Emergence of growth and dormancy from a kinetic model of the Escherichia coli central carbon metabolism

Physiological states of bacterial cells exhibit a wide spectrum of timescale. Under nutrient-rich conditions, most of the cells in an isogenic bacterial population grow at certain rates, while a small subpopulation sometimes stays in a dormant state where the growth rates slow down by orders of magnitude. For revealing the origins of such heterogeneity of timescales, we studied the kinetic model of Escherichia coli central carbon metabolism including the dynamics of the energy currency molecules. We found that the model robustly exhibits both the growing- and the dormant state. In order to unveil the mechanism of distinct behaviours, we developed a recursive method to simplify the model without changing the qualitative feature of the dynamics. Analytical and numerical studies of the 2-variable minimal model revealed the necessary conditions for the distinct behaviour, namely, the depletion of energy due to the futile cycle and its non-uniform impact on the kinetics because of the coexistence of the energy currency-coupled and uncoupled reactions as well as branching of the network. The result is consistent with the experimental reports that the dormant cells commonly exhibit low ATP levels, and provides a possible explanation for the appearance of dormant cells that causes antibiotic persistence.

Measuring NQO1 Bioactivation Using [2H7]Glucose

Treatment of cancers with β-lapachone causes NAD(P)H: quinone oxidoreductase 1 (NQO1) to generate an unstable hydroquinone that regenerates itself in a futile cycle while producing reactive oxygen species (ROS) in the form of superoxide and subsequently hydrogen peroxide. Rapid accumulation of ROS damages DNA, hyperactivates poly-ADP-ribose polymerase-I, causes massive depletion of NAD+/ATP, and hampers glycolysis. Cells overexpressing NQO1 subsequently die rapidly through an NAD+-keresis mechanism. Assessing changes in glycolytic rates caused by NQO1 bioactivation would provide a means of assessing treatment efficacy, potentially lowering the chemotherapeutic dosage, and reducing off-target toxicities. NQO1-mediated changes in glycolytic flux were readily detected in A549 (lung), MiaPaCa2 (pancreatic), and HCT-116 (colon) cancer cell lines by 2H-NMR after administration of [2H7]glucose. The deuterated metabolic products 2H-lactate and HDO were quantified, and linear relationships with glucose consumption for both products were observed. The higher concentration of HDO compared to 2H-lactate allows for more sensitive measurement of the glycolytic flux in cancer. Gas chromatography-mass spectrometry analysis agreed with the NMR results and confirmed downregulated energy metabolism in NQO1+ cells after β-lapachone treatment. The demonstrated method is ideal for measuring glycolytic rates, the effects of chemotherapeutics that target glycolysis, and has the potential for in vivo translation.

Thioesterase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a futile cycle that inhibits hepatic β-oxidation

Abstract2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing β-oxidation. In this study, the mechanism of β-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete β-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).

Emergence of growth and dormancy from a kinetic model of the Escherichia coli central carbon metabolism

Physiological states of bacterial cells exhibit a wide spectrum of timescale. Under the nutrient-rich conditions, most of the cells in an isogenic bacterial population grow at certain rates, while a small subpopulation sometimes stays in a dormant state where the growth rates slow down by orders of magnitude. What is the origin of such heterogeneity of timescales? Here we addressed this question by studying the kinetic model of Escherichia coli central carbon metabolism including the dynamics of the energy currency molecules, which have often been ignored. We found that the model robustly exhibits both the growing- and the dormant state. In order to unveil the mechanism of distinct behaviours, we developed a recursive method to simplify the model without changing the qualitative feature of the dynamics. Analytical and numerical studies of the 2-variable minimal model revealed the necessary conditions for the distinct behaviour, namely, the depletion of energy due to the futile cycle and its non-uniform impact to the kinetics because of the coexistence of the energy currency-coupled and uncoupled reactions as well as branching of the network. The result is consistent with the experimental evidences of the appearance of the futile cycle in mutants and provides a possible explanation for the appearance of dormant cells that causes antibiotic persistence.

