Metabolic modelling in a dynamic evolutionary framework predicts adaptive diversification of bacteria in a long-term evolution experiment

Background: Predicting adaptive trajectories is a major goal of evolutionary biology and useful for practical applications. Systems biology has enabled the development of genome-scale metabolic models. However, analysing these models via flux balance analysis (FBA) cannot predict many evolutionary outcomes including adaptive diversification, whereby an ancestral lineage diverges to fill multiple niches. Here we combine in silico evolution with FBA and apply this modelling framework, evoFBA, to a long-term evolution experiment with Escherichia coli. Results: Simulations predicted the adaptive diversification that occurred in one experimental population and generated hypotheses about the mechanisms that promoted coexistence of the diverged lineages. We experimentally tested and, on balance, verified these mechanisms, showing that diversification involved niche construction and character displacement through differential nutrient uptake and altered metabolic regulation. Conclusion: The evoFBA framework represents a promising new way to model biochemical evolution, one that can generate testable predictions about evolutionary and ecosystem-level outcomes

Bacilos Gram negativos entéricos y no fermentadores de la glucosa en pacientes con enfermedad periimplante

Introduction. Implants can be colonized by microorganisms from oral biofilms and may affect peri-implant tissues health. Among these bacteria, pathogens typically associated with periodontitis can be found, such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as Gram negative enteric bacilli not typically associated with periodontal diseases.Objective. Superinfecting bacteria were characterized from peri-implant lesions in patients with history of periodontitis.Materials and methods. Sixty-eight implants were studied in 55 patients; the average patient age was 56 years. Forty-nine implants had peri-implant lesions and 19 were considered stable. Subgingival samples were obtained in affected and stable implants. The samples were streaked on Mac-Conkey agar and incubated at 37°C for 24 hours. The colonies were identified with the kit-BD BBL Crystal E/NF®.Results. Superinfecting organisms were detected in 20 patients--they were seen more frequently at diseased implants (n=15) than at healthy implants (n=5). The prevalence of superinfecting bacteria on the selected implants was 33.8% (n=23/68). These bacteria were more prevalent among affected implants (n=17 or 25%) than those with stable implants n=6 (8.8%). Klebsiella pneumoniae was the most frequent Gram negative rod detected (n=12).Conclusions. One-third of the implants had superinfecting organisms. Implants with a peri-implant lesion had a higher frequency of superinfecting bacteria. Klebsiella pneumoniae was the most common superinfecting organism isolated. A multiple infection caused by superinfecting bacteria was present only at diseased implants. These microbial agents potentially affect implant stability.Introducción. Los implantes pueden ser colonizados por microorganismos de la biopelícula oral y así afectarse su salud. Entre los patógenos encontrados están los típicamente relacionados con periodontitis, como Aggregatibacter actinomycetemcomitans y Porphyromonas gingivalis, así como bacilos Gram negativos entéricos, los cuales no son clásicos de la enfermedad periodontal.Objetivo. Determinar el perfil de las bacterias sobreinfecciosas en lesiones periimplante (mucositis o periimplantitis), en pacientes con historia de periodontitis.Materiales y métodos. Se estudiaron 68 implantes en 55 pacientes; 49 implantes tuvieron lesión periimplante19 se consideraron estables. Se obtuvieron muestras subgingivales de implantes afectados y estables; éstas fueron sembradas en Agar MacConkey e incubadas a 37 °C por 24 horas. Las colonias aisladas fueron identificadas con el estuche BD BBL-Cristal E/NF®.Resultados. En 20 de los pacientes estudiados se detectaron bacterias sobreinfecciosas, pero, con mayor frecuencia en pacientes con implantes afectados (n=15) que en portadores de implantes sanos (n=5). La prevalencia de bacterias sobreinfecciosas en todos los implantes fue de 33,8 % (n=23/68), y también fue más frecuente su aislamiento en implantes afectados (n=17) (25%), que en estables (n=6) (8,8%). Klebsiella pneumoniae fue el microorganismo que se aisló con mayor frecuencia en todos los implantes (n=12).Conclusiones. Un tercio de los implantes estudiados presentaron organismos sobreinfecciosos. Los implantes con lesiones periimplante presentan una mayor frecuencia de bacterias sobreinfecciosas. K. pneumoniae es la especie sobreinfecciosa más frecuente en los implantes estudiados. Se presentó infección múltiple con dichas bacterias en los implantes afectados. Estos microorganismos pueden afectar la estabilidad de los implantes

