The accumulation of adaptive mutations is essential for survival in novel environments. However, in clonal populations with a high mutational supply, the power of natural selection is expected to be limited. This is due to clonal interference - the competition of clones carrying different beneficial mutations - which leads to the loss of many small effect mutations and fixation of large effect ones. If interference is abundant, then mechanisms for horizontal transfer of genes, which allow the immediate combination of beneficial alleles in a single background, are expected to evolve. However, the relevance of interference in natural complex environments, such as the gut, is poorly known. To address this issue, we have developed an experimental system which allows to uncover the nature of the adaptive process as Escherichia coli adapts to the mouse gut. This system shows the invasion of beneficial mutations in the bacterial populations and demonstrates the pervasiveness of clonal interference. The observed dynamics of change in frequency of beneficial mutations are consistent with soft sweeps, where different adaptive mutations with similar phenotypes, arise repeatedly on different haplotypes without reaching fixation. Despite the complexity of this ecosystem, the genetic basis of the adaptive mutations revealed a striking parallelism in independently evolving populations. This was mainly characterized by the insertion of transposable elements in both coding and regulatory regions of a few genes. Interestingly, in most populations we observed a complete phenotypic sweep without loss of genetic variation. The intense clonal interference during adaptation to the gut environment, here demonstrated, may be important for our understanding of the levels of strain diversity of E. coli inhabiting the human gut microbiota and of its recombination rate. Adaptation to novel environments involves the accumulation of beneficial mutations. If these are rare the process will proceed slowly with each one sweeping to fixation on its own. On the contrary if they are common in clonal populations, individuals carrying different beneficial alleles will experience intense competition and only those clones carrying the stronger effect mutations will leave a future line of descent. This phenomenon is known as clonal interference and the extent to which it occurs in natural environments is unknown. One of the most complex natural environments for E. coli is the mammalian intestine, where it evolves in the presence of many species comprising the gut microbiota. We have studied the dynamics of adaptation of E. coli populations evolving in this relevant ecosystem. We show that clonal interference is pervasive in the mouse gut and that the targets of natural selection are similar in independently E. coli evolving populations. These results illustrate how experimental evolution in natural environments allows us to dissect the mechanisms underlying adaptation and its complex dynamics and further reveal the importance of mobile genetic elements in contributing to the adaptive diversification of bacterial populations in the gut.

Viruses that infect bacteria, or bacteriophages, are among the most abundant entities in the gut microbiome. However, their role and the mechanisms by which they infect bacteria in the intestinal tract remain poorly understood. We recently reported that intestinal bacteria are an evolutionary force, driving the expansion of the bacteriophage host range by boosting the genetic variability of these viruses. Here, we expand these observations by studying antagonistic bacteriophage-bacteria coevolution dynamics and revealing that bacterial genetic variability is also increased under the pressure of bacteriophage predation. We propose a model showing how the expansion of bacteriophage-bacteria infection networks is relative to the opportunities for coevolution encountered in the intestinal tract. Our data suggest that predator-prey dynamics are perpetuated and differentiated in parallel, to generate and maintain intestinal microbial diversity and equilibrium.

The evolution of new strains within the gut ecosystem is poorly understood. We used a natural but controlled system to follow the emergence of intraspecies diversity of commensal Escherichia coli, during three rounds of adaptation to the mouse gut (∼1,300 generations). We previously showed that, in the first round, a strongly beneficial phenotype (loss-of-function for galactitol consumption; gat-negative) spread to >90% frequency in all colonized mice. Here, we show that this loss-of-function is repeatedly reversed when a gat-negative clone colonizes new mice. The regain of function occurs via compensatory mutation and reversion, the latter leaving no trace of past adaptation. We further show that loss-of-function adaptive mutants reevolve, after colonization with an evolved gat-positive clone. Thus, even under strong bottlenecks a regime of strong-mutation-strong-selection dominates adaptation. Coupling experiments and modeling, we establish that reverse evolution recurrently generates two coexisting phenotypes within the microbiota that can or not consume galactitol (gat-positive and gat-negative, respectively). Although the abundance of the dominant strain, the gat-negative, depends on the microbiota composition, gat-positive abundance is independent of the microbiota composition and can be precisely manipulated by supplementing the diet with galactitol. These results show that a specific diet is able to change the abundance of specific strains. Importantly, we find polymorphism for these phenotypes in indigenous Enterobacteria of mice and man. Our results demonstrate that natural selection can greatly overwhelm genetic drift at structuring the strain diversity of gut commensals and that competition for limiting resources may be a key mechanism for maintaining polymorphism in the gut.

The relative role of drift versus selection underlying the evolution of bacterial species within the gut microbiota remains poorly understood. The large sizes of bacterial populations in this environment suggest that even adaptive mutations with weak effects, thought to be the most frequently occurring, could substantially contribute to a rapid pace of evolutionary change in the gut. We followed the emergence of intra-species diversity in a commensal Escherichia coli strain that previously acquired an adaptive mutation with strong effect during one week of colonization of the mouse gut. Following this first step, which consisted of inactivating a metabolic operon, one third of the subsequent adaptive mutations were found to have a selective effect as high as the first. Nevertheless, the order of the adaptive steps was strongly affected by a mutational hotspot with an exceptionally high mutation rate of 10⁻⁵. The pattern of polymorphism emerging in the populations evolving within different hosts was characterized by periodic selection, which reduced diversity, but also frequency-dependent selection, actively maintaining genetic diversity. Furthermore, the continuous emergence of similar phenotypes due to distinct mutations, known as clonal interference, was pervasive. Evolutionary change within the gut is therefore highly repeatable within and across hosts, with adaptive mutations of selection coefficients as strong as 12% accumulating without strong constraints on genetic background. In vivo competitive assays showed that one of the second steps (focA) exhibited positive epistasis with the first, while another (dcuB) exhibited negative epistasis. The data shows that strong effect adaptive mutations continuously recur in gut commensal bacterial species. The relative contribution of random loss and migration versus de novo mutation to the overall diversity of the gut microbiota is far from understood. Population sizes of bacterial communities inhabiting the gut can be very large and therefore, both weak and strong effect beneficial mutations theoretically have the opportunity to contribute to adaptation. Here, by discretizing the adaptive steps that occur during colonization by a gut commensal, we uncover a mutational hotspot, large effect mutations and different forms of natural selection as the core ingredients to the emergence of diversity in this environment. We show that, on occasion, strong periodic selection can create and reduce diversity but then recurrent mutation generates new adaptive variants that compete for fixation. Selection for keeping particular adaptive variants at low frequency, balancing selection, was also shown to be pervasive. Unexpectedly, given the complexity of the gut ecosystem, we find a highly repeatable evolutionary process, motivated by a mutational hotspot and strong effect adaptive mutations continuously recurring in commensal bacterial species.