Comprendre l’adaptation de Lactococcus lactis par une\ud approche de biologie intégrative à l’échelle du génome

L’adaptation de Lactococcus lactis à différentes conditions de culture a été appréhendée grâce à une\ud démarche de biologie intégrative. Cette approche intègre les données issues de différents niveaux de\ud régulation et combine diverses techniques de mesure à l’échelle globale (transcriptome, protéome,\ud stabilité des ARN messagers) et locale (suivi des paramètres de culture). Plusieurs outils\ud mathématiques de modélisation (tels que la modélisation numérique et la modélisation statistique)\ud ont étés développés pour intégrer l’ensemble de ces données hétérogènes.\ud Une culture continue de L. lactis à différents taux de dilution a permis d’étudier l’influence du taux\ud de croissance sur la physiologie de la bactérie, un paramètre qui n’est jamais distingué de la réponse\ud au stress lors des études dynamiques de l’adaptation. La réponse à la variation du taux de croissance\ud implique majoritairement les fonctions associées à la biogenèse mais demeure extrêmement étendue\ud puisqu’elle affecte l’expression de 30 % des gènes de L. lactis. Cette réponse concerne les niveaux\ud d’ARN messagers et de protéines mais aussi les processus cellulaires majeurs que sont la\ud traduction, la dilution et la dégradation. Il a été montré, par une approche de modélisation, que les\ud efficacités de traduction et les vitesses de dégradation des protéines étaient en effet inversement\ud proportionnelles au taux de croissance. Au final, l’influence des différents processus cellulaires a pu\ud être quantifiée par des calculs de coefficients de contrôle.\ud L’imposition progressive d’une carence en isoleucine lors d’une culture discontinue en batch a\ud permis de caractériser la réponse, encore peu étudiée, de L. lactis à une carence en acide aminé.\ud L’adaptation à ce stress nutritionnel entraîne une vaste réorganisation de la physiologie cellulaire\ud qui se divise en trois types de réponses : une répression globale des principales fonctions\ud biologiques associées à la croissance, une réponse propre au stress imposé visant à lutter\ud spécifiquement contre la carence en isoleucine, ainsi qu’une activation inexpliquée de mécanismes\ud en lien avec le stress oxydatif. L’implication de différents mécanismes (réponse stringente,\ud mécanisme lié au taux de croissance, régulations par CodY, GlnR et CcpA) dans la régulation de\ud cette réponse a été évaluée par transcriptomique comparative.\ud Les déterminants majeurs des concentrations en protéines au sein de la cellule ont été recherchés\ud mathématiquement grâce à un algorithme de sélection de modèles de covariances. Le biais de\ud codons (CAI) s’est avéré être un paramètre majeur, plus important que les concentrations en ARN\ud messagers, suggérant l’existence d’un contrôle génétique prépondérant sur l’adaptation\ud transcriptionnelle. Enfin, il a pu être démontré que le degré d’implication des différents\ud déterminants varie en fonction du mode d’adaptation.\ud L’approche de biologie intégrative suivie au cours de cette thèse a permis une meilleure\ud compréhension des mécanismes d’adaptation de L. lactis et est aujourd’hui entièrement\ud généralisable à d’autres processus comme à d’autres microorganismes. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- A systems biology approach was implemented to study Lactococcus lactis adaptation to\ud various growing conditions. This method combines growth parameter monitoring and\ud genome-wide measurement technologies (transcriptome, proteome, messenger RNA\ud stability). Data from these diverse regulation levels were integrated thanks to mathematical\ud tools developed on purpose.\ud Growth rate influence on L. lactis physiology, which is never dissociated from stress\ud responses when studying dynamic adaptation processes, was analysed through continuous\ud culture at various growth rates. This widespread response mainly involves biogenesis-related\ud functions and affects the expression of 30 % of L. lactis genes. Both messenger RNA and\ud protein levels are modified but cellular processes such as translation, dilution and degradation\ud are also concerned. As a matter of fact, translation efficiency and protein degradation rates\ud were proved to be inversely proportional to growth rate by a modelling approach. Control\ud coefficient calculations enabled the quantification of cellular processes influences.\ud The dynamic response of L. lactis to isoleucine starvation was studied by the progressive\ud consumption of this amino-acid in a discontinuous batch fermentation. This poorly\ud characterized adaptation process triggers a wide reorganization of cellular physiology that\ud could be divided in three parts: a global repression of the main biological functions related to\ud growth, a response more specific to the encountered stress to struggle against isoleucine\ud starvation and an unexplained activation of oxidative stress-related cellular functions.\ud Comparative transcriptomics allowed the implication of various mechanisms to be quantified\ud in the regulation of this adaptation response (stringent response, growth rate adaptation\ud mechanism, CodY, GlnR and CcpA regulation).\ud The major biological determinants of protein intracellular concentration were mathematically\ud investigated thanks to a covariance model selection algorithm. Codons bias (CAI) was found\ud to be the most influent parameter, even more than mRNA concentrations, which suggests that\ud genetic control is stronger than transcriptional adaptation. The weight of the different\ud determinants was also found to depend on adaptation modes.\ud The systems biology approach applied in this work enabled a better understanding of L. lactis\ud adaptation mechanisms and will be entirely transposable to other cellular processes as well as\ud other microorganism