Ecological and molecular bases of adaptive diversification in Escherichia coli

Les événements de diversification adaptative sont des éléments primordiaux de l'évolution. En effet, ils engendrent des innovations phénotypiques telles que la colonisation de nouvelles niches écologiques et au final, la spéciation. Afin d'étudier les ressorts écologiques et moléculaires de la diversification adaptative, nous utilisons la plus longue des expériences d'évolution en cours. Depuis 1988, soit plus de 60 000 générations, douze populations indépendantes issues d'un ancêtre commun d'Escherichia coli sont propagées quotidiennement dans un milieu minimum comportant une faible quantité de glucose.Un événement unique de diversification s'est produit dans une des 12 populations (Ara–2). Deux lignées de phénotypes différents sont apparues après environ 6500 générations, les S pour «Small» et les L pour «Large», chacune présentant des tailles cellulaires différentes. Les deux lignées coexistent grâce à une sélection négative dépendant de la fréquence qui favorise la lignée la plus rare et permet de supplanter sa concurrente; ainsi, aucune des deux lignées ne s'éteint. Avant l’événement de diversification, la population Ara–2 a développé un phénotype hypermutateur suite à la mutation d'un gène de réparation de l'ADN. L'objectif de cette thèse est de caractériser les mécanismes écologiques, physiologiques et moléculaires sous-tendant l'émergence et la coexistence des lignées S et L.En premier lieu, nous avons utilisé un ensemble d'expériences d'évolution in vivo et in silico afin de déterminer les moteurs écologiques et physiologiques de l'émergence de ce polymorphisme. Plusieurs mécanismes écologiques, incluant les compromis (trade-off évolutifs), la saisonnabilité et les déplacements de caractères interviennent dans l'émergence et la persistance de la diversité au long terme. Nous avons montré que la lignée L, en produisant de l'acétate, créait une nouvelle opportunité écologique exploitée par les S. De plus, au cours du temps, les S et les L s'adaptent à leur niche écologique, respectivement l'acétate et le glucose.En second lieu, nous avons cultivé les S et les L séparément pour éliminer la compétition entre les deux lignées. Dans ces conditions, il y a perte des interactions dépendantes de la fréquence entre les S et les L. Ceci démontre l'importance de la compétition dans le maintien du polymorphisme.En troisième lieu, nous avons combiné des approches génétiques, physiologiques et biochimiques pour déterminer le rôle, dans l'émergence du polymorphisme, d'une mutation spécifique aux S survenant dans le gène arcA, codant un régulateur global. Nous avons montré que l'allèle évolué de arcA augmentait la transcription de gènes du métabolisme de l'acétate dans la lignée S. Au cours de cette étude, nous avons identifié une mutation supplémentaire dans le gène acs, impliqué dans le métabolisme de l'acétate, intervenant dans l'émergence de la lignée S. Nous avons aussi démontré que ces deux mutations étaient favorables à la lignée S au début de son émergence, puis que des mutations plus tardives agissaient de façon épistatiques avec les allèles évolués de acs et de arcA. Ainsi, ces résultats démontrent que l'établissement et le maintien du polymorphisme des S et des L est un processus en plusieurs étapes nécessitant des interactions épistatiques entre plusieurs mutations.En quatrième lieu, nous avons identifié la dynamique au long terme des taux de mutations dans cette population. L'apparition et l'invasion rapide du phénotype hypermutateur est suivie d'une réversion complète mais indépendante dans chacune des lignées S et L.L'émergence d'un polymorphisme bactérien durable reflète une restructuration complexe des réseaux métaboliques et de régulation dans ces lignées qui co-existent, ce qui aboutit à l'apparition et à l'exploitation de nouvelles opportunités écologiques. La compétition et l'évolution de l'utilisation de ressources différentes sont des forces sélectives permettant le maintien du polymorphisme.Diversification events are central issues in evolution since they generate phenotypic innovation such as colonization of novel ecological niches and, ultimately, speciation. To study the ecological and molecular drivers of adaptive diversification, we used the longest still-running evolution experiment. Twelve independent populations are propagated in a glucose limited minimal medium from a common ancestor of Escherichia coli by serial daily transfers since 1988 for more than 60,000 generations. In one of the twelve populations, called Ara–2, a unique diversification event occurred: two phenotypically-differentiated lineages, named S (Small) and L (Large) according to their cell size, emerged from a common ancestor at ~ 6500 generations. The two lineages co-exist ever since, owing to negative frequency-dependent selection whereby each lineage is favored and invades the other when rare, such that no lineage gets extinct. Moreover, and before the split between the two S and L lineages, the population Ara–2 evolved a hypermutator phenotype, owing to a defect in a DNA repair gene. The objective of this thesis is to characterize the ecological, physiological and molecular mechanisms that allowed the emergence and stable co-existence of the S and L lineages.First, we used a combination of in vivo and in silico experimental evolution to determine the ecological and physiological drivers of the emergence of the polymorphism. Several ecological mechanisms including tradeoff, seasonality and character displacement are involved in the emergence and long-term persistence of diversity. In particular, we showed that the L lineage secretes acetate which generates a new ecological opportunity that the S lineage exploited. In addition, the S and L lineages became fitter and fitter over time in their respective ecological niches, respectively acetate and glucose. Second, we propagated S and L clones separately to remove competition between the two lineages. In these conditions, frequency-dependent interactions between the S and L clones evolved separately were completely abolished, revealing the importance of competition in the maintenance of the polymorphism. Third, we combined genetic, physiological and biochemical approaches to determine the role of an S-specific mutation that was previously found in arcA, encoding a global regulator, in the emergence of the S and L polymorphism. We showed that the evolved arcA allele conferred to the S lineage the capacity to growth on acetate by increasing the transcription of target genes involved in acetate consumption. During this study, we found an additional mutation, in the acs gene involved in acetate metabolism, that was also involved in the emergence of the S lineage. We further showed that these two mutations were favorable to the S lineage early during its emergence, and that other mutations occurred later that interacted epistatically with the acs and arcA evolved alleles. Therefore, these data showed that the establishment and further maintenance of the S and L polymorphism was a multi-step process involving epistatic interactions between several mutations. Fourth, we identified the long-term dynamics of mutation rates in this divergent population. A first early rise of a hypermutator was followed by a full reversion of this mutator state twice independently in each of the two S and L lineages.The emergence of a long-term bacterial polymorphism reflects a complex restructuration of the metabolic and regulatory networks in the co-existing lineages, resulting in the generation and exploitation of a new ecological opportunity. Competition and evolution of divergent resource consumption were the selective forces driving the maintenance of the polymorphism

