The long noncoding RNA glycoLINC assembles a lower glycolytic metabolon to promote glycolysis.

Non-covalent complexes of glycolytic enzymes, called metabolons, were postulated in the 1970s, but the concept has been controversial. Here we show that a c-Myc-responsive long noncoding RNA (lncRNA) that we call glycoLINC (gLINC) acts as a backbone for metabolon formation between all four glycolytic payoff phase enzymes (PGK1, PGAM1, ENO1, and PKM2) along with lactate dehydrogenase A (LDHA). The gLINC metabolon enhances glycolytic flux, increases ATP production, and enables cell survival under serine deprivation. Furthermore, gLINC overexpression in cancer cells promotes xenograft growth in mice fed a diet deprived of serine, suggesting that cancer cells employ gLINC during metabolic reprogramming. We propose that gLINC makes a functional contribution to cancer cell adaptation and provide the first example of a lncRNA-facilitated metabolon.

The tumor niche can reprogram long-lived protumorigenic neutrophils.

The heterogeneity and plasticity of neutrophils in tumor-host interactions and how tumor signals induce reprogramming of neutrophil subpopulations need further investigation. Ng et al. recently reported that a hypoxic-glycolytic niche in mouse tumors could reprogram mature and immature neutrophils into a long-lived and terminally-differentiated subset, which promoted angiogenesis and tumor growth.

Small noncoding RNA interactome capture reveals pervasive, carbon source-dependent tRNA engagement of yeast glycolytic enzymes.

Small noncoding RNAs fulfill key functions in cellular and organismal biology, typically working in concert with RNA-binding proteins (RBPs). While proteome-wide methodologies have enormously expanded the repertoire of known RBPs, these methods do not distinguish RBPs binding to small noncoding RNAs from the rest. To specifically identify this relevant subclass of RBPs, we developed small noncoding RNA interactome capture (snRIC2C) based on the differential RNA-binding capacity of silica matrices (2C). We define the S. cerevisiae proteome of nearly 300 proteins that specifically binds to RNAs smaller than 200 nt in length (snRBPs), identifying informative distinctions from the total RNA-binding proteome determined in parallel. Strikingly, the snRBPs include most glycolytic enzymes from yeast. With further methodological developments using silica matrices, 12 tRNAs were identified as specific binders of the glycolytic enzyme GAPDH. We show that tRNA engagement of GAPDH is carbon source-dependent and regulated by the RNA polymerase III repressor Maf1, suggesting a regulatory interaction between glycolysis and RNA polymerase III activity. We conclude that snRIC2C and other 2C-derived methods greatly facilitate the study of RBPs, revealing previously unrecognized interactions.

New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer's disease.

Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of ∼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-ß-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.

Cytoskeleton Architecture Regulates Glycolysis Coupling Cellular Metabolism to Mechanical Cues.

Energy-demanding processes, such as cell growth, migration, and differentiation, are tension modulated, begging the question whether metabolism and mechanical tension are tightly linked. A recent report by Park et al. shows that stiffness in the extracellular matrix (ECM) promotes reorganization of actin, resulting in enhanced glycolysis.

Targeting HOXA11-AS to mitigate prostate cancer via the glycolytic metabolism: In vitro and in vivo.

As oncogenes or oncogene suppressors, long-stranded non-coding RNAs are essential for the formation and progression of human tumours. However, the mechanisms behind the regulatory role of RNA HOXA11-AS in prostate cancer (PCa) are unclear. PCa is a common malignant tumour worldwide, and an increasing number of studies have focused on its metabolic profile. Studies have shown that the long non-coding RNA (lncRNA) HOXA11-AS is aberrantly expressed in many tumours. However, the role of HOXA11-AS in PCa is unclear. This work aimed to determine how HOXA11-AS regulated PCa in vitro and in vivo. We first explored the clinical role of HOXA11-AS in PCa using bioinformatics methods, including single sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), and least absolute shrinkage and selection operator (LASSO)-logistics systematically. In this study, PCa cell lines were selected to assess the PCa regulatory role of HOXA11-AS overexpression versus silencing in vitro, and tumour xenografts were performed in nude mice to assess tumour suppression by HOXA11-AS silencing in vivo. HOXA11-AS expression was significantly correlated with clinicopathological factors, epithelial-mesenchymal transition (EMT) and glycolysis. Moreover, key genes downstream of HOXA11-AS exhibited good clinical diagnostic properties for PCa. Furthermore, we studied both in vitro and in vivo effects of HOXA11-AS expression on PCa. Overexpression of HOXA11-AS increased PCa cell proliferation, migration and EMT, while silencing HOXA11-AS had the opposite effect on PCa cells. In addition, multiple metabolites were downregulated by silencing HOXA11-AS via the glycolytic pathway. HOXA11-AS silencing significantly inhibited tumour development in vivo. In summary, silencing HOXA11-AS can inhibit PCa by regulating glucose metabolism and may provide a future guidance for the treatment of PCa.

