Effect of <i>lac</i> mutations on fitness.

<p>A) Fitness of <i>lacI</i> and <i>lacO1</i> mutants in the four evolution environments. Competitions were performed for <i>lacI</i> versus the ancestor, <i>lacO1</i> versus the ancestor and <i>lacI</i> versus <i>lacO1</i>. As a control, we included the ancestor competed against itself. The red dotted line indicates a relative fitness of 1 (no fitness difference). 95% confidence intervals are shown for each competition (n≥8).</p

Characterization of <i>lac</i> operon regulation.

<p>A) Examples of the range of evolved LacZ activity phenotypes present in the four evolution environments. Degree of blue coloration on TGX plates gives a qualitative measure of LacZ activity. B) Schematic of <i>lac</i> operon and reporters used to measure <i>lac</i> operon regulation. <i>lacZ</i> encodes the β-galactosidase responsible for lactose catabolism and <i>lacY</i> encodes a lactose permease. The expression of <i>lacZYA</i> is directly controlled by LacI and CRP. LacI is a negative regulator, binding to operator sites within the <i>lacZYA</i> promoter (P_lac). LacI binding is inhibited by lactose and gratuitous inducers, such as TMG. CRP is a positive regulator, activating <i>lacZYA</i> expression when cAMP levels are elevated in response to low glucose concentrations. High levels of glucose also repress <i>lac</i> expression by inhibiting import of lactose through LacY. Two reporters were designed to measure LacI and CRP inputs into <i>lac</i> operon regulation. The native <i>lac</i> promoter drives expression of GFP and is subject to regulation by both LacI and CRP. A second reporter utilizes a mutant <i>lac</i> promoter that cannot bind LacI to drive expression of DsRedExpress2. This reporter is only subject to regulation by CRP. Solid lines indicate positive (arrows) and negative (blunt arrow) regulatory interactions; dotted lines indicate the transfer of metabolites; blue lines indicate the production of proteins; open arrows indicate expression start sites. Figure adapted from Ozbudak et al. 2004. C) Ancestral inducer response profile. Shown are flow cytometry histograms for the ancestor grown in a range of TMG concentrations. P_lac-GFP and P_lac(O-)-RFP measurements were taken simultaneously from the same cultures.</p

Fitness effect of <i>lacI</i> and <i>lacO1</i> mutations depend on genetic background.

<p>The fitness effect of <i>lacO1</i> and <i>lacI</i> mutations were measured in the ancestor and the G+L3-1 evolved genetic backgrounds when competed in the G+L environment. Gray points indicate fitness effect in competitions against the corresponding progenitor strains that do not have the added <i>lac</i> mutation; black points indicate fitness of <i>lacI</i> and <i>lacO1</i> mutations competed directly against each other. Lines connect competitions of the same type but in different genetic backgrounds.</p

Changes in maximal <i>lac</i> expression for evolved clones.

<p>A) Steady state P_lac-GFP levels (RFU) are shown for evolved clones grown in the presence of 100 µM TMG. Clones are divided into categories by evolution environment: Glu, Lac, G+L and G/L. Markers of the same color denote clones recovered from the same evolved population. Data points are the average of two independent replicates. As a reference, data for six independent ancestral (Anc) samples is also shown. B) Native <i>lac</i> promoter activity for the ancestor, evolved clone G+L3-1 and the ancestor with <i>lacI</i> and <i>lacO1</i> mutations. Basal promoter activity was measured for strains grown in the absence of inducer, and maximum promoter activity was measured in the presence of saturating levels of inducer.</p

Physiological characterization of <i>lac</i> mutants.

<p>A) Growth curves for the ancestor (green), and reconstructed <i>lacI</i> (blue) and <i>lacO1</i> (purple) mutants. Conditions used were: Lac→Glu, Glu→Lac and G+L→G+L, where the sugars indicate pre-conditioning and measurement environments, respectively. These transitions correspond to those present in the G/L and G+L evolution environments. OD values are plotted on a log₁₀ scaled axis. B) LacZ expression time course for the ancestor and the ancestor with <i>lacI</i> and <i>lacO1</i> mutations during growth in the G+L evolution environment. Values are the average of two independent replicates with standard deviation shown.</p

Inducer response profiles of evolved clones.

<p>Heat maps show the mean response of the P_lac-GFP reporter (RFU) for evolved clones at different concentrations of inducer (TMG). Black squares indicate the concentration of TMG that gave half maximal expression (TMG_½Max) and red outlines indicate the presence of bimodal expression within the population. Inducer responses for clones with constitutive <i>lac</i> operons were only generated with four TMG concentrations because <i>lac</i> expression was independent of inducer concentration such that finer resolution measurements were redundant.</p

Contribution of <i>lac</i> mutations to evolved inducer responses.

<p>A) Inducer response histograms for reconstructed <i>lacI</i>^ΔTGGC and <i>lacO1</i>^G11A mutants. The <i>lacI</i> mutation confers constitutive <i>lac</i> expression, whereas the <i>lacO1</i> mutation confers a lower induction threshold and a graded response to inducer. B) Effect of the <i>lacO1</i> mutation on the maximum <i>lac</i> expression level. Mean P_lac-GFP expression levels during growth in saturating levels of inducer (100 µM TMG) are shown for the ancestor, evolved clone G+L3-1, <i>lacO1</i>^G11A single mutant and the G+L3-1 clone with <i>lacO1</i> reverted to the ancestral sequence (G+L3-1 <i>lacO1^anc</i>). Standard error is shown, n = 4.</p

Growth parameters for ancestor, <i>lacI</i> and <i>lacO1</i> mutants.

<p>*Significantly different than ancestor, <i>P</i><0.01 (two-tailed <i>t</i>-test, df = 10).</p><p>**Significantly different than ancestor, <i>P</i><0.001 (two-tailed <i>t</i>-test, df = 10).</p>#<p>μMax for <i>lacI</i> and <i>lacO1</i> is defined in an OD₆₀₀ range that is most comparable with the OD₆₀₀ range used to define μMax for the second exponential growth phase of the ancestor (μMax-2).</p

Identification <i>lac</i> mutations in evolved clones.

<p>Sequence alignments for evolved clones relative to the ancestor are shown for the <i>lacI</i> mutation hotspot region (left) and the primary <i>lacO1</i> operator (<i>right</i>). Clones were chosen that represent the diversity in <i>lac</i> genotypes within each population. Colored boxes by sequences indicate the inducer response type for each clone. Bimodal is shown as green, constitutive is shown as blue and lower threshold response is shown as purple. No sequence was obtained for the G/L5 clones. Sequence alignments are not shown for Glu clones since all have wt <i>lac</i> regulatory sequences.</p