Bases écologiques et moléculaires de la diversification adaptative chez Escherichia coli

Diversification events are central issues in evolution since they generate phenotypic innovation such as colonization of novel ecological niches and, ultimately, speciation. To study the ecological and molecular drivers of adaptive diversification, we used the longest still-running evolution experiment. Twelve independent populations are propagated in a glucose limited minimal medium from a common ancestor of Escherichia coli by serial daily transfers since 1988 for more than 60,000 generations. In one of the twelve populations, called Ara–2, a unique diversification event occurred: two phenotypically-differentiated lineages, named S (Small) and L (Large) according to their cell size, emerged from a common ancestor at ~ 6500 generations. The two lineages co-exist ever since, owing to negative frequency-dependent selection whereby each lineage is favored and invades the other when rare, such that no lineage gets extinct. Moreover, and before the split between the two S and L lineages, the population Ara–2 evolved a hypermutator phenotype, owing to a defect in a DNA repair gene. The objective of this thesis is to characterize the ecological, physiological and molecular mechanisms that allowed the emergence and stable co-existence of the S and L lineages.First, we used a combination of in vivo and in silico experimental evolution to determine the ecological and physiological drivers of the emergence of the polymorphism. Several ecological mechanisms including tradeoff, seasonality and character displacement are involved in the emergence and long-term persistence of diversity. In particular, we showed that the L lineage secretes acetate which generates a new ecological opportunity that the S lineage exploited. In addition, the S and L lineages became fitter and fitter over time in their respective ecological niches, respectively acetate and glucose. Second, we propagated S and L clones separately to remove competition between the two lineages. In these conditions, frequency-dependent interactions between the S and L clones evolved separately were completely abolished, revealing the importance of competition in the maintenance of the polymorphism. Third, we combined genetic, physiological and biochemical approaches to determine the role of an S-specific mutation that was previously found in arcA, encoding a global regulator, in the emergence of the S and L polymorphism. We showed that the evolved arcA allele conferred to the S lineage the capacity to growth on acetate by increasing the transcription of target genes involved in acetate consumption. During this study, we found an additional mutation, in the acs gene involved in acetate metabolism, that was also involved in the emergence of the S lineage. We further showed that these two mutations were favorable to the S lineage early during its emergence, and that other mutations occurred later that interacted epistatically with the acs and arcA evolved alleles. Therefore, these data showed that the establishment and further maintenance of the S and L polymorphism was a multi-step process involving epistatic interactions between several mutations. Fourth, we identified the long-term dynamics of mutation rates in this divergent population. A first early rise of a hypermutator was followed by a full reversion of this mutator state twice independently in each of the two S and L lineages.The emergence of a long-term bacterial polymorphism reflects a complex restructuration of the metabolic and regulatory networks in the co-existing lineages, resulting in the generation and exploitation of a new ecological opportunity. Competition and evolution of divergent resource consumption were the selective forces driving the maintenance of the polymorphism.Les événements de diversification adaptative sont des éléments primordiaux de l'évolution. En effet, ils engendrent des innovations phénotypiques