Investigating the dynamics of protein-protein interactions in plants.

Both stable and transient protein interactions play an important role in the complex assemblies required for the proper functioning of living cells. Several methods have been developed to monitor protein-protein interactions in plants. However, the detection of dynamic protein complexes is very challenging, with few technologies available for this purpose. Here, we developed a new platform using the plant UBIQUITIN promoter to drive transgene expression and thereby to detect protein interactions in planta. Typically, to decide which side of the protein to link the tags, the subcellular localization of the protein fused either N-terminal or C-terminal mCitrine was firstly confirmed by using eight different specific mCherry markers. Following stable or transient protein expression in plants, the protein interaction network was detected by affinity purification mass spectrometry. These interactions were subsequently confirmed by bimolecular fluorescence complementation (BiFC), bioluminescence resonance energy transfer and co-immunoprecipitation assays. The dynamics of these interactions were monitored by Förster resonance energy transfer (FRET) and split-nano luciferase, whilst the ternary protein complex association was monitored by BiFC-FRET. Using the canonical glycolytic metabolon as an example, the interaction between these enzymes was characterized under conditions that mimic physiologically relevant energy statuses.

The mutual interaction of glycolytic enzymes and RNA in post-transcriptional regulation.

About three decades ago, researchers suggested that metabolic enzymes participate in cellular processes that are unrelated to their catalytic activity, and the term "moonlighting functions" was proposed. Recently developed advanced technologies in the field of RNA interactome capture now unveil the unexpected RNA binding activity of many metabolic enzymes, as exemplified here for the enzymes of glycolysis. Although for most of these proteins a precise binding mechanism, binding conditions, and physiological relevance of the binding events still await in-depth clarification, several well explored examples demonstrate that metabolic enzymes hold crucial functions in post-transcriptional regulation of protein synthesis. This widely conserved RNA-binding function of glycolytic enzymes plays major roles in controlling cell activities. The best explored examples are glyceraldehyde 3-phosphate dehydrogenase, enolase, phosphoglycerate kinase, and pyruvate kinase. This review summarizes current knowledge about the RNA-binding activity of the ten core enzymes of glycolysis in plant, yeast, and animal cells, its regulation and physiological relevance. Apparently, a tight bidirectional regulation connects core metabolism and RNA biology, forcing us to rethink long established functional singularities.

On the coupling of intracellular K + ${{\rm{K}}}

We have investigated the interplay between glycolytic oscillations and intracellular K + ${{\rm{K}}}^{+}$ concentration in the yeast Saccharomyces cerevisiae. Intracellular K + ${{\rm{K}}}^{+}$ concentration was measured using the fluorophore potassium-binding benzofuranisophthalate (PBFI). We found that K + ${{\rm{K}}}^{+}$ is an essential ion for the occurrence of glycolytic oscillations and that intracellular K + ${{\rm{K}}}^{+}$ concentration oscillates synchronously with other variables such as nicotinamide adenine dinucleotide hydride (NADH), intracellular adenosine triphosphate (ATP), and mitochondrial membrane potential. We also investigated if glycolysis and intracellular K + ${{\rm{K}}}^{+}$ concentration oscillate in a number of yeast strains with mutations in K + ${{\rm{K}}}^{+}$ transporters in the plasma membrane, mitochondrial membrane and in the vacuolar membrane. Most of these strains are still capable of showing glycolytic oscillations, but two strains are not: (i) a strain with a deletion in the mitochondrial Mdm38p K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ transporter and (ii) a strain with deletion of the late endosomal Nhx1p K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ ( Na + ∕ H + ${\text{Na}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ ) transporter. In these two mutant strains intracellular K + ${{\rm{K}}}^{+}$ concentration seems to be low, indicating that the two transporters may be involved in transport of K + ${{\rm{K}}}^{+}$ into the cytosol. In the strain, Mdm38p Δ ${\rm{\Delta }}$ oscillations in glycolysis could be restored by addition of the K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ exchange ionophore nigericin. Furthermore, in two nonoscillating mutant strains with a defective V-ATPase and deletion of the Arp1p protein the intracellular K + ${{\rm{K}}}^{+}$ is relatively high, suggesting that the V-ATPase is essential for transport of K + ${{\rm{K}}}^{+}$ out of the cytosol and that the cytoskeleton may be involved in binding K + ${{\rm{K}}}^{+}$ to reduce the concentration of free ion in the cytosol. Analyses of the time series of oscillations of NADH, ATP, mitochondrial membrane potential, and potassium concentration using data-driven modeling corroborate the conjecture that K + ${{\rm{K}}}^{+}$ ion is essential for the emergence of oscillations and support the experimental findings using mutant strains.

