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

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

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

raw_competition_data

Text file containing raw data from all competition experiments. All competitions were performed over 2 transfer cycles. Fitness values from this file are summarized in 'genotype_fitness.txt', which is the input file for all analyses presented in Khan et al. (2011) Negative epistasis between beneficial mutations in an evolving bacterial population

genotype_fitness

Text file containing all replicate fitness measurements of each constructed genotype. Includes columns: strain, strain number; genotype, beneficial mutations present where r = rbs, t = topA, s = spoT, g = glmUS and p = pykF; mutations, number of beneficial mutations in the genotype; w, fitness estimate; block, experimental block; Plate, physical container used for the competition. This file is the input file for all analyses presented in Khan et al. (2011) Negative epistasis between beneficial mutations in an evolving bacterial population

Khan_etal_Fig4

R script to generate figure 4 of Khan et al. (2011). Takes as input the file: genotype_fitness.txt

Khan_etal_analysis

R script used to: estimate beneficial mutation effects and epistasis parameters, perform statistical tests and generate all figures presented in Khan et al. (2011), except figures 1 and 4. Takes as input the file: genotype_fitness.txt

Khan_etal_Fig1

R script to generate figure 1 of Khan et al. (2011). Takes as input the file: genotype_fitness.txt

fig1.b R script

R script to generate figure 1

fig4_mutations_in_founder_also_in_replay

Input file for fig. 4 R script. Summarizes mutations present in founders that also occurred in some replay clones. These mutations are omitted from calculation of the convergence metric