telles que la colonisation de nouvelles niches écologiques et au final, la spéciation. Afin d'étudier les ressorts écologiques et moléculaires de la diversification adaptative, nous utilisons la plus longue des expériences d'évolution en cours. Depuis 1988, soit plus de 60 000 générations, douze populations indépendantes issues d'un ancêtre commun d'Escherichia coli sont propagées quotidiennement dans un milieu minimum comportant une faible quantité de glucose.Un événement unique de diversification s'est produit dans une des 12 populations (Ara–2). Deux lignées de phénotypes différents sont apparues après environ 6500 générations, les S pour «Small» et les L pour «Large», chacune présentant des tailles cellulaires différentes. Les deux lignées coexistent grâce à une sélection négative dépendant de la fréquence qui favorise la lignée la plus rare et permet de supplanter sa concurrente; ainsi, aucune des deux lignées ne s'éteint. Avant l’événement de diversification, la population Ara–2 a développé un phénotype hypermutateur suite à la mutation d'un gène de réparation de l'ADN. L'objectif de cette thèse est de caractériser les mécanismes écologiques, physiologiques et moléculaires sous-tendant l'émergence et la coexistence des lignées S et L.En premier lieu, nous avons utilisé un ensemble d'expériences d'évolution in vivo et in silico afin de déterminer les moteurs écologiques et physiologiques de l'émergence de ce polymorphisme. Plusieurs mécanismes écologiques, incluant les compromis (trade-off évolutifs), la saisonnabilité et les déplacements de caractères interviennent dans l'émergence et la persistance de la diversité au long terme. Nous avons montré que la lignée L, en produisant de l'acétate, créait une nouvelle opportunité écologique exploitée par les S. De plus, au cours du temps, les S et les L s'adaptent à leur niche écologique, respectivement l'acétate et le glucose.En second lieu, nous avons cultivé les S et les L séparément pour éliminer la compétition entre les deux lignées. Dans ces conditions, il y a perte des interactions dépendantes de la fréquence entre les S et les L. Ceci démontre l'importance de la compétition dans le maintien du polymorphisme.En troisième lieu, nous avons combiné des approches génétiques, physiologiques et biochimiques pour déterminer le rôle, dans l'émergence du polymorphisme, d'une mutation spécifique aux S survenant dans le gène arcA, codant un régulateur global. Nous avons montré que l'allèle évolué de arcA augmentait la transcription de gènes du métabolisme de l'acétate dans la lignée S. Au cours de cette étude, nous avons identifié une mutation supplémentaire dans le gène acs, impliqué dans le métabolisme de l'acétate, intervenant dans l'émergence de la lignée S. Nous avons aussi démontré que ces deux mutations étaient favorables à la lignée S au début de son émergence, puis que des mutations plus tardives agissaient de façon épistatiques avec les allèles évolués de acs et de arcA. Ainsi, ces résultats démontrent que l'établissement et le maintien du polymorphisme des S et des L est un processus en plusieurs étapes nécessitant des interactions épistatiques entre plusieurs mutations.En quatrième lieu, nous avons identifié la dynamique au long terme des taux de mutations dans cette population. L'apparition et l'invasion rapide du phénotype hypermutateur est suivie d'une réversion complète mais indépendante dans chacune des lignées S et L.L'émergence d'un polymorphisme bactérien durable reflète une restructuration complexe des réseaux métaboliques et de régulation dans ces lignées qui co-existent, ce qui aboutit à l'apparition et à l'exploitation de nouvelles opportunités écologiques. La compétition et l'évolution de l'utilisation de ressources différentes sont des forces sélectives permettant le maintien du polymorphisme

Use of Operational Training Simulation in the Study of Ethanol Operating Conditions: A Powerful Tool for Education and Research Performance Improvement