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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The price of informality : how informal finance schemes defaulted in China, 1989–2015.

The default of a large number of informal finance schemes in China has caused enormous financial losses, and therefore has potential social and political significance. Analysing 354 defaulted schemes from 1989 to 2015, this study defines how they differ from other types of informal finance. It also produces an ideal-type representation of the default process and concludes that the default results from greed, increasing financial pressure at the individual level and private enterprises’ restricted access to state bank loans at the institutional level. China’s financial system should be more flexible in order to prevent further financial losses through informal financial relations

Transcriptome and Proteome Exploration to Model Translation Efficiency and Protein Stability in Lactococcus lactis

This genome-scale study analysed the various parameters influencing protein levels in cells. To achieve this goal, the model bacterium Lactococcus lactis was grown at steady state in continuous cultures at different growth rates, and proteomic and transcriptomic data were thoroughly compared. Ratios of mRNA to protein were highly variable among proteins but also, for a given gene, between the different growth conditions. The modeling of cellular processes combined with a data fitting modeling approach allowed both translation efficiencies and degradation rates to be estimated for each protein in each growth condition. Estimated translational efficiencies and degradation rates strongly differed between proteins and were tested for their biological significance through statistical correlations with relevant parameters such as codon or amino acid bias. These efficiencies and degradation rates were not constant in all growth conditions and were inversely proportional to the growth rate, indicating a more efficient translation at low growth rate but an antagonistic higher rate of protein degradation. Estimated protein median half-lives ranged from 23 to 224 min, underlying the importance of protein degradation notably at low growth rates. The regulation of intracellular protein level was analysed through regulatory coefficient calculations, revealing a complex control depending on protein and growth conditions. The modeling approach enabled translational efficiencies and protein degradation rates to be estimated, two biological parameters extremely difficult to determine experimentally and generally lacking in bacteria. This method is generic and can now be extended to other environments and/or other micro-organisms

Comprendre l adaptation de Lactococcus lactis par une approche de biologie intégrative à l échelle du génome

