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  How bias in the production of phenotypic variation shapes and is shaped by adaptive evolution

Barnett, M. (2022). How bias in the production of phenotypic variation shapes and is shaped by adaptive evolution. PhD Thesis, Faculty of Mathematics and Natural Sciences at Kiel University, Kiel.

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Barnett, Michael1, Author           
Rainey, Paul B.1, Advisor           
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1Department Microbial Population Biology, Max Planck Institute for Evolutionary Biology, Max Planck Society, ou_2421699              

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 Abstract: This thesis presents evolution experiments using a bacterial model organism that examine how the processes that generate phenotypic variation interact with the process of natural selection. More specifically, the experiments investigate in what ways the systems that define the variability of an organism – the genotype-phenotype map (G-P map) and the mutational processes by which this map is re-configured – create biases in the production of variation and how these biases both shape and are shaped by the course of adaptive evolution. In Chapter 2, I examine whether the classical Darwinian notion of the ‘survival of the fittest’ is sufficient to understand the phenotypes that arise in the course of adaptation or whether the outcome is also determined by the likelihood that a phenotype is generated by mutation. To this end, I conducted an experiment that repeatedly challenged replicate populations to adapt to the same environment, while continually precluding the evolved adaptive solutions via genetic engineering. By re-playing evolution with an engineered strain in which previously discovered phenotypes were unavailable, evolution was forced to find new viable phenotypic solutions during each round of adaptation. Through this process I found that the phenotypes that evolution initially favoured were not necessarily the fittest possibilities available; latent phenotypes of higher fitness existed yet evolution did not make use of them. Reconstruction of the mutational paths required to reach each phenotype, and evaluation of both fitness and likelihood of each mutational step, revealed that the likelihood of the phenotypes being manifest by mutation played a key role in determining their use by natural selection. These results revealed that adaptive evolution may at times proceed along the most likely mutational path to a sub-optimal phenotype: the ‘survival of the likeliest’. As biases in the production of variation are themselves the product of evolution, the possibility exists that they can be shaped by natural selection to facilitate adaptation – a controversial notion known as the ‘evolution of evolvability’. In Chapter 3, I present an evolution experiment that explicitly selected for evolvability. Specifically, bacterial lineages were challenged to repeatedly activate and then inactivate a single focal phenotype that was in turn beneficial then deleterious across time. Failure to reach the target phenotypic state at any one point resulted in extinction (death) of that lineage and replacement (birth) by a contemporary extant lineage from the population. This lineage- level death-birth process allowed natural selection to operate on an entity reproducing beyond the generation time of individual cells and therefore over a timescale where variation in evolutionary potential, or evolvability, was visible to selection. Through extensive whole-genome sequencing I identified each mutation that occurred along the trajectory of the evolving lineages, revealing how both the G-P map and mutational biases had shaped and were shaped by evolution, as well as how this influenced the success or failure of a given lineage. I found that the most successful lineages increased their evolvability through the establishment of a mutational bias that facilitated rapid switching between the target phenotypic states. Moreover, due to the increased speed with which the target phenotype was generated in these lineages, additional adaptive steps became possible that optimized cell fitness with respect to other aspects of the environment – a possibility that was stalled in lineages not possessing the rapid-switching ability. These results represent the clearest experimental evidence yet for the capacity of evolution to act in a self-facilitating manner.

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Language(s): eng - English
 Dates: 20222022
 Publication Status: Published in print
 Pages: 128
 Publishing info: Kiel : Faculty of Mathematics and Natural Sciences at Kiel University
 Table of Contents: Table of Contents



