechnology in the design of emerging biomaterials, putatively of high interest to reduce our dependence on fossil carbon resources. In this way, cellulose, a renewable carbon based polysaccharide and derived products, displays unique properties used in many industrial applications. Although the functionalization of cellulose is common, it is however limited in terms of number and type of functions. In this work, a Carbohydrate-Binding Module (CBM) was used as a central core to provide a versatile strategy to bring a large diversity of functions to cellulose surfaces. CBM3a from Clostridium thermocellum, which has a high affinity for crystalline cellulose, was flanked through linkers with a streptavidin domain and an azide group introduced through a non-canonical amino acid. Each of these two extra domains was effectively produced and functionalized with a variety of biological and chemical molecules. Structural properties of the resulting tripartite chimeric protein were investigated using molecular modelling approaches, and its potential for the multi-functionalization of cellulose was confirmed experimentally. As a proof of concept, we show that cellulose can be labelled with a fluorescent version of the tripartite protein grafted to magnetic beads and captured using a magnet.

Genome-wide gene expression profiling and a forward genetic screen show that differential expression of the sodium ion transporter Ena21 contributes to the differential tolerance of Candida albicans and Candida dubliniensis to osmotic stress.

Candida albicans is more pathogenic than Candida dubliniensis. However, this disparity in virulence is surprising given the high level of sequence conservation and the wide range of phenotypic traits shared by these two species. Increased sensitivity to environmental stresses has been suggested to be a possible contributory factor to the lower virulence of C. dubliniensis. In this study, we investigated, in the first comparison of C. albicans and C. dubliniensis by transcriptional profiling, global gene expression in each species when grown under conditions in which the two species exhibit differential stress tolerance. The profiles revealed similar core responses to stresses in both species, but differences in the amplitude of the general transcriptional responses to thermal, salt and oxidative stress. Differences in the regulation of specific stress genes were observed between the two species. In particular, ENA21, encoding a sodium ion transporter, was strongly induced in C. albicans but not in C. dubliniensis. In addition, ENA21 was identified in a forward genetic screen for C. albicans genomic sequences that increase salt tolerance in C. dubliniensis. Introduction of a single copy of CaENA21 was subsequently shown to be sufficient to confer salt tolerance upon C. dubliniensis.

Pseudomonas aeruginosa secreted factors impair biofilm development in Candida albicans.

Signal-mediated interactions between the human opportunistic pathogens Pseudomonas aeruginosa and Candida albicans affect virulence traits in both organisms. Phenotypic studies revealed that bacterial supernatant from four P. aeruginosa strains strongly reduced the ability of C. albicans to form biofilms on silicone. This was largely a consequence of inhibition of biofilm maturation, a phenomenon also observed with supernatant prepared from non-clinical bacterial species. The effects of supernatant on biofilm formation were not mediated via interference with the yeast-hyphal morphological switch and occurred regardless of the level of homoserine lactone (HSL) produced, indicating that the effect is HSL-independent. A transcriptome analysis to dissect the effects of the P. aeruginosa supernatants on gene expression in the early stages of C. albicans biofilm formation identified 238 genes that exhibited reproducible changes in expression in response to all four supernatants. In particular, there was a strong increase in the expression of genes related to drug or toxin efflux and a decrease in expression of genes associated with adhesion and biofilm formation. Furthermore, expression of YWP1, which encodes a protein known to inhibit biofilm formation, was significantly increased. Biofilm formation is a key aspect of C. albicans infections, therefore the capacity of P. aeruginosa to antagonize this has clear biomedical implications.

Genomewide Stabilization of mRNA during a "Feast-to-Famine" Growth Transition in Escherichia coli.

