Adaptation dynamics between copy-number and point mutations
- Institute of Science and Technology Austria, Austria
Figures
Figure 1 with 1 supplement
An experimental system to study gene duplication and divergence in strains with different duplication rates.
(A) Cartoon of chromosomal selection and reporter cassette. The galK-yfp gene fusion does not have a functional promoter, but instead a random sequence, P0 (thin arrow), drives very low levels of …
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Figure 1—source data 1
Contains an R script along with optical density measurments to plot Figure 1B.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig1-data1-v3.zip
Figure 1—figure supplement 1
An experimental system to study gene duplication and divergence in strains with different duplication rates.
(A–C) Fluorescence phenotype and copy-number as measured by qPCR of bacterial clones with different levels of galK expression and copy-number grown on LB agar. Point colour indicates P0 sequence of …
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Figure 1—figure supplement 1—source data 1
Contains an R script along with qPCR and fluorescence intensity data to plot Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig1-figsupp1-data1-v3.zip
Figure 2 with 3 supplements
Evolutionary dynamics depend on galactose concentration and duplication rate.
(A) Daily measurements of normalized CFP fluorescence as a proxy for gene copy-number of 96 populations of IS+ (black) and IS- (red) strains growing in three different galactose concentrations (% …
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Figure 2—source data 1
Contains an R script along with optical density and fluorescence data to plot Figure 2A-B.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig2-data1-v3.zip
Figure 2—figure supplement 1
Number of amplified populations and their copy-number depends on the gene expression demand of the environment.
(A) Data replotted from Figure 2A. Green line indicates threshold to classify as population as amplified (CFP/OD600 exceeds the mean ancestral CFP/OD600 by four standard deviations). (B) Using the …
Figure 2—figure supplement 2
Evolutionary dynamics depend on galactose concentration.
(A) Additional evolution experiment with daily measurements of normalized CFP fluorescence as a proxy for gene copy-number of 96 populations of the IS+ strain growing in three different galactose …
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Figure 2—figure supplement 2—source data 1
Contains an R script along with optical density measurments to plot Figure 2—figure supplement 2B.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig2-figsupp2-data1-v3.zip
Figure 2—figure supplement 3
YFP-only amplifications occur in IS- populations evolved in 0.1% galactose.
(A) Normalized YFP fluorescence as a proxy for galK expression of 96 populations in the IS- strain growing in 0.1% galactose. Populations with increased YFP fluorescence are highlighted. (B) GalK …
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Figure 2—figure supplement 3—source data 1
Contains an R script along with qPCR data to plot Figure 2—figure supplement 3B.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig2-figsupp3-data1-v3.zip
Figure 3
Confirming the presence of a combination of copy-number and point mutations in intermediate and high galactose.
(A–B) Log plot of YFP and CFP fluorescence of all 96 IS+ populations during evolution in 0.1% (A) and 1% (B) galactose (black points), respectively. Data replotted from Figure 2B for an overview of …
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Figure 3—source data 1
Contains an alignment of sequencing data for Figure 3E.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig3-data1-v3.txt
Figure 4 with 2 supplements
Confirming the presence of mutually exclusive mutations in low galactose.
(A) Representative flow cytometry density plot showing YFP fluorescence (upper left and right panel) and CFP fluorescence (lower left and right panel) of IS+ populations B3 (left panels) and B1 …
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Figure 4—source data 1
Contains an R script along with colony fluorescence intensity data over time to plot Figure 4D.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig4-data1-v3.zip
Figure 4—figure supplement 1
Adaptation to the low galactose environment is dominated by gene amplification.
(A) Flow cytometry density plot showing YFP fluorescence and (B) CFP fluorescence of IS+ populations over time (grey – ancestral, black – day 4, dark blue – day 8, purple – day 12).
Figure 4—figure supplement 2
Monitoring population fluorescence under neutral conditions with respect to galK expression reveals small increases in YFP fluorescence in the absence of amplification.
(A) Representative images of CFP (left panel) and YFP (middle panel) fluorescence of populations patched onto LB agar, which allows comparing population fluorescence in the absence of …
Figure 5 with 1 supplement
Amplicon deep sequencing of P0 in pooled evolved populations.
(A) (Left panel) Number of reads carrying a P0 sequence with two adaptive SNPs 30 and 37 bp upstream of galK, respectively (‘T>A + C>T’ in blue) or its respective single SNPs (‘T>A’ in green, ‘C>T’ …
Figure 5—figure supplement 1
Total number of sequencing reads for all replicates.
(A) Log plot of total read numbers showing contamination of P0 amplicons with P02 amplicons stemming from pooled samples of the 0.1% galactose populations of both promoter sequences (blue rectangles;…
Figure 6 with 1 supplement
Evolutionary dynamics for different random P0 sequences in 0.1% galactose.