Currently, virtual education has reached a significant boom in higher education institutions. The latter, considering health emergency due to coronavirus pandemic, which makes it difficult to re-open physical infrastructures. As a consequence of the above, there is concern about experimental development taught by higher education and research institutions, since the execution of the experimental component is still considered a challenge due to uncertainly of on-going infections. Currently, the University of Bremen, Germany has implemented an operational training simulator for the study of bioprocess based on mathematical equations and experimental confirmations. The software enables teaching of virtual handling and operation of bioreactors. Students and engineers can deal with real situations that arise in the plant operation. Fermentations in different modes to reflect its effects on productivity could be studied. The main purpose of this research is determining the best operating conditions for the ethanol continuous production using the BioProcess Trainer software. One of the most important findings suggest a bioreactor feed rate of 5 mL/min charged with 20 g/L of glucose to reach a productivity of 0.037 g/L.h. Results found here demonstrates the Bioprocess Trainer potential capacity not only in the study of industrial plant operating conditions but also regarding educational institutions since mentioned tool improves engineers’ expertise

In Silico Experimental Evolution Highlights the Influence of Environmental Seasonality on Bacterial Diversification

International audienceExperimental evolution, where fast replicating organisms are evolved in controlled environments for thousands of generations, has shown that microorganisms are able to evolve at an amazing speed: in virtually all experimental frameworks that use bacteria or viruses, important phenotypic innovations have emerged in only a few tens of generations [1], and ecological diversifications are commonly observed [2]. Experimental evolution, by providing a variety of data from genetic mutations to ecological interactions, is an excellent tool to study multilevel evolution. Unfortunately, those experiments remain a long and costly process. As an alternative, computational models of In Silico Experimental Evolution (ISEE), where artificial organisms are evolved in a computer for thousands of generations [3], have already explored a lot of theoretical questions [4,5,6]. However, these models usually include only two or three scales (typically the genome, the phenotype and the environment), strongly limiting their possibility to mimic in vivo experiments, since evolution of real microorganisms implies the interaction of a wide range of biological structures and levels. We developed a multiscale framework of ISEE. In this model, bacterial-like organisms own a genome encoding a genetic regulation network and a metabolic network, and evolve on a virtual medium for tens of thousands of generations. By up-taking nutrients and releasing by-products, organisms modify their environment , possibly leading to complex ecosystem evolution. Thus, our individual based model evolves complex genotype-to-phenotype mappings and fitness landscapes. This model allows us to study a large variety of questions raised by experimental evolution, e.g. the evolution of the genome and the genetic regulation network, the evolution of ecological interactions, and so on [3]. A more complete description of the model is available in [7] as well as on the EvoEvo project website (http://www.evoevo.eu). The Long Term Experimental Evolution (LTEE), the longest bacterial experimental evolution experiment to date [8] has revealed an ecological diversification based on a niche construction associated to a negative frequency-dependent interaction [9]. By performing ISEE experiments with our model, we studied the environmental conditions in which such a diversification could occur. More precisely , we let initial random viable populations evolve during 500,000 time steps (∼40,000 generations) in three different environments

Environmental seasonality drives digital populations towards stable cross-feeding

International audienceStable bacterial cross-feeding interactions, where one strain feeds on the waste of the other, are important to understand, as they can be a first step towards bacterial speciation. Their emergence is commonly observed in laboratory experiments using Escherichia coli as a model organism. Yet it is not clear how cross-feeding interactions can resist the invasion of a fitter mutant when the environment contains a single resource since there seems to be a single ecological niche. Here, we used digital organisms to tackle this question, allowing for detailed and fast investigations, and providing a way to disentangle generic evolutionary mechanisms from speci-ficities associated with E. coli. Digital organisms with evolv-able genomes and metabolic networks compete for resources in conditions mimicking laboratory evolution experiments. In chemostat simulations, although cross-feeding interactions regularly emerged, selective sweeps regularly purged the population of its diversity. By contrast, batch culture allowed for much more stable cross-feeding interactions, because it includes seasons and thus distinct temporal niches, thereby favoring the adaptive diversification of proto-species

Beware batch culture: Seasonality and niche construction predicted to favor bacterial adaptive diversification