Bacteria have to continuously adjust to nutrient fluctuations from favorable to less-favorable conditions and in response to carbon starvation. The glucose-acetate transition followed by carbon starvation is representative of such carbon fluctuations observed in Escherichia coli in many environments. Regulation of gene expression through fine-tuning of mRNA pools constitutes one of the regulation levels required for such a metabolic adaptation. It results from both mRNA transcription and degradation controls. However, the contribution of transcript stability regulation in gene expression is poorly characterized. Using combined transcriptome and mRNA decay analyses, we investigated (i) how transcript stability changes in E. coli during the glucose-acetate-starvation transition and (ii) if these changes contribute to gene expression changes. Our work highlights that transcript stability increases with carbon depletion. Most of the stabilization occurs at the glucose-acetate transition when glucose is exhausted, and then stabilized mRNAs remain stable during acetate consumption and carbon starvation. Meanwhile, expression of most genes is downregulated and we observed three times less gene expression upregulation. Using control analysis theory on 375 genes, we show that most of gene expression regulation is driven by changes in transcription. Although mRNA stabilization is not the controlling phenomenon, it contributes to the emphasis or attenuation of transcriptional regulation. Moreover, upregulation of 18 genes (33% of our studied upregulated set) is governed mainly by transcript stabilization. Because these genes are associated with responses to nutrient changes and stress, this underscores a potentially important role of posttranscriptional regulation in bacterial responses to nutrient starvation.IMPORTANCE The ability to rapidly respond to changing nutrients is crucial for E. coli to survive in many environments, including the gut. Reorganization of gene expression is the first step used by bacteria to adjust their metabolism accordingly. It involves fine-tuning of both transcription (transcriptional regulation) and mRNA stability (posttranscriptional regulation). While the forms of transcriptional regulation have been extensively studied, the role of mRNA stability during a metabolic switch is poorly understood. Investigating E. coli genomewide transcriptome and mRNA stability during metabolic transitions representative of the carbon source fluctuations in many environments, we have documented the role of mRNA stability in the response to nutrient changes. mRNAs are globally stabilized during carbon depletion. For a few genes, this leads directly to expression upregulation. As these genes are regulators of stress responses and metabolism, our work sheds new light on the likely importance of posttranscriptional regulations in response to environmental stress.

Availability of the Molecular Switch XylR Controls Phenotypic Heterogeneity and Lag Duration during Escherichia coli Adaptation from Glucose to Xylose.

The glucose-xylose metabolic transition is of growing interest as a model to explore cellular adaption since these molecules are the main substrates resulting from the deconstruction of lignocellulosic biomass. Here, we investigated the role of the XylR transcription factor in the length of the lag phases when the bacterium Escherichia coli needs to adapt from glucose- to xylose-based growth. First, a variety of lag times were observed when different strains of E. coli were switched from glucose to xylose. These lag times were shown to be controlled by XylR availability in the cells with no further effect on the growth rate on xylose. XylR titration provoked long lag times demonstrated to result from phenotypic heterogeneity during the switch from glucose to xylose, with a subpopulation unable to resume exponential growth, whereas the other subpopulation grew exponentially on xylose. A stochastic model was then constructed based on the assumption that XylR availability influences the probability of individual cells to switch to xylose growth. The model was used to understand how XylR behaves as a molecular switch determining the bistability set-up. This work shows that the length of lag phases in E. coli is controllable and reinforces the role of stochastic mechanism in cellular adaptation, paving the way for new strategies for the better use of sustainable carbon sources in bioeconomy.IMPORTANCE For decades, it was thought that the lags observed when microorganisms switch from one substrate to another are inherent to the time required to adapt the molecular machinery to the new substrate. Here, the lag duration was found to be the time necessary for a subpopulation of adapted cells to emerge and become the main population. By identifying the molecular mechanism controlling the subpopulation emergence, we were able to extend or reduce the duration of the lags. This work is of special importance since it demonstrates the unexpected complexity of monoclonal populations during growth on mixed substrates and provides novel mechanistic insights with regard to bacterial cellular adaptation.

A multifunctional, synthetic Gaussia princeps luciferase reporter for live imaging of Candida albicans infections.