(A) YFP versus CFP fluorescence normalized to the ancestral value of 96 populations of IS+ (black) and IS- (red) strain each harbouring a different random sequence upstream of galK (‘P0’, ‘P0-1’, …
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Figure 6—source data 1
Contains an R script along with optical density and fluorescence intensity measurments to plot Figure 6A-B.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig6-data1-v3.zip
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Figure 6—source data 2
Contains an R script along with optical density and fluorescence intensity measurments to plot Figure 6D-E.
- https://cdn.elifesciences.org/articles/82240/elife-82240-fig6-data2-v3.zip
Figure 6—figure supplement 1
Rapid amplification of IS+ populations with P02.
(A) CFP/OD600 as a proxy for copy-number plotted over the course of the evolution experiment for IS+ with P0-2 populations in 0.1% galactose and control populations in 0% galactose (grey). Green …
Figure 7 with 1 supplement
Frequent copy-number mutation can hinder adaptation by point mutations.
Genotype-fitness map (‘fitness landscape’) illustrating the difference between adaptive trajectories of a high demand (A) and low demand (B) environment, which differ solely by the increase in gene …
Figure 7—figure supplement 1
Amplification hindrance is consistent with negative epistasis under conditions of low gene expression demand.
(A). Maximal OD600 (‘yield’) as a function of different induction levels of galK expression in four different concentrations of galactose. Expression of a synthetic para-galK cassette is induced by …
Author response image 1
Replicates of the Experiment shown in Figure 1b, where growth rate is plotted against arabinose concentration used to induce galK expression for different galactose concentrations (line colors, see legend).
Left panels show all tested galactose concentrations, while right panels show the relevant galactose concentrations used in the experiments of the manuscript.
Author response image 2
Histogram of population B1 CFP fluorescence intensity [a.u.] plotted on a log scale to visualize the small amplified subpopulation (black=day 4, blue=day 8, purple = day 12).
Author response image 3
Example growth curves in M9 evolution medium and a 2D gradient of galactose and arabinose (as an inducer of galK expression).
These measurements are the basis for Figure 1b. The top left panel shows growth in 1% glycerol (control) instead of galactose.
Tables
Table 1
Sequencing and phenotypic analysis of all YFP+IS+ populations evolved in 0.01% galactose (Figure 4D – red triangles).
Increase in fluorescence relative to ancestral (anc) phenotype indicated by YFP+ and CFP+. Results shown for day 12 populations unless otherwise noted (d4, d8).
| Population | Seq (all YFP+) | Flow cytometry phenotype | Agar streak | Comment |
|---|---|---|---|---|
| A6 | –30T>A | YFP+, v. few CFP+ (mixed populations) | YFP+, few CFP+ | |
| B1 | –30T>A, –37C>T (“mutation H5”) | YFP+, CFP+ (mixed populations) | Few YFP+, few CFP+, mixed pop | |
| B2 | –30T>A | YFP+ | YFP+, v. few CFP+ | |
| C1 | –30T>A | YFP+ (d12) | YFP+, v. few CFP+ | |
| C9 | – | Ancestral YFP (d8), only CFP+(d12) | Few CFP+ | Incorrectly classified as YFP+ (Figure 4D – grey triangle) |
| D2 | –30T>A | YFP+ (d12) | YFP+ only | |
| D9 | anc | YFP+ (d8, d12) | YFP+ only | |
| E10 | –30T>A | YFP+ (d12) | YFP+ only | |
| F6 | – | YFP+ (d4), CFP+(d12) | CFP+ subpopulation | YFP+ at d8, then amplified population (Figure 5D – blue triangle) |
| F10 | –30T>A | YFP+, CFP+, anc (mixed populations) | YFP+, CFP+, mixed pop | qPCR confirmed |
| G1 | –30T>A | YFP+(d4–8), v. few CFP+ (d12) | YFP+, v. few CFP+ | |
| G12 | –30T>A | YFP+ (d8) | YFP+, no CFP+ (d12) | FACS CFP+ carry over |
Table 2
Mutations of P0-2 underlying increased YFP fluorescence in IS+ and IS- populations evolved in 0.1% galactose.
| IS+ clones | IS- clones | ||
|---|---|---|---|
| P02-A11 | –131_–144del | P02-A7 | –100C>T |
| P02-B10 | –122_–134del | P02-H12 | –100C>T |
| P02-F4 | –100C>T | P02-C3 | –100C>T |
| P02-F4 | –100C>T, poor quality read | P02-H9 | –122_–134del |
| P02-F2 | –122_–134del | ||
| P02-D1 | –100C>T | ||
| P02-E2 | –100C>T | ||
| P02-A1 | Bigger band, maps to insD1 coding sequence | ||
| P02-E5 | –41del | ||
| P02-C5 | 201 bp deletion leaving 20 bp of P02 | ||
| P02-H5 | 201 bp deletion leaving 20 bp of P02 | ||
| (seven different kinds of mutations) | |||
