Scatter Plots Showing Effects of the <i>crp</i> Deletion on Expression Profiles of the Ancestor and Two Evolved Lines

<div><p>All axes are log₁₀-transformed standardized expression levels. Points falling above (below) the diagonal have higher (lower) expression in the absence of <i>crp</i>.</p><p>(A) Comparison of ancestor and its <i>crp</i>⁻ derivative.</p><p>(B) Comparison of evolved Ara-1 strain and its <i>crp</i>⁻ derivative.</p><p>(C) Comparison of evolved Ara+1 strain and its <i>crp</i>⁻ derivative. Genes whose expression was significantly (<i>p</i> < 0.05) affected by the <i>crp</i> deletion are highlighted as follows.</p><p>(A) Solid black symbols, dependent on <i>crp</i> in the ancestor (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0040035#pgen-0040035-g001" target="_blank">Figure 1</a>, regions D, E, F, and G).</p><p>(B,C) Solid black symbols, <i>crp</i>-dependent in the ancestor and corresponding evolved line (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0040035#pgen-0040035-g001" target="_blank">Figure 1</a>, regions D and E for Ara-1, regions E and F for Ara+1); hollow black symbols, <i>crp</i>-dependent in ancestor only (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0040035#pgen-0040035-g001" target="_blank">Figure 1</a>, regions F and G for Ara-1, regions D and G for Ara+1); solid red symbols, newly <i>crp</i>-dependent in both evolved lines (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0040035#pgen-0040035-g001" target="_blank">Figure 1</a>, region B); hollow red symbols, newly <i>crp</i>-dependent in only one evolved line (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0040035#pgen-0040035-g001" target="_blank">Figure 1</a>, region A for Ara-1, region C for Ara+1). Genes that were not <i>crp</i>-dependent in any of the three backgrounds are shown as gray dots in all three panels.</p></div

Venn Diagram Showing the Relationship among the CRP Regulons Inferred for Three Genetic Backgrounds Based on Their Whole-Genome Transcription Profiles

<p>Ara-1 and Ara+1 are two lineages independently derived from the Ancestor by evolution for 20,000 generations in glucose-limited minimal medium. The union of all three circles defines the <i>crp</i> meta-regulon, while their intersection (region E) defines the ‘core' regulon. Those genes in the intersection between the two evolved lines (region B) have independently evolved dependence on <i>crp</i>. The size of each circle is proportional to the number of genes in the corresponding inferred CRP regulon. The numbers of genes in each section of the diagram are as follows: A, 569; B, 117; C, 232; D, 30; E, 25; F, 18; G, 98. See text for further details.</p

Regulon-Level Analysis of Evolved Changes in <i>crp</i>-dependent Gene Expression

<p>Regulons include sets of genes that are known to be directly controlled by a particular regulatory gene. The <i>y</i>-axis shows the significance level of the change in a regulon's dependence on <i>crp</i> in an evolved background relative to the ancestor, where a double Z-score value of 2 corresponds to a <i>p-</i>value of ∼0.05. The 135 regulons characterized in E. coli are arranged alphabetically along the <i>x</i>-axis, but only those regulons that changed significantly in both evolved lines are listed by name. Blue and red symbols indicate regulons that became significantly more or less sensitive, respectively, to the <i>crp</i> deletion in an evolved background. See text for additional information.</p

Viruses Evolved under Low Co-Infection Are More Robust than Those Evolved under High Co-Infection

<p>Each point is the mean (<i>n</i> = 3, ± standard error of the mean) change in log₁₀ fitness (Δlog₁₀<i>W</i>) resulting from mutation accumulation, for an independent lineage founded by a virus clone evolved under low level of co-infection (circles) or high level of co-infection (squares). Horizontal lines are grand means for Δlog₁₀<i>W</i> among lineages within a treatment, and the dashed lines indicate one standard deviation away from the grand mean.</p

Differences in Virus Robustness Are Not Confounded by Increased Mutation Frequencies in Viruses Evolved under High Co-Infection

<p>Mean mutation frequency (± 95% confidence interval) was assayed for wild-type ϕ6 (ANC), and for one pre-bottleneck clone isolated from each population evolved under low co-infection (L1–L3) and high co-infection (H1–H3). Assays on <i>P. tomato</i> (circles) and <i>P. atrofaciens</i> (squares) were replicated 5-fold. Assays for <i>P. atrofaciens</i> mutants challenged with growth on <i>P. tomato</i> (diamonds) were replicated 6-fold, except for H3 (<i>n</i> = 2).</p