Real-time monitoring of the spatial and temporal progression of infection/gene expression in animals will contribute greatly to our understanding of host-pathogen interactions while reducing the number of animals required to generate statistically significant data sets. Sensitive in vivo imaging technologies can detect low levels of light emitted from luciferase reporters in vivo, but the existing reporters are not optimal for fungal infections. Therefore, our aim was to develop a novel reporter system for imaging Candida albicans infections that overcomes the limitations of current luciferase reporters for this major fungal pathogen. This luciferase reporter was constructed by fusing a synthetic, codon-optimized version of the Gaussia princeps luciferase gene to C. albicans PGA59, which encodes a glycosylphosphatidylinositol-linked cell wall protein. Luciferase expressed from this PGA59-gLUC fusion (referred to as gLUC59) was localized at the C. albicans cell surface, allowing the detection of luciferase in intact cells. The analysis of fusions to strong (ACT1 and EFT3), oxidative stress-induced (TRX1, TRR1, and IPF9996), and morphogenesis-dependent (HWP1) promoters confirmed that gLUC59 is a convenient and sensitive reporter for studies of gene regulation in yeast or hyphal cells, as well as a flexible screening tool. Moreover, the ACT1-gLUC59 fusion represented a powerful tool for the imaging of disease progression in superficial and subcutaneous C. albicans infections. gLUC59 and related cell surface-exposed luciferase reporters might find wide applications in molecular biology, cell biology, pathobiology, and high-throughput screens.

Role of the Hog1 stress-activated protein kinase in the global transcriptional response to stress in the fungal pathogen Candida albicans.

The resistance of Candida albicans to many stresses is dependent on the stress-activated protein kinase (SAPK) Hog1. Hence we have explored the role of Hog1 in the regulation of transcriptional responses to stress. DNA microarrays were used to characterize the global transcriptional responses of HOG1 and hog1 cells to three stress conditions that activate the Hog1 SAPK: osmotic stress, oxidative stress, and heavy metal stress. This revealed both stress-specific transcriptional responses and a core transcriptional response to stress in C. albicans. The core transcriptional response was characterized by a subset of genes that responded in a stereotypical manner to all of the stresses analyzed. Inactivation of HOG1 significantly attenuated transcriptional responses to osmotic and heavy metal stresses, but not to oxidative stress, and this was reflected in the role of Hog1 in the regulation of C. albicans core stress genes. Instead, the Cap1 transcription factor plays a key role in the oxidative stress regulation of C. albicans core stress genes. Our data show that the SAPK network in C. albicans has diverged from corresponding networks in model yeasts and that the C. albicans SAPK pathway functions in parallel with other pathways to regulate the core transcriptional response to stress.

Impact of the unfolded protein response upon genome-wide expression patterns, and the role of Hac1 in the polarized growth, of Candida albicans.

The unfolded protein response (UPR) regulates the expression of genes involved in the protein secretory pathway and in endoplasmic reticulum (ER) stress in yeasts and filamentous fungi. We have characterized the global transcriptional response of Candida albicans to ER stresses (dithiothreitol and tunicamycin) and established the impact of the transcription factor Hac1 upon this response. Expression of C. albicans Hac1, which is the functional homologue of Saccharomyces cerevisiae Hac1p, is predicted to be translationally regulated via an atypical mRNA splicing event during ER stress. C. albicans genes involved in secretion, vesicle trafficking, stress responses and cell wall biogenesis are up-regulated in response to ER stress, and translation and ribosome biogenesis genes are down-regulated. Hac1 is not essential for C. albicans viability, but plays a major role in this stress-related transcriptional response and is required for resistance to ER stress. In addition, we show that Hac1 plays an important role in regulating the morphology of C. albicans and in the expression of genes encoding cell surface proteins during ER stress, factors that are important in virulence of this fungal pathogen.

Global roles of Ssn6 in Tup1- and Nrg1-dependent gene regulation in the fungal pathogen, Candida albicans.