Exogenous methylglyoxal alleviates drought-induced 'plant diabetes' and leaf senescence in maize.

Drought-induced leaf senescence is associated with high sugar levels, which bears some resemblance to the syndrome of diabetes in humans; however, the underlying mechanisms of such 'plant diabetes' on carbon imbalance and the corresponding detoxification strategy are not well understood. Here, we investigated the regulatory mechanism of exogenous methylglyoxal (MG) on 'plant diabetes' in maize plants under drought stress applied via foliar spraying during the grain-filling stage. Exogenous MG delayed leaf senescence and promoted photoassimilation, thereby reducing the yield loss induced by drought by 14%. Transcriptome and metabolite analyses revealed that drought increased sugar accumulation in leaves through inhibition of sugar transporters that facilitate phloem loading. This led to disequilibrium of glycolysis and overaccumulation of endogenous MG. Application of exogenous MG up-regulated glycolytic flux and the glyoxalase system that catabolyses endogenous MG and glycation end-products, ultimately alleviating 'plant diabetes'. In addition, the expression of genes facilitating anabolism and catabolism of trehalose-6-phosphate was promoted and suppressed by drought, respectively, and exogenous MG reversed this effect, implying that trehalose-6-phosphate signaling in the mediation of 'plant diabetes'. Furthermore, exogenous MG activated the phenylpropanoid biosynthetic pathway, promoting the production of lignin and phenolic compounds, which are associated with drought tolerance. Overall, our findings indicate that exogenous MG activates defense-related pathways to alleviate the toxicity derived from 'plant diabetes', thereby helping to maintain leaf function and yield production under drought.

Exposure to toxicologically relevant atrazine concentrations impair the glycolytic function of mouse Sertoli cells through the downregulation of lactate dehydrogenase.

Atrazine (ATZ), a widely used herbicide with potent endocrine-disrupting properties, has been implicated in hormonal disturbances and fertility issues. Sertoli cells (SCs) play a crucial role in providing mechanical and nutritional support of spermatogenesis. Herein, we aimed to study the effects of environmentally relevant ATZ concentrations on the nutritional support of spermatogenesis provided by SCs. For that, mouse SCs (TM4) were exposed to increasing ATZ concentrations (in µg/L: 0.3, 3, 30, 300, or 3000). After 24 h, cellular proliferation and metabolic activity were assessed. Mitochondrial activity and endogenous reactive oxygen species (ROS) production were evaluated using JC-1 and CM-H2DCFDA probes, respectively. We also analyzed protein levels of lactate dehydrogenase (LDH) using Western Blot and live cells glycolytic function through Seahorse XF Glycolysis Stress Test Kit. ATZ exposure decreased the activity of oxidoreductases in SCs, suggesting a decreased metabolic activity. Although ATZ is reported to induce oxidative stress, we did not observe alterations in mitochondrial membrane potential and ROS production across all tested concentrations. When we evaluated the glycolytic function of SCs, we observed that ATZ significantly impaired glycolysis and the glycolytic capacity at all tested concentrations. These results were supported by the decreased expression of LDH in SCs. Overall, our findings suggest that ATZ impairs the glycolytic function of SCs through LDH downregulation. Since lactate is the preferential energetic substrate for germ cells, exposure to ATZ may detrimentally impact the nutritional support crucial for spermatogenesis, hinting for a relationship between ATZ exposure and male infertility.

