The interplay between nutrition and the microbial communities colonizing the gastrointestinal tract (i.e., gut microbiota) determines juvenile growth trajectory. Nutritional deficiencies trigger developmental delays, and an immature gut microbiota is a hallmark of pathologies related to childhood undernutrition. However, how host-associated bacteria modulate the impact of nutrition on juvenile growth remains elusive. Here, using gnotobiotic Drosophila melanogaster larvae independently associated with Acetobacter pomorum WJL (Ap WJL) and Lactobacillus plantarum NC8 (Lp NC8), 2 model Drosophila-associated bacteria, we performed a large-scale, systematic nutritional screen based on larval growth in 40 different and precisely controlled nutritional environments. We combined these results with genome-based metabolic network reconstruction to define the biosynthetic capacities of Drosophila germ-free (GF) larvae and its 2 bacterial partners. We first established that Ap WJL and Lp NC8 differentially fulfill the nutritional requirements of the ex-GF larvae and parsed such difference down to individual amino acids, vitamins, other micronutrients, and trace metals. We found that Drosophila-associated bacteria not only fortify the host's diet with essential nutrients but, in specific instances, functionally compensate for host auxotrophies by either providing a metabolic intermediate or nutrient derivative to the host or by uptaking, concentrating, and delivering contaminant traces of micronutrients. Our systematic work reveals that beyond the molecular dialogue engaged between the host and its bacterial partners, Drosophila and its associated bacteria establish an integrated nutritional network relying on nutrient provision and utilization.

BackgroundPredicting 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.ResultsSimulations 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.ConclusionThe evoFBA framework represents a promising new way to model biochemical evolution, one that can generate testable predictions about evolutionary and ecosystem-level outcomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0733-x) contains supplementary material, which is available to authorized users.

HIGHLIGHTS L. plantarum feeds lactate to A. pomorum A. pomorum supplies essential amino acids and vitamins to L. plantarum Microbiota metabolic dialogue boosts Drosophila's larval growth Lactate utilization by Acetobacter releases anabolic metabolites to larvae

Insertion sequences (IS) are ubiquitous bacterial mobile genetic elements, and the mutations they cause can be deleterious, neutral, or beneficial. The long-term dynamics of IS elements and their effects on bacteria are poorly understood, including whether they are primarily genomic parasites or important drivers of adaptation by natural selection. Here, we investigate the dynamics of IS elements and their contribution to genomic evolution and fitness during a long-term experiment with Escherichia coli. IS elements account for ~35% of the mutations that reached high frequency through 50,000 generations in those populations that retained the ancestral point-mutation rate. In mutator populations, IS-mediated mutations are only half as frequent in absolute numbers. In one population, an exceptionally high ~8-fold increase in IS150 copy number is associated with the beneficial effects of early insertion mutations; however, this expansion later slowed down owing to reduced IS150 activity. This population also achieves the lowest fitness, suggesting that some avenues for further adaptation are precluded by the IS150-mediated mutations. More generally, across all populations, we find that higher IS activity becomes detrimental to adaptation over evolutionary time. Therefore, IS-mediated mutations can both promote and constrain evolvability.

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Metabolic 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 Evo2Sim, 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 evolvable 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 polymorphism, 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.

SUMMARYThe gut microbiota shapes animal growth trajectory in stressful nutritional environments, but the molecular mechanisms behind such physiological benefits remain poorly understood. The gut microbiota is mostly composed of bacteria, which construct metabolic networks among themselves and with the host. Until now, how the metabolic activities of the microbiota contribute to host juvenile growth remains unknown. Here, using Drosophila as a host model, we report that two of its major bacterial partners, Lactobacillus plantarum and Acetobacter pomorum engage in a beneficial metabolic dialogue that boosts host juvenile growth despite nutritional stress. We pinpoint that lactate, produced by L. plantarum, is utilized by A. pomorum as an additional carbon source, and A. pomorum provides essential amino-acids and vitamins to L. plantarum. Such bacterial cross-feeding provisions a set of anabolic metabolites to the host, which may foster host systemic growth despite poor nutrition.GRAPHICAL ABSTRACTHIGHLIGHTSL. plantarum feeds lactate to A. pomorumA. pomorum supplies essential amino acids and vitamins to L. plantarumMicrobiota metabolic dialogue boosts Drosophila’s larval growthLactate utilization by Acetobacter releases anabolic metabolites to larvae

12The interplay between nutrition and the microbial communities colonizing the gastro-13 intestinal tract (i.e. gut microbiota) determines juvenile growth trajectory. Nutritional 14 deficiencies trigger developmental delays, and an immature gut microbiota is a 15 hallmark of pathologies related to childhood undernutrition. However, how 16 commensal bacteria modulate the impact of nutrition on juvenile growth remains 17 elusive. Here, using gnotobiotic Drosophila melanogaster larvae independently 18 associated with two model commensal bacterial strains, Acetobacter pomorum WJL 19 (Ap WJL ) and Lactobacillus plantarum NC8 (Lp NC8 ), we performed a large-scale, 20 systematic nutritional screen based on larval growth in 40 different and precisely 21 controlled nutritional environments. We combined these results with genome-based 22 metabolic network reconstruction to define the biosynthetic capacities of Drosophila 23 germ-free (GF) larvae and its two commensal bacteria. We first established that 24 Ap WJL and Lp NC8 differentially fulfills the nutritional requirements of the ex-GF larvae 25 and parsed such difference down to individual amino acids, vitamins, other 26 micronutrients and trace metals. We found that Drosophila commensal bacteria not 27 only fortify the host's diet with essential nutrients but, in specific instances, 28 functionally compensate for host auxotrophies, by either providing a metabolic 29 intermediate or nutrient derivative to the host or by uptaking, concentrating and 30 sparing contaminant traces of micronutrients. Our systematic work reveals that, 31 beyond the molecular dialogue engaged between the host and its commensal 32 partners, Drosophila and its facultative bacterial partners establish an integrated 33 nutritional network relying on nutrients sparing and utilization. 34 4 pathways increase plasma biomarkers and levels of mediators of growth, bone 61 formation, neurodevelopment, and immune function in children with moderate acute 62 malnutrition [7]. These studies clearly show that microbes strongly impact how 63 organisms respond to changes in their nutritional environment. 64Diverse animal models are employed to decipher the physiological, ecological, 65 genetic and molecular mechanisms underpinning host/microbiota/diet interactions. 66Among them, Drosophila melanogaster is frequently chosen to study the impact of 67 the nutritional environment on growth and development thanks to its short growth 68 period as well as easy and cost-effective rearing conditions. During the juvenile 69 phase of the Drosophila life cycle, larvae feed constantly and increase their body 70 mass ~200 times until entry into metamorphosis [8]. However, the pace and duration 71 of larval growth can be altered by the nutritional context and the host-associated 72 microbes [9][10][11]. Like other animals, Drosophila live in constant association with 73 commensal microbes, including bacteria and yeast [12]. The impact of the host-74 associated microbes can be systematically assessed by generating gnotobiotic ...