L adaptation de Lactococcus lactis à différentes conditions de culture a été appréhendée grâce à une démarche de biologie intégrative. Cette approche intègre les données issues de différents niveaux de régulation et combine diverses techniques de mesure à l échelle globale (transcriptome, protéome, stabilité des ARN messagers) et locale (suivi des paramètres de culture). Plusieurs outils mathématiques de modélisation (tels que la modélisation numérique et la modélisation statistique) ont étés développés pour intégrer l ensemble de ces données hétérogènes. Une culture continue de L. lactis à différents taux de dilution a permis d étudier l influence du taux de croissance sur la physiologie de la bactérie, un paramètre qui n est jamais distingué de la réponse au stress lors des études dynamiques de l adaptation. La réponse à la variation du taux de croissance implique majoritairement les fonctions associées à la biogenèse mais demeure extrêmement étendue puisqu elle affecte l expression de 30 % des gènes de L. lactis. Cette réponse concerne les niveaux d ARN messagers et de protéines mais aussi les processus cellulaires majeurs que sont la traduction, la dilution et la dégradation. Il a été montré, par une approche de modélisation, que les efficacités de traduction et les vitesses de dégradation des protéines étaient en effet inversement proportionnelles au taux de croissance. Au final, l influence des différents processus cellulaires a pu être quantifiée par des calculs de coefficients de contrôle. L imposition progressive d une carence en isoleucine lors d une culture discontinue en batch a permis de caractériser la réponse, encore peu étudiée, de L. lactis à une carence en acide aminé. L adaptation à ce stress nutritionnel entraîne une vaste réorganisation de la physiologie cellulaire qui se divise en trois types de réponses : une répression globale des principales fonctions biologiques associées à la croissance, une réponse propre au stress imposé visant à lutter spécifiquement contre la carence en isoleucine, ainsi qu une activation inexpliquée de mécanismes en lien avec le stress oxydatif. L implication de différents mécanismes (réponse stringente, mécanisme lié au taux de croissance, régulations par CodY, GlnR et CcpA) dans la régulation de cette réponse a été évaluée par transcriptomique comparative. Les déterminants majeurs des concentrations en protéines au sein de la cellule ont été recherchés mathématiquement grâce à un algorithme de sélection de modèles de covariances. Le biais de codons (CAI) s est avéré être un paramètre majeur, plus important que les concentrations en ARN messagers, suggérant l existence d un contrôle génétique prépondérant sur l adaptation transcriptionnelle. Enfin, il a pu être démontré que le degré d implication des différents déterminants varie en fonction du mode d adaptation. L approche de biologie intégrative suivie au cours de cette thèse a permis une meilleure compréhension des mécanismes d adaptation de L. lactis et est aujourd hui entièrement généralisable à d autres processus comme à d autres microorganismesA systems biology approach was implemented to study Lactococcus lactis adaptation to various growing conditions. This method combines growth parameter monitoring and genome-wide measurement technologies (transcriptome, proteome, messenger RNA stability). Data from these diverse regulation levels were integrated thanks to mathematical tools developed on purpose. Growth rate influence on L. lactis physiology, which is never dissociated from stress responses when studying dynamic adaptation processes, was analysed through continuous culture at various growth rates. This widespread response mainly involves biogenesis-related functions and affects the expression of 30 % of L. lactis genes. Both messenger RNA and protein levels are modified but cellular processes such as translation, dilution and degradation are also concerned. As a matter of fact, translation efficiency and protein degradation rates were proved to be inversely proportional to growth rate by a modelling approach. Control coefficient calculations enabled the quantification of cellular processes influences. The dynamic response of L. lactis to isoleucine starvation was studied by the progressive consumption of this amino-acid in a discontinuous batch fermentation. This poorly characterized adaptation process triggers a wide reorganization of cellular physiology that could be divided in three parts: a global repression of the main biological functions related to growth, a response more specific to the encountered stress to struggle against isoleucine starvation and an unexplained activation of oxidative stress-related cellular functions. Comparative transcriptomics allowed the implication of various mechanisms to be quantified in the regulation of this adaptation response (stringent response, growth rate adaptation mechanism, CodY, GlnR and CcpA regulation). The major biological determinants of protein intracellular concentration were mathematically investigated thanks to a covariance model selection algorithm. Codons bias (CAI) was found to be the most influent parameter, even more than mRNA concentrations, which suggests that genetic control is stronger than transcriptional adaptation. The weight of the different determinants was also found to depend on adaptation modes. The systems biology approach applied in this work enabled a better understanding of L. lactis adaptation mechanisms and will be entirely transposable to other cellular processes as well as other microorganismsTOULOUSE-INSA (315552106) / SudocSudocFranceF

PNPase is involved in the coordination of mRNA degradation and expression in stationary phase cells of Escherichia coli

Background: Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives in the stationary phase. The genome-wide mRNA half-lives were determined by a dynamic analysis of transcriptomes after transcription arrest. We have combined the analysis of mRNA half-lives with the steady-state concentrations (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. Results: The values of mRNA half-lives demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. Conclusions: This work had identified PNPase as a central player associated with mRNA degradation in stationary phase