Glycolytic enzyme PGK1 promotes M1 macrophage polarization and induces pyroptosis of acute lung injury via regulation of NLRP3.

Acute lung injury (ALI) is characterized by an unregulated inflammatory reaction, often leading to severe morbidity and ultimately death. Excessive inflammation caused by M1 macrophage polarization and pyroptosis has been revealed to have a critical role in ALI. Recent study suggests that glycolytic reprogramming is important in the regulation of macrophage polarization and pyroptosis. However, the particular processes underlying ALI have yet to be identified. In this study, we established a Lipopolysaccharide(LPS)-induced ALI model and demonstrated that blocking glycolysis by using 2-Deoxy-D-glucose(2-DG) significantly downregulated the expression of M1 macrophage markers and pyroptosis-related genes, which was consistent with the in vitro results. Furthermore, our research has revealed that Phosphoglycerate Kinase 1(PGK1), an essential enzyme in the glycolysis pathway, interacts with NOD-, LRR- and pyrin domain-containing protein 3(NLRP3). We discovered that LPS stimulation improves the combination of PGK1 and NLRP3 both in vivo and in vitro. Interestingly, the absence of PGK1 reduces the phosphorylation level of NLRP3. Based on in vitro studies with mice bone marrow-derived macrophages (BMDMs), we further confirmed that siPGK1 plays a protective role by inhibiting macrophage pyroptosis and M1 macrophage polarization. The PGK1 inhibitor NG52 suppresses the occurrence of excessive inflammation in ALI. In general, it is plausible to consider a therapeutic strategy that focuses on modulating the relationship between PGK1 and NLRP3 as a means to mitigate the activation of inflammatory macrophages in ALI.

Tunnel-like region observed as a potential allosteric site in Staphylococcus aureus Glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzing the sixth step of glycolysis has been investigated for allosteric features that might be used as potential target for specific inhibition of Staphylococcus aureus (S.aureus). X-ray structure of bacterial enzyme for which a tunnel-like opening passing through the center previously proposed as an allosteric site has been subjected to six independent 500 ns long Molecular Dynamics simulations. Harmonic bond restraints were employed at key residues to underline the allosteric feature of this region. A noticeable reduction was observed in the mobility of NAD+ binding domains when restrictions were applied. Also, a substantial decrease in cross-correlations between distant Cα fluctuations was detected throughout the structure. Mutual information (MI) analysis revealed a similar decrease in the degree of correspondence in positional fluctuations in all directions everywhere in the receptor. MI between backbone and side-chain torsional variations changed its distribution profile and decreased considerably around the catalytic sites when restraints were employed. Principal component analysis clearly showed that the restrained state sampled a narrower range of conformations than apo state, especially in the first principal mode due to restriction in the conformational flexibility of NAD+ binding domain. Clustering the trajectory based on catalytic site residues displayed a smaller repertoire of conformations for restrained state compared to apo. Representative snapshots subjected to k-shortest pathway analysis revealed the impact of bond restraints on the allosteric communication which displayed distinct optimal and suboptimal pathways for two states, where observed frequencies of critical residues Gln51 and Val283 at the proposed site changed considerably.

Transcriptional regulation and post-translational modifications in the glycolytic pathway for targeted cancer therapy.

Cancer cells largely rely on aerobic glycolysis or the Warburg effect to generate essential biomolecules and energy for their rapid growth. The key modulators in glycolysis including glucose transporters and enzymes, e.g. hexokinase 2, enolase 1, pyruvate kinase M2, lactate dehydrogenase A, play indispensable roles in glucose uptake, glucose consumption, ATP generation, lactate production, etc. Transcriptional regulation and post-translational modifications (PTMs) of these critical modulators are important for signal transduction and metabolic reprogramming in the glycolytic pathway, which can provide energy advantages to cancer cell growth. In this review we recapitulate the recent advances in research on glycolytic modulators of cancer cells and analyze the strategies targeting these vital modulators including small-molecule inhibitors and microRNAs (miRNAs) for targeted cancer therapy. We focus on the regulation of the glycolytic pathway at the transcription level (e.g., hypoxia-inducible factor 1, c-MYC, p53, sine oculis homeobox homolog 1, N6-methyladenosine modification) and PTMs (including phosphorylation, methylation, acetylation, ubiquitination, etc.) of the key regulators in these processes. This review will provide a comprehensive understanding of the regulation of the key modulators in the glycolytic pathway and might shed light on the targeted cancer therapy at different molecular levels.

Liver X receptor inverse agonist SR9243 attenuates rheumatoid arthritis via modulating glycolytic metabolism of macrophages.

Liver X receptors (LXRs) which link lipid metabolism and inflammation, were overexpressed in experimental rheumatoid arthritis (RA) rats as observed in our previous studies, while suppression of LXRα by silybin ameliorates arthritis and abnormal lipid metabolism. However, the role of LXRs in RA remains undefined. In this study, we investigated the inhibition role of LXRs in the polarization and activation of M1 macrophage by using a special LXRs inverse agonist SR9243, which led to ameliorating the progression of adjuvant-induced arthritis (AIA) in rats. Mechanistically, SR9243 disrupted the LPS/IFN-γ-induced Warburg effect in M1 macrophages, while glycolysis inhibitor 2-DG attenuated the inhibition effect of SR9243 on M1 polarization and the cytokines expression of M1 macrophages including iNOS, TNF-α, and IL-6 in vitro. Furthermore, SR9243 downregulated key glycolytic enzymes, including LDH-A, HK2, G6PD, GLUT1, and HIF-1α in M1 macrophages, which is mediated by increased phosphorylation of AMPK (Thr172) and reduced downstream phosphorylation of mTOR (Ser2448). Importantly, gene silencing of LXRs compromises the inhibition effect of SR9243 on M1 macrophage polarization and activation. Collectively, for the first time, our findings suggest that the LXR inverse agonist SR9243 mitigates adjuvant-induced rheumatoid arthritis and protects against bone erosion by inhibiting M1 macrophage polarization and activation through modulation of glycolytic metabolism via the AMPK/mTOR/HIF-1α pathway.

FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism.

The proportions of the various muscle fiber types are important in the regulation of skeletal muscle metabolism, as well as animal meat production. Four-and-a-half LIM domain protein 3 (FHL3) is highly expressed in fast glycolytic muscle fibers and differentially regulates the expression of myosin heavy chain (MyHC) isoforms at the cellular level. Whether FHL3 regulates the transformation of muscle fiber types in vivo and the regulatory mechanism is unclear. In this study, muscle-specific FHL3 transgenic mice were generated by random integration, and lentivirus-mediated gene knockdown or overexpression in muscles of mice or pigs was conducted. Functional analysis showed that overexpression of FHL3 in muscles significantly increased the proportion of fast-twitch myofibers and muscle mass but decreased muscle succinate dehydrogenase (SDH) activity and whole-body oxygen consumption. Lentivirus-mediated FHL3 knockdown in muscles significantly decreased muscle mass and the proportion of fast-twitch myofibers. Mechanistically, FHL3 directly interacted with the Yin yang 1 (YY1) DNA-binding domain, repressed the binding of YY1 to the fast glycolytic MyHC2b gene regulatory region, and thereby promoted MyHC2b expression. FHL3 also competed with EZH2 to bind the repression domain of YY1 and reduced H3K27me3 enrichment in the MyHC2b regulatory region. Moreover, FHL3 overexpression reduced glucose tolerance by affecting muscle glycolytic metabolism, and its mRNA expression in muscle was positively associated with hemoglobin A1c (HbA1c) in patients with type 2 diabetes. Therefore, FHL3 is a novel potential target gene for the treatment of muscle metabolism-related diseases and improvement of animal meat production.

Moonlighting enzymes: when cellular context defines specificity.

It is not often realized that the absolute protein specificity is an exception rather than a rule. Two major kinds of protein multi-specificities are promiscuity and moonlighting. This review discusses the idea of enzyme specificity and then focusses on moonlighting. Some important examples of protein moonlighting, such as crystallins, ceruloplasmin, metallothioniens, macrophage migration inhibitory factor, and enzymes of carbohydrate metabolism are discussed. How protein plasticity and intrinsic disorder enable the removing the distinction between enzymes and other biologically active proteins are outlined. Finally, information on important roles of moonlighting in human diseases is updated.

Enhanced glycolysis in the myometrium with ectopic endometrium of patients with adenomyosis: a preliminary study.

OBJECTIVES: The objective of this study was to investigate the glycolytic activity of adenomyosis, which is characterized by malignant biological behaviors including abnormal cell proliferation, migration, invasion, cell regulation, and epithelial-mesenchymal transition. METHODS: From January 2021 to August 2022, a total of 15 patients who underwent total hysterectomy for adenomyosis and 14 patients who had non-endometrial diseases, specifically with cervical squamous intraepithelial neoplasia and uterine myoma, were included in this study. Myometrium with ectopic endometrium from patients with adenomyosis while normal myometrium from patients in the control group were collected. All samples were confirmed by a histopathological examination. The samples were analyzed by liquid chromatography-mass spectrometry (LC-MS), real-time quantitative PCR, NAD+/NADH assay kit as well as the glucose and lactate assay kits. RESULTS: Endometrial stroma and glands could be observed within the myometrium of patients in the adenomyosis group. We found that the mRNA expressions of HK1, PFKFB3, glyceraldehyde-3-phospate dehydrogenase (GAPDH), PKM2, and PDHA as well as the protein expressions of PFKFB3 were elevated in ectopic endometrial tissues of the adenomyosis group as compared to normal myometrium of the control group. The level of fructose 1,6-diphosphate was increased while NAD + and NAD+/NADH ratio were decreased compared with the control group. Besides, increased glucose consumption and lactate production were observed in myometrium with ectopic endometrium. CONCLUSIONS: We concluded that altered glycolytic phenotype of the myometrium with ectopic endometrium in women with adenomyosis may contribute the development of adenomyosis.

Regulation of lactose, glucose and sucrose metabolisms in S. thermophilus.

Streptococcus thermophilus is a bacterium widely used in the production of yogurts and cheeses, where it efficiently ferments lactose, the saccharide naturally present in milk. It is also employed as a starter in dairy- or plant-based fermented foods that contain saccharides other than lactose (e.g., sucrose, glucose). However, little is known about how saccharide use is regulated, in particular when saccharides are mixed. Here, we determine the effect of the 5 sugars that S. thermophilus is able to use, at different concentration and when they are mixed on the promoter activities of the C-metabolism genes. Using a transcriptional fusion approach, we discovered that lactose and glucose modulated the activity of the lacS and scrA promoters in a concentration-dependent manner. When mixed with lactose, glucose also repressed the two promoter activities; when mixed with sucrose, lactose still repressed scrA promoter activity. We determined that catabolite control protein A (CcpA) played a key role in these dynamics. We also showed that promoter activity was linked with glycolytic flux, which varied depending on saccharide type and concentration. Overall, this study identified key mechanisms in carbohydrate metabolism - autoregulation and partial hierarchical control - and demonstrated that they are partly mediated by CcpA.

Intercellular communication induces glycolytic synchronization waves between individually oscillating cells.

Many organs have internal structures with spatially differentiated and sometimes temporally synchronized groups of cells. The mechanisms leading to such differentiation and coordination are not well understood. Here we design a diffusion-limited microfluidic system to mimic a multicellular organ structure with peripheral blood flow and test whether a group of individually oscillating yeast cells could form subpopulations of spatially differentiated and temporally synchronized cells. Upon substrate addition, the dynamic response at single-cell level shows glycolytic oscillations, leading to wave fronts traveling through the monolayered population and to synchronized communities at well-defined positions in the cell chamber. A detailed mechanistic model with the architectural structure of the flow chamber incorporated successfully predicts the spatial-temporal experimental data, and allows for a molecular understanding of the observed phenomena. The intricate interplay of intracellular biochemical reaction networks leading to the oscillations, combined with intercellular communication via metabolic intermediates and fluid dynamics of the reaction chamber, is responsible for the generation of the subpopulations of synchronized cells. This mechanism, as analyzed from the model simulations, is experimentally tested using different concentrations of cyanide stress solutions. The results are reproducible and stable, despite cellular heterogeneity, and the spontaneous community development is reminiscent of a zoned cell differentiation often observed in multicellular organs.