<i>Ex Uno Plures</i>: Clonal Reinforcement Drives Evolution of a Simple Microbial Community

<div><p>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 <i>E. coli</i> 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 <u>S</u>ingle <u>N</u>ucleotide <u>P</u>olymorphism 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.</p></div

Gene expression and SNPs among loci that mediate glycolysis and fermentation.

<p>Mutations that may affect glycolysis and fermentation are restricted to the glucose-scavenger CV103. Green denotes lower transcription level in monoculture chemostats relative to the ancestor JA122. Red indicates higher transcript levels and grey denotes no transcript level change relative to JA122. Blue colored genes are those that show variable expression; normalized log₂ expression ratios of the evolved strain relative to JA122 are shown in the inset table (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#s3" target="_blank">Materials and Methods</a>). Positive values indicate increased expression in the evolved isolates while negative values denote decreased expression. Stars and corresponding text denote the location and type of particular SNPs. Consistent with strain representations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g001" target="_blank">Figure 1</a>, light blue stars indicate ancestral mutations present in JA122, purple indicates SNPs present in CV101, yellow indicates SNPs in CV103, green indicates those in CV115/116, brown indicates SNPs shared by CV101/CV103/CV115/CV116 and pink denotes SNPs shared by CV101 and CV115/116.</p

Strain characteristics.

1<p>Ref. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen.1004430-Adams1" target="_blank">[149]</a>.</p>2<p>Data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen.1004430-Helling1" target="_blank">[3]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-t001" target="_blank">Table 1</a>.</p>3<p>Data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen.1004430-Rosenzweig2" target="_blank">[31]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-t002" target="_blank">Table 2</a>.</p

Types of Single Nucleotide Polymorphisms (SNPs) and their distribution among evolved strains.

<p>Types of Single Nucleotide Polymorphisms (SNPs) and their distribution among evolved strains.</p

Gene expression and SNPs among loci that mediate glycerol and acetate uptake/metabolism.

<p>As in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g002" target="_blank">Figure 2</a>, a green color indicates that the gene has a lower transcription level in monoculture chemostats relative to the ancestor JA122. A red color denotes elevated monoculture transcript levels while grey denotes no change in transcript level. Expression levels of loci that vary significantly in a strain-specific manner are shown in blue; for these genes, the normalized log₂ expression ratios of the evolved strain relative to JA122 are shown in the inset table (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#s3" target="_blank">Materials and Methods</a>). Positive values indicate increased expression in the evolved isolates while negative values denote decreased expression. Transcript ratios for these genes, relative to the ancestor JA122 grown under identical conditions, are presented in the inset table. Stars and corresponding text denote the location and type of particular SNPs. Consistent with strain representations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g001" target="_blank">Figure 1</a>, light blue stars indicate ancestral mutations present in JA122, purple indicates SNPs present in CV101, yellow indicates SNPs in CV103, green indicates those in CV115/116, brown indicates SNPs shared by CV101/CV103/CV115/CV116 and pink denotes SNPs shared by CV101 and CV115/116.</p

SNPs in genes involved phospholipid biosynthesis and that contribute to glycerol synthesis.

<p>Three possible routes to the production of glycerol in <i>E. coli</i> are shown along with corresponding gene names and SNPs. CV103 carries mutations that affect (A) phospholipid biosynthesis (B) the interconversion of glycerol-3-phosphate and glycerol and (C) the interconversion of glycerol-1-phosphate and glycerol. A red color denotes elevated monoculture transcript levels while grey denotes no change in transcript level. Stars (★) and corresponding text denote the location and type of particular SNPs. Consistent with strain representations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g001" target="_blank">Figure 1</a> light blue stars indicate ancestral mutations present in JA122, purple indicates SNPs in CV101, yellow indicates SNPs in CV103, green indicates those in CV115/116, brown indicates SNPs shared by CV101/CV103/CV115/CV116 and pink denotes SNPs shared by CV101 and CV115/116.</p

Gene expression and SNPs among loci in the TCA cycle and glyoxylate shunt.