Zusammenfassung.................................................................................................................................II
Abstract.................................................................................................................................................IV
Chapter 1. Introduction...................................................................................................................1
1.1 Internal versus external forces of evolution......................................................................................2
1.1.1 Fisher’s arguments for gradualism.....................................................................................4
1.1.1.1 The infinitesimal model……………………………………………………………………………………………….4
1.1.1.2 The opposing forces argument…………………………………………………………………………………….5
1.1.1.3 The geometric model…………………………………………………………………………………………………..5
1.1.2 Responses to Fisher............................................................................................................5
1.1.2.1 Responses to Fisher: the infinitesimal model argument..................................................6
1.1.2.2 Responses to Fisher: the opposing forces argument........................................................6
1.1.2.3 Responses to Fisher: the geometric model argument......................................................7
1.2 Bias in the production of phenotypic variation....................................................................................9
1.2.1 Mutational bias……………………………...……………………………………………………………………………………………..9
1.2.2 The genotype-phenotype map……………………………………………………………………………………………………….9
1.2.3 G-P maps and mutational bias both shape and are shaped by evolution……………………………………. 12
1.3 Technology, microbes, and experimental evolution.........................................................................13
Chapter 2. Disentangling fitness from likelihood in adaptive evolution………………………………………. 15
2.1 Introduction......................................................................................................................................16
2.1.1 The problem of observing counterfactual outcomes……………………………………………………………………16
2.1.2 Approaches using microbial experimental evolution……………………………………………………………………16
2.1.3 The Wrinkly Spreader phenotype…………………………………………………………………………………………………17
2.1.4 Alternative paths to the Wrinkly Spreader phenotype………………………………………………………………….17
2.1.5 Alternative phenotypes……………………………………………………………………………………………………………….18
2.2 Results & Discussion.........................................................................................................................20
2.2.1 Discovery of additional phenotypes capable of colonizing the ALI……………………………… 21
2.2.2.1 The discovery of three additional ALI-colonizing phenotypes…………………………………….22
2.2.2.2 The colanic acid-producing phenotype..........................................................................22
2.2.2.3 The fimbria phenotype ..................................................................................................23
2.2.2.4 The PSL-Wrinkly Spreader phenotype...........................................................................24
2.2.2.5 Summary of discovery....................................................................................................24
2.2.3 Reconstruction of mutational paths, fitness assays, and understanding the genetic basis
of phenotypes to inform likelihood estimations......................................................................25


VII



2.2.4 ‘CAP-producing’ phenotype: genetic basis, reconstruction, and fitness...........................27
2.2.4.1 Duplication of pflu3655-3657 is sufficient to generate the first-step to CAPP and
recapitulates the effects of the larger evolved duplications......................................................28
2.2.4.2 Substitutions in Pflu3677 are not equivalent to gene-wide LoF.....................................29
2.2.4.3 CAPP summary...............................................................................................................31
2.2.5 ‘Fimbria’ phenotype: genetic basis, reconstruction, and fitness.....................................31
2.2.5.1 Fitness trajectories to Fim.............................................................................................32
2.2.5.2 The first-step Pflu1605 mutation is predicted to increase transcription of the adjacent
fimbria structural genes.............................................................................................................32
2.2.5.3 Mutation to Pflu1605 is not equivalent to gene-wide LoF............................................33
2.2.5.4 Insights from PvrSR/RcsCB function...............................................................................34
2.2.5.5 RcsC/Pflu1605 is involved in both positive and negative regulation of the fimbria
structural gene cluster.............................................................................................................. 34
2.2.5.6 Collecting additional Fim-causing mutations in Pflu1605............................................. 35
2.2.5.7 The first-step mutation to Fim requires LoF to a highly constrained extragenic negative
regulator....................................................................................................................................36
2.2.5.8 The second-step mutation to Fim requires a GoF mutation to a structural gene.........36
2.2.5.9 Fim Summary.................................................................................................................37
2.2.6 ‘PSL-Wrinkly Spreader’ phenotype: genetic basis, reconstruction, and fitness................37
2.2.6.1 Two PSL-WS mutations are predicted to target expression of the sigma factor RpoS..38
2.2.6.2 PSL-WS cannot be re-created in the ancestral background due to an overlapping
regulon with PGA...................................................................................................................... 39
2.2.6.3 Fitness trajectories to PSL-WS...................................................................................... 39
2.2.6.4 PSL-WS Summary..........................................................................................................40
2.2.7 Comparing fitness............................................................................................................41
2.2.7.1 Confirming the precluding effects of FS through an extended evolution experiment..43
2.2.8 Comparing likelihood.......................................................................................................45
2.2.8.1 Likelihood of FS..............................................................................................................46
2.2.8.2 Likelihood of Fim............................................................................................................46
2.2.8.3 Likelihood of CAPP.........................................................................................................47
2.2.8.4 Summary of Likelihood..................................................................................................48
2.3 Conclusion.........................................................................................................................................48
Chapter 3. The evolution of evolvability via lineage selection.........................................................52
3.1 Introduction......................................................................................................................................53
3.1.1 Examples of modifying traits............................................................................................................53
3.1.2 Objections........................................................................................................................................ 55
3.1.3 Experimental challenges...................................................................................................................56
3.1.4 The life cycle experiment..................................................................................................................56


