The <i>gat</i> locus is a mutational hotspot.

<p>The graph shows the log₁₀ mutation rate (μ) (per locus, per generation) for each phenotype: <i>gat</i>-negative (grey bar) and furazolidone resistance (black bar) and their respective confidence intervals. Resistance to furazolidone is conferred by the inactivation of the <i>nfsA</i> gene. The values of μ were estimated by performing fluctuation assays and analyzing the results using the maximum likelihood approach implemented in FALCOR [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006420#pgen.1006420.ref025" target="_blank">25</a>]. The mean frequency of spontaneous <i>gat-</i>negative mutants is ~300 times higher than the frequency of spontaneous resistants to furazolidone. After correcting for the difference between loci sizes the frequency of <i>gat</i>-negative phenotype is still ~38 times higher than the frequency of furazolidone resistance.</p

Clonal interference and periodic selection co-occur during adaptation of <i>E</i>. <i>coli</i> to the mouse gut.

<p>Muller plot of the frequencies of newly generated haplotypes during 24 days (corresponding to 432 generations) of evolution inside the mouse gut (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006420#pgen.1006420.s006" target="_blank">S4 Table</a> for numeric data). Shaded areas are proportional to the frequencies of each haplotype. Yellow and blue shaded areas represent the two sub-populations of bacteria labeled either with <i>cfp</i> or <i>yfp</i> alleles. Increasingly darker tones of yellow or blue represent the accumulation of target beneficial mutations in clones with a given fluorescence background (either <i>cfp</i> or <i>yfp</i>). In cases where the fluorescent background is not known, the new mutation (as assayed by whole genome population sequencing) is indicated without change in the shading tone. Evidence for positive Darwinian selection in the gut: clonal interference, where clones carrying different adaptive mutations at the same and in different loci compete for fixation in the gut in population 2.7 (A); periodic selection results in the rapid replacement of a clone, <i>i</i>.<i>e</i>. a hard selective sweep in which the haplotype <i>radA</i>-dup reaches close to 100% frequency in the population 2.10 (B). Dashed lines mark the time points in days (lower axis) or generations (lower axis) where the sampling of clones (in black) or WGS (in red) of the population took place. For population 2.7, between 40 and 50 clones were analyzed at each time point, whereas in population 2.10, between 20 and 60 clones were analyzed each day (please refer to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006420#sec009" target="_blank">Methods</a> for the further details of this analysis). The effects of two second step mutations (<i>radA-</i>dup and <i>arcA</i>) were estimated through their frequency increase in the populations where they emerged (C). This quantification was only possible because additional mutations are very unlikely to be segregating at the same time. Only mutations observed more than once in any of the sequenced clones are represented here. Population 2.7 has one additional mutation not represented in the figure and population 2.10 has two other mutations (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006420#pgen.1006420.s006" target="_blank">S4 Table</a>).</p

Transposable elements modulate gene expression.

<p>The graph represents the log₂ relative expression rate of each gene in the evolved clone in relation to that of the ancestral clone. This was obtained by RT-qPCR and analyzed according to the method described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006420#pgen.1006420.ref019" target="_blank">19</a>], using <i>hfq</i> as an housekeeping gene. The expression rate is above 0 for evolved clones where a SNP in <i>srlR</i> (A) or an IS insertion in the depicted genes (B) caused an increase in gene expression (in relation to the ancestor); if this value is below 0 gene expression was decreased (in comparison to the ancestor). Asterisks indicate significant differences at the level <i>P</i> < 0.05 (unpaired T-test). Effects of IS insertions on gene expression were conditional on oxygen availability. Error bars represent 2 times the standard error. Representative clones used for RT-qPCR measurements were the following (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006420#pgen.1006420.s003" target="_blank">S1 Table</a>): 18YFP (<i>focA srlR</i>), 22YFP (<i>dcuB</i>), 25YFP (<i>yjjP/yjjQ radA insX-insA</i>), 29CFP (<i>arcA</i>) and the ancestral strains DM08-YFP and DM09-CFP.</p

The genetic basis of the 2^nd step of adaptation involved 7 parallel mutational targets.

<p>Mutations were identified by whole genome sequencing of 29 evolved clones: 14 independently evolved clones from an ancestral strain (first colonization) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006420#pgen.1006420.ref008" target="_blank">8</a>] and 15 independently evolved clones from an adapted clone carrying a single beneficial mutation (second colonization). For simplicity, the genomes are represented linearly (vertical bars). The genomic context of the parallel mutations is represented on the right. Type and position of mutations are shown as triangles for insertions and deletions and small vertical bars denote single nucleotide polymorphisms (SNPs). Four duplications are depicted as red horizontal bars. See the symbol legend for other events. Regions of parallel mutation are highlighted. Numbers above marked mutations represent the number of clones where a particular mutation was detected at the same position.</p

The mutations occurring during the second step of adaptation show beneficial effects in the wild type background (<i>gat</i>-positive).

<p>Graphical representation of the frequencies of each mutant (labelled with a <i>yfp</i> allele) carrying one of the second step mutations: (A) <i>arcA</i>, (B) <i>focA</i>, (C) <i>yjjP</i>, (D) <i>dcuB</i>, (E) <i>oppB</i> and (F) <i>srlR</i> and the ancestor (labelled with a <i>cfp</i> allele). Shaded areas are proportional to the frequencies of each phenotype. Increasingly darker tones of yellow or blue represent newly generated <i>gat</i>-negative phenotypic mutants in the respective fluorescent background. <i>gat</i>-negative mutants (likely originating from multiple mutations and typed for the phenotype) invade both genetic backgrounds, indicating that its beneficial effect could be observed both in the background of the first and second step mutations. In this sense no historical contingency along the adaptive walk is expected since the fixation of any of the second step mutations could not prevent the emergence and spread of the <i>gat</i>-negative phenotype.</p

Emergency of morphological diversity in the bacterial populations adapting to MΦ.

<p>(A) Examples of the variability for colony morphology that emerged in <i>E. coli</i> populations adapting to MΦ, from left to right – ANC stands for morphology of ancestral, SCV for the small colony variants morphology and MUC for the mucoid colony morphology. (B) Dynamics of frequency change of the evolved phenotypes in each replicate evolving populations (M1 to M6): white squares indicate ANC, black triangles SCV, black circles MUC phenotypes.</p

Predictions of model of clonal interference for changes in mucoid frequencies with time.

<p>Simulations of the adaptive dynamics over the period of the experiment (30 days). The frequencies of mucoid phenotypes are plotted and can be compared to those observed in the experiments (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003802#ppat-1003802-g001" target="_blank">Fig. 1B</a>). The values of parameters used and the dynamics of haplotypes that compete for fixation are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003802#ppat.1003802.s009" target="_blank">Figure S9</a>.</p

<i>In vitro</i> evolved <i>E. coli</i> show increased virulence <i>in vivo</i>.

<p><b>A</b>) Survival of mice infected with different doses of ancestral (ANC, in blue), mucoid bacteria evolved in the presence of MΦ (MUC, in red) or bacteria evolved in the absence of MΦ (CON, in green). The number of mice are shown inside the bars, <b>B</b>) Survival probability of mice infected with ANC, MUC and CON, represented as lines from the fit of a binomial General Linear Model used to infer LD₅₀, <b>C</b>) Kaplan-Meier curves and <b>D</b>) % maximum reduction in temperature or weight at the LD₅₀ dose for the MUC (n = 10), ANC (n = 11) and CON (n = 5) (Error bars correspond to 2SE, * indicates p<0.05).</p

Mutations acquired by evolved clones identified through whole genome re-sequencing (WGS).

<p>Mutations in intergenic regions have the two flanking genes listed (e.g., <i>clpX</i>/<i>lon</i>). SNPs are represented by an arrow between the ancestral and the evolved nucleotide. Whenever a SNP gives rise to a non-synonymous mutation the amino acid replacement is also indicated. The symbol Δ means a deletion. For intergenic mutations, the numbers in the Mutation row represent nucleotides relative to each of the neighboring genes, here + indicates the distance downstream of the stop codon of a gene and − indicates the distance upstream of the gene, that is relative to the start codon. Insertions of IS elements are denoted by the specific IS element followed by the number of repeated bases caused by its insertion.</p

Genetic characterization of adaptive mutations and the dynamics of their appearance.

<p>(A) Mutations identified in MUC1 to MUC6 clones isolated from M1 to M6 populations (evolved for 450 generations), represented along the <i>E. coli</i> chromosome. For simplicity, the genomes are represented linearly and are horizontally drawn. The types of mutations are represented in the following way: SNPs are shown as crosses, IS insertions as inverted triangles and deletions as triangles. Filled symbols represent mutation in the coding region of the gene and empty symbols in the regulatory region. (B) Emergence and spread of adaptive mutations in M1 to M6 populations. Dynamics of haplotype frequencies in evolving populations at different days of evolution experiment are represented by circles. The color and symbol (IS insertions are represented as circles and other mutations as crosses) of each sector represents different haplotypes and the area of the circle their frequency in the population. Grey area represents the frequency of clones in the population that were typed for existing mutations in the population and did not differ from ancestral haplotype.</p