Investigation of the adaptation of Lactococcus lactis to isoleucine starvation integrating dynamic transcriptome and proteome information

Background: Amino acid assimilation is crucial for bacteria and this is particularly true for Lactic Acid Bacteria (LAB) that are generally auxotroph for amino acids. The global response of the Lmodel Lactococcus lactis ssp. lactis was characterized during progressive isoleucine starvation in batch culture using a chemically defined medium in which isoleucine concentration was fixed so as to become the sole limiting nutriment. Dynamic analyses were performed using transcriptomic and proteomic approaches and the results were analysed conjointly with fermentation kinetic data. Results: The response was first deduced from transcriptomic analysis and corroborated by proteomic results. It occurred progressively and could be divided into three major mechanisms: (i) a global down-regulation of processes linked to bacterial growth and catabolism (transcription, translation, carbon metabolism and transport, pyrimidine and fatty acid metabolism), (ii) a specific positive response related to the limiting nutrient (activation of pathways of carbon or nitrogen metabolism and leading to isoleucine supply) and (iii) an unexpected oxidative stress response (positive regulation of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms during this adaptation was analysed on the basis of transcriptomic data comparisons. The global regulator CodY seemed specifically dedicated to the regulation of isoleucine supply. Other regulations were massively related to growth rate and stringent response. Conclusion: This integrative biology approach provided an overview of the metabolic pathways involved during isoleucine starvation and their regulations. It has extended significantly the physiological understanding of the metabolism of L. lactis ssp. lactis. The approach can be generalised to other conditions and will contribute significantly to the identification of the biological processes involved in complex regulatory networks of micro-organisms

Growth rate regulated genes and their wide involvement in the Lactococcus lactis stress responses

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A nonlinear mixed-effects approach for the mechanistic interpretation of time-series transcriptomics data

Mechanistic models are essential to unravel the molecular mechanisms driving cellular responses. However, the integration of high-throughput data with mechanistic knowledge is limited by the availability of scalable computational approaches able to disentangle biological and technical sources of variation. Results: We present an approach based on nonlinear mixed-effects modelling for the parameter estimation of large-scale mechanistic models from time-series transcriptomics data. It allows to factor out technical variability, to compensate for the limited number of conditions and time points by a population approach, and it incorporates mechanistic details to gain insight on the molecular causes of biological variability. We applied our approach for the biological interpretation of microarray and RNA-Seq gene expression profiles, with different levels of technical noise, but it is generalisable to numerous types of data. When integrated in a model describing the degradation kinetics of all cellular mRNAs, the data allowed to identify the targets of post-transcriptional regulatory mechanisms. Our approach paves the way for the interpretation of high-throughput biological data with more comprehensive mechanistic models. Availability: The Monolix script for estimation and output files are freely available at https://gitlab.inria. fr/tetienne/eccb_script, together with the microarray data. The RNA-Seq dataset is being prepared for publication (Roux et al., in preparation) and will be made available on demand upon acceptance of the article

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

International audienceIn the bacterium Escherichia coli, the posttranscriptional regulatory system Csr was postulated to influence the transition from glycolysis to gluconeogene-sis. 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 me-tabolite 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

On-line optimization of glutamate production based on balanced metabolic control by RQ

In glutamate fermentations by Corynebacterium glutamicum, higher glutamate concentration could be achieved by constantly controlling dissolved oxygen concentration (DO) at a lower level; however, by-product lactate also severely accumulated. The results of analyzing activities changes of the two key enzymes, glutamate and lactate dehydrogenases involved with the fermentation, and the entire metabolic network flux analysis showed that the lactate overproduction was because the metabolic flux in TCA cycle was too low to balance the glucose glycolysis rate. As a result, the respiratory quotient (RQ) adaptive control based “balanced metabolic control” (BMC) strategy was proposed and used to regulate the TCA metabolic flux rate at an appropriate level to achieve the metabolic balance among glycolysis, glutamate synthesis, and TCA metabolic flux. Compared with the best results of various DO constant controls, the BMC strategy increased the maximal glutamate concentration by about 15% and almost completely repressed the lactate accumulation with competitively high glutamate productivity

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum

BACKGROUND: Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates. RESULTS: Quantification of the fluxes of the elementary modes in the metabolism of C. glutamicum was formulated as linear programming. The analysis demonstrated that the solution was dependent on the criteria of objective function when less than four accumulation rates of the external metabolites were considered. The analysis yielded feasible ranges of fluxes of elementary modes that satisfy the experimental accumulation rates. In C. glutamicum, the elementary modes relating to biomass synthesis through glycolysis and TCA cycle were predominantly operational in the initial growth phase. At a later time, the elementary modes contributing to lysine synthesis became active. The oxygen and ammonia uptake rates were shown to be bounded in the phenotypic space due to the stoichiometric constraint of the elementary modes. CONCLUSION: We have demonstrated the use of elementary modes and the linear programming to quantify a metabolic network. We have used the methodology to quantify the network of C. glutamicum, which evaluates the set of operational elementary modes at different phases of fermentation. The methodology was also used to determine the feasible solution space for a given set of substrate uptake rates under specific optimization criteria. Such an approach can be used to determine the optimality of the accumulation rates of any metabolite in a given network

A Glutamic Acid-Producing Lactic Acid Bacteria Isolated from Malaysian Fermented Foods

l-glutamaic acid is the principal excitatory neurotransmitter in the brain and an important intermediate in metabolism. In the present study, lactic acid bacteria (218) were isolated from six different fermented foods as potent sources of glutamic acid producers. The presumptive bacteria were tested for their ability to synthesize glutamic acid. Out of the 35 strains showing this capability, strain MNZ was determined as the highest glutamic-acid producer. Identification tests including 16S rRNA gene sequencing and sugar assimilation ability identified the strain MNZ as Lactobacillus plantarum. The characteristics of this microorganism related to its glutamic acid-producing ability, growth rate, glucose consumption and pH profile were studied. Results revealed that glutamic acid was formed inside the cell and excreted into the extracellular medium. Glutamic acid production was found to be growth-associated and glucose significantly enhanced glutamic acid production (1.032 mmol/L) compared to other carbon sources. A concentration of 0.7% ammonium nitrate as a nitrogen source effectively enhanced glutamic acid production. To the best of our knowledge this is the first report of glutamic acid production by lactic acid bacteria. The results of this study can be further applied for developing functional foods enriched in glutamic acid and subsequently γ-amino butyric acid (GABA) as a bioactive compound