How subtle changes in 3D structure can create large changes in transcription

Animal genomes are organized into topologically associated domains (TADs). TADs are thought to contribute to gene regulation by facilitating enhancer-promoter (E-P) contacts within a TAD preventing these contacts across TAD borders. However, the absolute difference in contact frequency across TAD boundaries is usually less than two-fold, even though disruptions of TAD borders can change gene expression by ten-fold. Existing models fail to explain this hypersensitive response. Here, we propose a futile cycle model of enhancer-mediated regulation that can exhibit hypersensitivity through bistability and hysteresis. Consistent with recent experiments, this regulation does not exhibit strong correlation between enhancer-promoter contact and promoter activity, even though regulation occurs through contact. Through mathematical analysis and stochastic simulation, we show that this system can create an illusion of enhancer-promoter biochemical specificity and explain the importance of weak TAD boundaries. It also offers a mechanism to reconcile apparently contradictory results from recent global TAD disruption with local TAD boundary deletion experiments. Together, these analyses advance our understanding of cis-regulatory contacts in controlling gene expression, and suggest new experimental directions.

Crosstalk between repair pathways elicits Double Strand Breaks in alkylated DNA and implications for the action of temozolomide

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG). Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-Methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We show that in this system, mismatch repair (MMR) proteins are enriched on MNU-treated DNA and we observe robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increased linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving BER acting at a nearby N-alkylation adducts. We propose a new, replication-independent mechanism of action of TMZ, that operates in addition to the well-studied cell cycle dependent mode of action.

Yeast adaptive response to acetic acid stress involves structural alterations and increased stiffness of the cell wall

This work describes a coordinate and comprehensive view on the time course of the alterations occurring at the level of the cell wall during adaptation of a yeast cell population to sudden exposure to a sub-lethal stress induced by acetic acid. Acetic acid is a major inhibitory compound in industrial bioprocesses and a widely used preservative in foods and beverages. Results indicate that yeast cell wall resistance to lyticase activity increases during acetic acid-induced growth latency, corresponding to yeast population adaptation to sudden exposure to this stress. This response correlates with: (i) increased cell stiffness, assessed by atomic force microscopy (AFM); (ii) increased content of cell wall β-glucans, assessed by fluorescence microscopy, and (iii) slight increase of the transcription level of the GAS1 gene encoding a β-1,3-glucanosyltransferase that leads to elongation of (1→3)-β-d-glucan chains. Collectively, results reinforce the notion that the adaptive yeast response to acetic acid stress involves a coordinate alteration of the cell wall at the biophysical and molecular levels. These alterations guarantee a robust adaptive response essential to limit the futile cycle associated to the re-entry of the toxic acid form after the active expulsion of acetate from the cell interior.

Photorespiration: The Futile Cycle?

Photorespiration, or C2 photosynthesis, is generally considered a futile cycle that potentially decreases photosynthetic carbon fixation by more than 25%. Nonetheless, many essential processes, such as nitrogen assimilation, C1 metabolism, and sulfur assimilation, depend on photorespiration. Most studies of photosynthetic and photorespiratory reactions are conducted with magnesium as the sole metal cofactor despite many of the enzymes involved in these reactions readily associating with manganese. Indeed, when manganese is present, the energy efficiency of these reactions may improve. This review summarizes some commonly used methods to quantify photorespiration, outlines the influence of metal cofactors on photorespiratory enzymes, and discusses why photorespiration may not be as wasteful as previously believed.

Retroactivity induced operating regime transition in an enzymatic futile cycle

Activated phosphorylation-dephosphorylation biochemical reaction cycles are a class of enzymatic futile cycles. A futile cycle such as a single MAPK cascade governed by two underlying enzymatic reactions permits Hyperbolic (H), Signal transducing (ST), Threshold-hyperbolic (TH) and Ultrasensitive (U) operating regimes that characterize input-output behaviour. Retroactive signalling caused by load due to sequestration of phosphorylated or unphosphorylated form of the substrate in a single enzymatic cascade without explicit feedback can introduce two-way communication, a feature not possible otherwise. We systematically characterize the operating regimes of a futile cycle subject to retroactivity in either of the substrate forms. We demonstrate that increasing retroactivity strength, which quantifies the downstream load, can trigger five possible regime transitions. Retroactivity strength is a reflection of the fraction of the substrate sequestered by its downstream target. Remarkably, the minimum required retroactivity strength to evidence any sequestration triggered regime transition demands 23% of the substrate bound to its downstream target. This minimum retroactivity strength corresponds to the transition of the dose-response curve from ST to H regime. We show that modulation of the saturation and unsaturation levels of the enzymatic reactions by retroactivity is the fundamental mechanism governing operating regime transition.

Thioesterase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a futile cycle that inhibits hepatic β-oxidation

ABSTRACT2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing β-oxidation. In this study, the mechanism of β-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete β-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).