IsoCor is distributed under OpenSource license at http://metasys.insa-toulouse.fr/software/isocor/

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.

Mass spectrometry (MS) is widely used for isotopic studies of metabolism in which detailed information about biochemical processes is obtained from the analysis of isotope incorporation into metabolites. The biological value of such experiments is dependent on the accuracy of the isotopic measurements. Using MS, isotopologue distributions are measured from the quantitative analysis of isotopic clusters. These measurements are prone to various biases, which can occur during the experimental workflow and/or MS analysis. The lack of relevant standards limits investigations of the quality of the measured isotopologue distributions. To meet that need, we developed a complete theoretical and experimental framework for the biological production of metabolites with fully controlled and predictable labeling patterns. This strategy is valid for different isotopes and different types of metabolisms and organisms, and was applied to two model microorganisms, Pichia augusta and Escherichia coli, cultivated on (13)C-labeled methanol and acetate as sole carbon source, respectively. The isotopic composition of the substrates was designed to obtain samples in which the isotopologue distribution of all the metabolites should give the binomial coefficients found in Pascal's triangle. The strategy was validated on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform by quantifying the complete isotopologue distributions of different intracellular metabolites, which were in close agreement with predictions. This strategy can be used to evaluate entire experimental workflows (from sampling to data processing) or different analytical platforms in the context of isotope labeling experiments.

The metabolism of microorganisms is regulated through two main mechanisms: changes of enzyme capacities as a consequence of gene expression modulation (“hierarchical control”) and changes of enzyme activities through metabolite-enzyme interactions. An increasing body of evidence indicates that hierarchical control is insufficient to explain metabolic behaviors, but the system-wide impact of metabolic regulation remains largely uncharacterized. To clarify its role, we developed and validated a detailed kinetic model of Escherichia coli central metabolism that links growth to environment. Metabolic control analyses confirm that the control is widely distributed across the network and highlight strong interconnections between all the pathways. Exploration of the model solution space reveals that several robust properties emerge from metabolic regulation, from the molecular level (e.g. homeostasis of total metabolite pool) to the overall cellular physiology (e.g. coordination of carbon uptake, catabolism, energy and redox production, and growth), while allowing a large degree of flexibility at most individual metabolic steps. These properties have important physiological implications for E. coli and significantly expand the self-regulating capacities of its metabolism.

In-cell NMR of macromolecules has gained momentum over the last ten years as an approach that might bridge the branches of cell biology and structural biology. In this review, we put it in the context of earlier efforts that aimed to characterize by NMR the cellular environment of live cells and their intracellular metabolites. Although technical aspects distinguish these earlier in vivo NMR studies and the more recent in cell NMR efforts to characterize macromolecules in a cellular environment, we believe that both share major concerns ranging from sensitivity and line broadening to cell viability. Approaches to overcome the limitations in one subfield thereby can serve the other one and vice versa. The relevance in biomedical sciences might stretch from the direct following of drug metabolism in the cell to the observation of target binding, and thereby encompasses in-cell NMR both of metabolites and macromolecules. We underline the efforts of the field to move to novel biological insights by some selected examples.

Summary Mass spectrometry (MS) is widely used for isotopic studies of metabolism and other (bio)chemical processes. Quantitative applications in systems and synthetic biology require to correct the raw MS data for the contribution of naturally occurring isotopes. Several tools are available to correct low-resolution MS data, and recent developments made substantial improvements by introducing resolution-dependent correction methods, hence opening the way to the correction of high-resolution MS (HRMS) data. Nevertheless, current HRMS correction methods partly fail to determine which isotopic species are resolved from the tracer isotopologues and should thus be corrected. We present an updated version of our isotope correction software (IsoCor) with a novel correction algorithm which ensures to accurately exploit any chemical species with any isotopic tracer, at any MS resolution. IsoCor v2 also includes a novel graphical user interface for intuitive use by end-users and a command-line interface to streamline integration into existing pipelines. Availability and implementation IsoCor v2 is implemented in Python 3 and was tested on Windows, Unix and MacOS platforms. The source code and the documentation are freely distributed under GPL3 license at https://github.com/MetaSys-LISBP/IsoCor/ and https://isocor.readthedocs.io/.

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The role of the post-transcriptional carbon storage regulator (Csr) system in nutrient utilization and in the control of the central metabolism in E. coli reference commensal strain Nissle 1917 was investigated. Analysis of the growth capabilities of mutants altered for various components of the Csr system (csrA51, csrB, csrC and csrD mutations) showed that only the protein CsrA - the key component of the system - exerts a marked role in carbon nutrition. Attenuation of CsrA activity in the csrA51 mutant affects the growth efficiency on a broad range of physiologically relevant carbon sources, including compounds utilized by the Entner-Doudoroff (ED) pathway. Detailed investigations of the metabolomes and fluxomes of mutants and wild-type cells grown on carbon sources representative of glycolysis and of the ED pathway (glucose and gluconate, respectively), revealed significant re-adjusting of central carbon metabolism for both compounds in the csrA51 mutant. However, the metabolic re-adjusting observed on gluconate was strikingly different from that observed on glucose, indicating a nutrient-specific control of metabolism by the Csr system.