advances.sciencemag.org/cgi/content/full/2/1/e1500921/DC1
Supplementary Materials for
Environmental changes bridge evolutionary valleys
Barrett Steinberg and Marc Ostermeier
Published 22 January 2016, Sci. Adv. 2, e1500921 (2016)
DOI: 10.1126/sciadv.1500921
This PDF file includes:
Fig. S1. Heat map of the matrix of Hamming similarity between unique proteins
found from 47 of the most resistant alleles from each selection regimen following
round 8.
Fig. S2. Comparison of the differences in sequences obtained using the different
selection regimen.
Fig. S3. Structural mapping of mutations found in each selection scheme
following round 8.
Fig. S4. Heat map of the frequency of mutations observed during the negative
evolution regimen, as analyzed by deep sequencing.
Fig. S5. Stacked frequency distribution of mutations found by deep sequencing at
each step in the negative evolution regimen as a function of codon position over
eight rounds.
Fig. S6. Stacked distribution of mutations found in the top 47 alleles from all
selection strategies following round 8 as a function of codon position in lactamase.
Fig. S7. Structural mapping of mutations found in the BS-NEG-4 allele, which
confers a high resistance to cefotaxime.
Fig. S8. Pathways between clusters found in SWAG analysis.
Fig. S9. Two-dimensional SWAG landscape of pathways between TEM-15 and
BS-NEG-4 with the nine single mutants indicated.
Fig. S10. Change in pairwise epistasis (3) between two mutations on the
pathway from TEM-15 to BS-NEG-4 in response to the addition of F230S.
Other Supplementary Material for this manuscript includes the following:
(available at advances.sciencemag.org/cgi/content/full/2/1/e1500921/DC1)
Data S1. Evolutionary history of selection schemes.
Data S2. Sequences resulting from the positive selection regimen (TEM-1).
Data S3. Sequences resulting from the positive selection regimen (TEM-15).
Data S4. Sequences resulting from the neutral selection regimen.
Data S5. Sequences resulting from the negative selection regimen.
Data S6. Sequences resulting from the oscillating selection regimen.
Data S7. Fitness and epistasis values for BS-NEG-4 pathways.
Data S8. Additional MIC data.
Figure S1: Heat map of the matrix of Hamming similarity between unique proteins found
from 47 of the most resistant alleles from each selection regimen following round 8. Red
color indicates a higher similarity in amino acid mutations between two given sequences
up to the full 287 amino acids of the protein, while blue indicates a greater number of
differences in amino acid mutations.
Figure S2: Comparison of the differences in sequences obtained using the different
selection regimen. (A) Mean Hamming distance at the protein level between TEM-15
and unique protein sequences taken from 47 most resistant alleles resulting from each
selection type after eight rounds of evolution. Negative selection resulted in alleles with
greater average evolutionary distance from the starting point as compared with all other
selection types (p<0.005 by Student’s T-test). (B) Diversity of proteins within each
selection type as measured by Hamming distance. Oscillating selection results in the
lowest diversity (p<0.005 each by Student’s T-test relative to all other selection
strategies).
Positive (TEM-15)
Positive (TEM-1)
Neutral
Negative
Oscillating
Combined
Figure S3: Structural mapping of mutations found in each selection scheme following
round 8. The selection scheme demonstrating most frequent mutation rate at each site is
depicted in the combined panel.
Figure S4: Heat map of the frequency of mutations observed during the negative
evolution regimen, as analyzed by deep sequencing. Each plot indicates all possible
amino acid mutations (y-axis) found in a given round across position (x-axis) in the
protein. Color indicates the frequency of that mutation from low (blue) to high (yellow).
White indicates no observations of the indicated mutation. Rounds 1-3 were negative
selections, while rounds 4-8 were positive selections in this selection regimen.
Figure S5: Stacked frequency distribution of mutations found by deep sequencing at
each step in the negative evolution regimen as a function of codon position over eight
rounds.
Figure S6: Stacked distribution of mutations found in the top 47 alleles from all
selection strategies following round 8 as a function of codon position in β-lactamase.
F230
E104
G238
Figure S7: Structural mapping of mutations found in the BS-NEG-4 allele, which confers
a high resistance to cefotaxime. Mutation sites are shown in green and with stick models.
The active site is shown in blue. E104 and G238 are noted in yellow stick models.
Figure S8: Pathways between clusters found in SWAG analysis. (A) Heat map of the
frequency of connections within and between clusters. (B) Heat map of the frequency of
connections within and between clusters weighted by the selection strength of each
connection.
Figure S9: Two-dimensional SWAG landscape of pathways between TEM-15 and BSNEG-4 with the nine single mutants indicated.
Figure S10: Change in pairwise epistasis (3) between two mutations on the pathway
from TEM-15 to BS-NEG-4 in response to the addition of F230S. (A) Histogram of all
values of 3 measured. (B) Heatmap of mean 3 values between pairs of mutations
upon the addition of F230S.