VIII

3.1.5 The emergence of a life-cycle..........................................................................................................56
3.1.6 The LCE as a means to examine the evolution of evolvability.........................................................59
3.1.7 A modified LCE.................................................................................................................................59
3.1.8 Possible mutational paths to activate and inactivate WS................................................................60
3.1.9 Persistence and adaptation..............................................................................................................63
3.2 Results & Discussion..........................................................................................................................64
3.2.1 Fates of the four lineage populations through extended evolution in the modified LCE..64
3.2.2 The line 54 population.....................................................................................................65
3.2.3 Overview of sequencing results from the line 54 population..........................................66
3.2.4 The spectra and biases of mutations...............................................................................67
3.2.5 Overview of mutational targets and their function.........................................................68
3.2.5.1 Mutational targets and their function: Pflu0185 and other cyclic di-GMP regulators..68
3.2.5.2 Mutational targets and their function: Wss cellulose synthase.....................................70

3.2.5.3 Mutational targets and their function: other components of WS genetic architecture..70

3.2.5.4 Mutational targets and their function: odd loci............................................................ 71
3.2.5.5 Mutational targets and their function: double and triple mutant(s)............................72
3.2.6 A map of subsequent sections.........................................................................................73
3.2.7 Lineage persistence.........................................................................................................74
3.2.7.1 The genetic causes of extinction...................................................................................77
3.2.7.2 Repairing LoF mutations in the wss operon..................................................................80
3.2.7.3 Intergenic compensation to Wss loss-of-function.........................................................81
3.2.7.3.1 WssE function.............................................................................................84
3.2.7.3.2 The ‘scaffold-adjustment’ hypothesis to explain intergenic
WssE compensation...................................................................................................86
3.2.7.3.3 An alternative hypothesis for WssE compensation via co-option of the Lpt
porin.......................................................................................................................... 86
3.2.8 Lineage adaptation.......................................................................................................... 88
3.2.8.1 The mechanism of the genetic switch...........................................................................89
3.2.8.2 An inverted switch.........................................................................................................90
3.2.8.3 Rare expansion of the GGTGCCC repeat leads to observable increase
in mutation rate.........................................................................................................................91

3.2.8.4 Understanding the frequency of the initial GGTGCCC duplication................................92

3.2.8.5 Local sequence context and DNA secondary structure.................................................93
3.2.8.6 The possibility of transcription-associated mutagenesis affecting
pflu0185 mutability...................................................................................................................94
3.2.9 Secondary adaptive mutations........................................................................................96


IX



3.2.9.1 Secondary adaptive mutations target motility and chemotaxis regulators....96
3.2.9.2 Additional secondary adaptive mutation candidates......................................97
3.2.9.3 Adaptive significance of secondary mutations...............................................99
3.2.9.4 Phenotypic effects of secondary adaptive mutations.....................................99
3.2.9.4.1 Phenotypic effects of secondary adaptive mutations: Aer & FimV............100
3.2.9.4.2 Phenotypic effects of secondary adaptive mutations: Pflu1687...............101
3.3 Conclusion.......................................................................................................................................103
3.3.1 The birth of a genetic switch..........................................................................................................103
3.3.2 Secondary adaptive mutations.......................................................................................................104
3.4 Appendices......................................................................................................................................106
Chapter 4. General Materials & Methods.....................................................................................107
4.1 Bacterial strains and culture conditions......................................................................................... 107
4.2 DNA extraction and purification for sequencing and molecular cloning........................................108
4.3 Sequencing and detection of mutations.........................................................................................109
4.4 Strain construction......................................................................................................................... 109
4.5 Transposon mutagenesis................................................................................................................110
4.6 Invasion fitness assays....................................................................................................................111
4.7 The modified LCE............................................................................................................................111
4.8 Software used.................................................................................................................................112
Bibliography........................................................................................................................................113
Acknowledgments..............................................................................................................................127
Declaration.........................................................................................................................................128
 Rev. Type: -
 Identifiers: Other: Diss/13511
 Degree: PhD

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