Ex Uno Plures: Clonal Reinforcement Drives Evolution of a Simple Microbial Community

Abstract

A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of E. coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community's underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones. Underlying CV103's phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an “engine” generating diversity by creating new metabolic niches, but not the occupants themselves.

Stability of Cross-Feeding Polymorphisms in Microbial Communities

“…This is consistent with the findings of [28] and is further confirmed by measuring cell redox balance (NADH/NAD+) in chemostat monocultures of the two cell types (S1 Supporting Information). …”

“…Also, beneficial mutations that enable increased glucose assimilation may arise in a lineage before it acquires the capacity to scavenge acetate. This clearly happened in the Kinnersley et al experiments [28]. Phylogenetic analyses suggest that the glucose specialist and acetate specialist clades diverged early in evolutionary process: they share only one derived Single Nucleotide Polymorphism, while they differ by hundreds of SNPs, including different mutations that increase expression of LamB glycoporin.…”

“…Genetic changes that influence glucose assimilation arose early in this population [28], and similar changes appear to have arisen in replicate populations [4]. Mutants with improved glucose assimilation, termed glucose specialists, were found to be the “engine” generating diversity, not because they produce new genetic variants, but because they create new niches via the wasteful consumption of the limiting primary resource, glucose [28]. The creation of new niches in the form of overflow metabolites opens the door to specialists that can profitably use these resources.…”

The spectrum of adaptive mutations in experimental evolution

“…Sequencing of endpoint clones can miss much of the genetic variation in the population. In particular, sequencing of clones will fail to detect subpopulations which may be common in experimental evolution due to balancing selection (Kinnersley et al 2014; Lang et al 2011; Mcdonald et al 2009; Traverse et al 2013). In addition, sequencing of clones will identify a random subset of low-frequency mutations that happened to be present in the clone that was selected to be representative of the population.…”

Experimental Microbial Evolution of Extremophiles

“…Furthermore, experimental evolution provides an advantage over reverse geneticsbased approaches that employ targeted activation or inactivation of genes in that ALE can result in occurrence of mutations of unexpected composition that provide gain of function for the organism (Conrad et al 2011). Growing knowledge about the biochemical and physiological natures of some of the model organisms along with the use of genetic and mapping techniques developed in the 1980s and 1990s, has opened up new avenues (Helling et al 1987;Rosenzweig et al 1994;Treves et al 1998;Kinnersley et al 2014;Ferenci 2007;Adams et al 1992). These same approaches have been used to test both experimentally-evolved and naturally occurring microorganisms for genome amplification, deletion, insertion and rearrangement of genes or sequences (Cooper et al 2003;Philippe et al 2007;Kadam et al 2008;Bachmann et al 2012;Gresham et al 2008;Wenger et al 2011).…”