<p>Loci that are part of the TCA cycle are associated with SNPs in CV103, CV115/CV116 and the ancestor JA122. Transcriptional profiling SAM analysis shows that many of the TCA cycle genes are up-regulated in the evolved strains relative to their comon ancestor, while two genes that control the TCA/glyoxylate switch point (<i>icd</i>, <i>acnB</i>) are expressed at a lower level in CV103. Blue colored genes are those that show variable expression among strains; normalized log₂ expression ratios of the evolved strain relative to JA122 are shown in the inset table (for details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#s3" target="_blank">Materials and Methods</a>). Color coding and symbols are the same as for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g002" target="_blank">Figures 2</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g005" target="_blank">5</a>. Stars and corresponding text denote the location and type of particular SNPs. Consistent with strain representations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g001" target="_blank">Figure 1</a> light blue stars indicate ancestral mutations present in JA122, purple indicates SNPs in CV101, yellow indicates SNPs in CV103, green indicates those in CV115/116, brown indicates SNPs shared by CV101/CV103/CV115/CV116 and pink denotes SNPs shared by CV101 and CV115/116.</p

Gene expression and SNPs among loci that mediate glucose uptake.

<p>Several SNPs occur in or upstream of genes known or suspected to be involved in glucose uptake. Loci shown in green have lower monoculture transcription levels in all evolved isolates compared to the ancestor JA122, while loci shown in red have elevated monoculture transcript levels. Grey denotes no change in transcript level detected. Loci depicted in blue have different gene expression levels depending on the strain tested, and for these genes, the normalized log₂ expression ratios of the evolved strain relative to JA122 are shown in the inset table (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#s3" target="_blank">Materials and Methods</a>). Positive values indicate increased expression in the evolved isolates while negative values denote decreased expression. SNPs thought to affect proteins involved in glucose transport are indicated by stars with strain and mutation details as indicated. Consistent with strain representations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g001" target="_blank">Figure 1</a> light blue stars indicate ancestral mutations present in JA122, purple indicates SNPs in CV101, yellow indicates SNPs in CV103, green indicates those in CV115/116, brown indicates SNPs shared by CV101/CV103/CV115/CV116 and pink denotes SNPs shared by CV101 and CV115/116.</p

Distribution of SNPs in pathways that mediate pyruvate catabolism.

<p>Several SNPs were detected in genes involved in the conversion of pyruvate into acetate/acetyl Co-A in CV103. The four routes with their respective cofactors are shown on the left. Genes involved in the conversion are shown on the right with asterisks denoting those genes that have a SNP in CV103. Stars and corresponding text denote the location and type of particular SNPs. Consistent with strain representations in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004430#pgen-1004430-g001" target="_blank">Figure 1</a> light blue stars indicate ancestral mutations present in JA122, purple indicates SNPs present in CV101, yellow indicates SNPs in CV103, green indicates those in CV115/116, brown indicates SNPs shared by CV101/CV103/CV115/CV116 and pink denotes SNPs shared by CV101 and CV115/116.</p

Development of a Multi Kilogram-Scale, Tandem Cyclopropanation Ring-Expansion Reaction en Route to Hedgehog Antagonist IPI-926

The formation of the d-homocyclopamine ring system in IPI-926 is the key step in its semisynthesis and proceeds via a chemoselective cyclopropanation followed by a stereoselective acid-catalyzed carbocation rearrangement. In order to perform large-scale cyclopropanation reactions, we developed new iodomethylzinc bis­(aryl)­phosphate reagents that were found to be both effective and safe. These soluble reagents can be prepared under mild conditions and are stable during the course of the reaction. Importantly, they have favorable energetics relative to other cyclopropanating agents such as EtZnCH₂I. Herein, we describe the process optimization studies that led to successful large-scale production of the d-homocyclopamine core necessary for IPI-926