International audienceMetabolic cross-feeding interactions between microbial strains are common in nature, and emerge during evolution experiments in the laboratory, even in homogeneous environments providing a single carbon source. In sympatry, when the environment is well-mixed, the reasons why emerging cross-feeding interactions may sometimes become stable and lead to monophyletic genotypic clusters occupying specific niches, named ecotypes, remain unclear. As an alternative to evolution experiments in the laboratory, we developed Evo 2 Sim, a multi-scale model of in silico experimental evolution, equipped with the whole tool case of experimental setups, competition assays, phylogenetic analysis, and, most importantly, allowing for evolvable ecological interactions. Digital organisms with an evolv-able genome structure encoding an evolvable metabolic network evolved for tens of thousands of generations in environments mimicking the dynamics of real controlled environments, including chemostat or batch culture providing a single limiting resource. We show here that the evolution of stable cross-feeding interactions requires seasonal batch conditions. In this case, adaptive diversification events result in two stably co-existing ecotypes, with one feeding on the primary resource and the other on by-products. We show that the regularity of serial transfers is essential for the maintenance of the polymor-phism, as it allows for at least two stable seasons and thus two temporal niches. A first season is externally generated by the transfer into fresh medium, while a second one is internally generated by niche construction as the provided nutrient is replaced by secreted by-products derived from bacterial growth. In chemostat conditions, even if cross-feeding interactions emerge, they are not stable on the long-term because fitter mutants eventually invade the whole population. We also show that the long-term evolution of the two stable ecotypes leads to character displacement, at the level of the metabolic network but also of the genome structure. This difference of genome structure between both ecotypes impacts the stability of the cross-feeding interaction, when the population is propagated in chemostat conditions. This study shows the crucial role played by seasonality in temporal niche partitioning and in promoting cross-feeding subgroups into stable ecotypes, a premise to sympatric speciation

Environmental seasonality drives digital populations towards stable cross-feeding

International audienceStable bacterial cross-feeding interactions, where one strain feeds on the waste of the other, are important to understand, as they can be a first step towards bacterial speciation. Their emergence is commonly observed in laboratory experiments using Escherichia coli as a model organism. Yet it is not clear how cross-feeding interactions can resist the invasion of a fitter mutant when the environment contains a single resource since there seems to be a single ecological niche. Here, we used digital organisms to tackle this question, allowing for detailed and fast investigations, and providing a way to disentangle generic evolutionary mechanisms from speci-ficities associated with E. coli. Digital organisms with evolv-able genomes and metabolic networks compete for resources in conditions mimicking laboratory evolution experiments. In chemostat simulations, although cross-feeding interactions regularly emerged, selective sweeps regularly purged the population of its diversity. By contrast, batch culture allowed for much more stable cross-feeding interactions, because it includes seasons and thus distinct temporal niches, thereby favoring the adaptive diversification of proto-species

In Silico Experimental Evolution Highlights the Influence of Environmental Seasonality on Bacterial Diversification

International audienceExperimental evolution, where fast replicating organisms are evolved in controlled environments for thousands of generations, has shown that microorganisms are able to evolve at an amazing speed: in virtually all experimental frameworks that use bacteria or viruses, important phenotypic innovations have emerged in only a few tens of generations [1], and ecological diversifications are commonly observed [2]. Experimental evolution, by providing a variety of data from genetic mutations to ecological interactions, is an excellent tool to study multilevel evolution. Unfortunately, those experiments remain a long and costly process. As an alternative, computational models of In Silico Experimental Evolution (ISEE), where artificial organisms are evolved in a computer for thousands of generations [3], have already explored a lot of theoretical questions [4,5,6]. However, these models usually include only two or three scales (typically the genome, the phenotype and the environment), strongly limiting their possibility to mimic in vivo experiments, since evolution of real microorganisms implies the interaction of a wide range of biological structures and levels. We developed a multiscale framework of ISEE. In this model, bacterial-like organisms own a genome encoding a genetic regulation network and a metabolic network, and evolve on a virtual medium for tens of thousands of generations. By up-taking nutrients and releasing by-products, organisms modify their environment , possibly leading to complex ecosystem evolution. Thus, our individual based model evolves complex genotype-to-phenotype mappings and fitness landscapes. This model allows us to study a large variety of questions raised by experimental evolution, e.g. the evolution of the genome and the genetic regulation network, the evolution of ecological interactions, and so on [3]. A more complete description of the model is available in [7] as well as on the EvoEvo project website (http://www.evoevo.eu). The Long Term Experimental Evolution (LTEE), the longest bacterial experimental evolution experiment to date [8] has revealed an ecological diversification based on a niche construction associated to a negative frequency-dependent interaction [9]. By performing ISEE experiments with our model, we studied the environmental conditions in which such a diversification could occur. More precisely , we let initial random viable populations evolve during 500,000 time steps (∼40,000 generations) in three different environments