Research ArticleMOLECULAR EVOLUTION

Environmental changes bridge evolutionary valleys

See allHide authors and affiliations

Science Advances  22 Jan 2016:
Vol. 2, no. 1, e1500921
DOI: 10.1126/sciadv.1500921
  • Fig. 1 Simplified model representation of how evolution in alternating environments that modulate the fitness landscape can lead to the crossing of fitness valleys.

    The xy plane represents sequence space, whereas the z axis represents fitness. The solid red arrow indicates an evolutionary pathway following increasing positive selection pressure. (A) A sequence (circle) sits at an optimum separated from global optima by a fitness valley. (B) A change in the environment alters the landscape such that evolution brings the gene to a new sequence that previously resided in the valley. (C) Upon return to the original environment, the sequence resides in the valley but can now evolve to the global optimum, which lies uphill.

  • Fig. 2 Schematic and course of experimental evolution on the β-lactamase gene.

    (A) General schematic. After a comprehensive saturation mutagenesis, selection proceeds in one of three manners depending on the environment. Positive selection (blue) enriches for sequences that provide resistance above a certain level. Neutral selection (yellow) enriches for sequences conferring about the same resistance as the starting gene. Negative selection (red) enriches for sequences conferring very low but above-background resistance. The latter two selection schemes are enabled by an engineered bandpass gene circuit present in the cell (22) and require the addition of the antibiotic tetracycline and different levels of cefotaxime, which constitute the difference in environment from the positive selection. (B to E) Course of the evolution experiments. Experiments differ in the first three rounds in which the bacteria underwent (B) positive selection, (C) neutral selection, (D) negative selection, or (E) oscillating selection. Each round represents both mutagenesis (line segments) and selection at the indicated level of cefotaxime (endpoints). Red circles indicate bandpass selections, and blue circles indicate positive selections. The dashed horizontal line indicates the resistance level conferred by TEM-15. Graphed resistance values indicate a lower limit on the resistance values of the entire population during selection (that is, the concentration at which the selection was performed).

  • Fig. 3 Diversity and conferred resistance of alleles after the eighth round of evolution.

    (A) Unrooted tree of Hamming distances between randomly selected alleles. Branches are color-coded by the evolution experiment as indicated. Scale bar indicates 1 unit of Hamming distance (that is, one missense mutation). (B) Increase in resistance conferred by seven alleles from each selection strategy as compared to TEM-1. Resistance was measured using the MIC assay. The seven alleles were selected from among those depicted in the tree to represent diverse sequences. The dashed line represents resistance conferred by the GKTS allele, whereas the TEM-15 allele conferred a 64-fold higher resistance relative to TEM-1.

  • Fig. 4 SWAG of the possible evolutionary intermediates between TEM-15 and BS-NEG-4.

    Nodes are sequences, and edges connect nodes that differ by one missense mutation. Forces between connected nodes are weighted proportional to the selection strength, as determined by the node’s fitness values. (A) Two-dimensional SWAG. Edges are curved to aid visualization. (B to D) Three-dimensional SWAG has protein fitness as the z axis and is color-coded by (B) cluster, (C) whether the sequence contains F230S, and (D) protein fitness value.

  • Fig. 5 Radial graph of all analyzed pathways from TEM-15 to BS-NEG-4.

    Each unit branch extending from the center indicates the gain of one of the nine amino acids from TEM-15 until the endpoint containing the full set of mutations is reached. (A) The line color indicates the log of the fitness change from the addition of the mutation. This graph pictorially shows that most pathways have steps with decreased fitness. (B) Same data as in (A), but with the color indicating the fitness relative to TEM-15 after the addition of the mutation. This graph pictorially shows that early steps in most pathways pass through mutants with fitness values less than that of TEM-15. Data is the same in A and B; only color coding is different.

  • Fig. 6 Three-dimensional SWAG of all evolutionary pathways in which fitness increases monotonically with each additional mutation.

    Fitness is represented by height on the z axis. Color indicates clusters. Each node is sized by the likelihood of arrival, as measured by PageRank algorithm weighted by selection strength between nodes.

  • Fig. 7 Protein fitness and epistasis along possible evolutionary pathways between TEM-15 and BS-NEG-4.

    (A) Protein fitness values of all possible evolutionary intermediates. (B) N-order epistasis between all mutations as a function of the number of mutations. Proteins containing F230S are noted in red, whereas all other proteins are shown in blue. Points are jittered for clarity. Larger points indicate the mean for each data set, with bars extending to the SD.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/1/e1500921/DC1

    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 β-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 (Δε3) between two mutations on the pathway from TEM-15 to BS-NEG-4 in response to the addition of F230S.

    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.

  • Supplementary Materials

    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 β-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 (Δε3) between two mutations on the pathway from TEM-15 to BS-NEG-4 in response to the addition of F230S.

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • 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.

    Download Data S1 to S8

    Files in this Data Supplement: