Section: New Results

Models of carbon metabolism in bacteria

Adaptation of bacterial growth to changes in environmental conditions, such as the availability of specific carbon sources, is triggered at the molecular level by the reorganization of metabolism and gene expression: the concentration of metabolites is adjusted, as well as the concentration and activities of enzymes, the rate of metabolic reactions, the transcription and translation rates, and the stability of proteins and RNAs. This reprogramming of the bacterial cell is carried out by i) specific interactions involving regulatory proteins or RNAs that specifically respond to the change of environmental conditions and ii) global regulation involving changes in the concentration of RNA polymerase, ribosomes, and metabolite pools that globally affect the rates of transcription, translation, and degradation of all RNAs and proteins. While these phenomena have been well studied in steady-state growth conditions, recent works by IBIS members and collaborators support the view that regulatory mechanisms of growth adaptation are best observed in dynamical conditions.

A first study concerns the second messenger cAMP in E. coli and its role in carbon catabolite repression, the mechanism by which bacterial cells select their preferred carbon source for growth. Studies performed in steady-state conditions have questioned the importance of cAMP, leading to a controversy on its physiological role, more than fifty years after its discovery. In a recently submitted journal paper, reporting work started during the PhD thesis of Valentin Zulkower and continued over the past two years, we argue that in order to properly assess the role of cAMP one should shift the focus from steady-state to dynamical conditions. We show, by a combination of fluorescent reporter gene assays and quantitative modeling, that a transient peak in the expression of cAMP-dependent genes leads to the accumulation of proteins necessary for growth on a variety of alternative carbon sources. In the long run, the expression of genes cognate to the alternative carbon source present in the environment is maintained by dedicated positive feedback circuits. Our results thus demonstrate that carbon catabolite repression and diauxic growth need to be understood from a dynamical perspective within the context of a hierarchical regulatory network.

A quantitative description and understanding of this complex network, cutting across metabolism, gene expression, and signalling, can be accessed through mathematical modelling only. In collaboration with Andreas Kremling, professor at TU München and former visiting scientist in the IBIS project-team, Hans Geiselmann, Delphine Ropers and Hidde de Jong developed an ensemble of variants of a simple core model of carbon catabolite repression. The model variants, with two substrate assimilation pathways and four intracellular metabolites only, differ from one another in only a single aspect, each breaking the symmetry between the two pathways in a different manner. Interestingly, all model variants are able to reproduce the data from a reference diauxic growth experiment. For each of the model variants, we predicted the behaviour in two new experimental conditions. When qualitatively comparing these predictions with experimental data, a number of models could be excluded while other model variants are still not discriminable. The best-performing model variants are based on inducer inclusion and activation of enzymatic genes by a global transcription factor, but the other proposed factors may complement these well-known regulatory mechanisms. The model ensemble, which was described in a journal paper recently submitted for publication, offers a better understanding of the variety of mechanisms that have been proposed to play a role in carbon catabolite repression, but is also useful as an educational resource for systems biology.

The same focus on the dynamics of physiological processes has shaped a project on the post-transcriptional control of carbon central metabolism in E. coli. In the framework of the PhD thesis of Manon Morin, supported by a Contrat Jeune Scientifique INRA-Inria, the collaboration of Delphine Ropers with Muriel Cocaign-Bousquet and Brice Enjalbert at INRA/INSA Toulouse has demonstrated the key role played by the post-transcriptional regulatory system CSR in growth transitions. In a multi-scale analysis of several wild-type and mutant strains of the CSR system, a variety of experimental data have been acquired in relevant conditions, including growth parameters, gene expression levels and metabolite pools. Data integration through the estimation of fermentation fluxes and flux balance analysis, using the method described above (Section 6.3), have elucidated the role of post-transcriptional regulation in the dynamics of glycogen storage and consumption, as well as the key role of the latter compound for bacterial fitness, through the regulation of intracellular energy levels. A paper summarizing the work has been published in mBio [20].

The collaboration with INRA/INSA de Toulouse is continued in the context of the PhD thesis of Thibault Etienne, funded by an INRA-Inria PhD grant, with the objective of developing models able to explain how cells coordinate their physiology and the functioning of the transcription, translation, and degradation machineries following changes in the availability of carbon sources in the environment.