In budding yeast, Tup1 and Ssn6/Cyc8 form a corepressor that regulates a large number of genes. This Tup1-Ssn6 corepressor appears to be conserved from yeast to man. In the pathogenic fungus Candida albicans, Tup1 regulates cellular morphogenesis, phenotypic switching, and metabolism, but the role of Ssn6 remains unclear. We show that there are clear differences in the morphological and invasive phenotypes of C. albicans ssn6 and tup1 mutants. Unlike Tup1, Ssn6 depletion promoted morphological events reminiscent of phenotypic switching rather than filamentous growth. Transcript profiling revealed minimal overlap between the Ssn6 and Tup1 regulons. Hypha-specific genes, which are repressed by Tup1 and Nrg1, were not derepressed in ssn6 cells under the conditions studied. In contrast, the phase specific gene WH11 was derepressed in ssn6 cells, but not in tup1 or nrg1 cells. Hence Ssn6 and Tup1 play distinct roles in C. albicans. Nevertheless, both Ssn6 and Tup1 were required for the Nrg1-mediated repression of an artificial NRE promoter, and lexA-Nrg1 mediated repression in the C. albicans one-hybrid system. These observations are explained in models that are generally consistent with the Tup1-Ssn6 paradigm in budding yeast.

Stress-induced gene expression in Candida albicans: absence of a general stress response.

We used transcriptional profiling to investigate the response of the fungal pathogen Candida albicans to temperature and osmotic and oxidative stresses under conditions that permitted >60% survival of the challenged cells. Each stress generated the transient induction of a specific set of genes including classic markers observed in the stress responses of other organisms. We noted that the classical hallmarks of the general stress response observed in Saccharomyces cerevisiae are absent from C. albicans; no C. albicans genes were significantly induced in a common response to the three stresses. This observation is supported by our inability to detect stress cross-protection in C. albicans. Similarly, in C. albicans there is essentially no induction of carbohydrate reserves like glycogen and trehalose in response to a mild stress, unlike the situation in S. cerevisiae. Thus C. albicans lacks the strong general stress response exhibited by S. cerevisiae.

Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway.

Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible process. However, dynamic 13C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E. coli and its environment. The Pta-AckA pathway was found to be central for both flux directions, while alternative routes (Acs or PoxB) play virtually no role in glucose consumption. Kinetic modelling of the Pta-AckA pathway predicted that its flux is thermodynamically controlled by the extracellular acetate concentration in vivo. Experimental validations confirmed that acetate production can be reduced and even reversed depending solely on its extracellular concentration. Consistently, the Pta-AckA pathway can rapidly switch from acetate production to consumption. Contrary to current knowledge, E. coli is thus able to co-consume glucose and acetate under glucose excess. These metabolic capabilities were confirmed on other glycolytic substrates which support the growth of E. coli in the gut. These findings highlight the dual role of the Pta-AckA pathway in acetate production and consumption during growth on glycolytic substrates, uncover a novel regulatory mechanism that controls its flux in vivo, and significantly expand the metabolic capabilities of E. coli.

The Csr System Regulates Escherichia coli Fitness by Controlling Glycogen Accumulation and Energy Levels.

In the bacterium Escherichia coli, the posttranscriptional regulatory system Csr was postulated to influence the transition from glycolysis to gluconeogenesis. Here, we explored the role of the Csr system in the glucose-acetate transition as a model of the glycolysis-to-gluconeogenesis switch. Mutations in the Csr system influence the reorganization of gene expression after glucose exhaustion and disturb the timing of acetate reconsumption after glucose exhaustion. Analysis of metabolite concentrations during the transition revealed that the Csr system has a major effect on the energy levels of the cells after glucose exhaustion. This influence was demonstrated to result directly from the effect of the Csr system on glycogen accumulation. Mutation in glycogen metabolism was also demonstrated to hinder metabolic adaptation after glucose exhaustion because of insufficient energy. This work explains how the Csr system influences E. coli fitness during the glycolysis-gluconeogenesis switch and demonstrates the role of glycogen in maintenance of the energy charge during metabolic adaptation.IMPORTANCE Glycogen is a polysaccharide and the main storage form of glucose from bacteria such as Escherichia coli to yeasts and mammals. Although its function as a sugar reserve in mammals is well documented, the role of glycogen in bacteria is not as clear. By studying the role of posttranscriptional regulation during metabolic adaptation, for the first time, we demonstrate the role of sugar reserve played by glycogen in E. coli Indeed, glycogen not only makes it possible to maintain sufficient energy during metabolic transitions but is also the key component in the capacity of cells to resume growth. Since the essential posttranscriptional regulatory system Csr is a major regulator of glycogen accumulation, this work also sheds light on the central role of posttranscriptional regulation in metabolic adaptation.