Effects of single mutations from experimental evolution of microbial proteins: Thermostability in phi-6 Cystovirus and toxin diversification in Escherichia coli
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Experimental and directed evolution using microbes offer powerful methods for uncovering processes of evolution across the tree of life. The goal of such experiments is to generate mutational diversity, either through propagation of microbes in stressful conditions (experimental evolution) or through artificial introduction of mutations into their genomes (directed evolution). In the case of multiple resulting mutations, each is then reverse engineered into the ancestral genotype individually to determine how it changes the phenotype of interest. This thesis presents the results of one experimental evolution project (evolution of viral thermostability under increasing temperatures) and one directed evolution project (diversification of toxin-antitoxin protein pairs in bacteria), including both evolutionary and single-mutation analyses. In both cases, I found that mutations may persist in a population due to their pleiotropic effects on traits other than the focal one of the study. My thesis emphasizes the usefulness of laboratory evolution of microbes to guide new hypotheses about evolutionary processes. Chapter 1: Adaptations of an RNA virus to increasing thermal stress. In an incrementally changing environment, a shift from one environmental state to another occurs over multiple organismal generations. The rate at which the environment changes is expected to influence both how and how well populations adapt to the ultimate environment. To investigate this question, I evolved the lytic RNA bacteriophage phi-6 for greater thermostability by exposing viral populations to heat shocks that increased to a maximum temperature at different rates. I observed increases in the ability of many heat-shocked populations to survive high temperature heat shocks, although the survival of populations at the highest temperature and the number of mutations per population did not vary significantly according to the rate of thermal change. I then engineered specific mutations from the endpoint populations into the ancestral genotype and evaluated the effects of these mutations on viral thermostability and growth. As expected, some mutations increased viral thermostability. However, other mutations decreased thermostability but increased growth rate, suggesting that benefits of an increased replication rate may have sometimes outweighed the benefits of enhanced thermostability. This work highlights the importance of considering the effects of multiple selective pressures, even in environments where a single factor is changing. Chapter 2: Colicin mutation confers resistance to colicins in Escherichia coli. Colicins are toxic proteins produced by Escherichia coli that target and kill other E. coli cells. To prevent death by clone-mates, colicinogenic cells also express an immunity protein that neutralizes their own colicin by binding to it tightly and specifically. Although disruption of this binding interaction can be lethal, the colicin-immunity complex has diversified multiple times. Diversification is typically thought to occur in an immunity-led manner, through a promiscuous immunity protein that can bind multiple colicins. I aimed to test colicin-immunity diversification though directed mutagenesis of the immunity and colicin genes and screening for novel colicin-immunity pairs. I isolated a novel colicin that killed cells that expressed the ancestral immunity protein. Unexpectedly, when this novel colicin was combined with the ancestral immunity protein in the same cell, not only did the cells survive, but they also demonstrated resistance to a wide range of other colicins. Through deeper investigation of the novel colicin, I demonstrate a mechanism of colicin resistance that depends only on the colicin genotype. That a colicin can itself protect cells from its toxic effects furthermore suggests that colicin-immunity diversification might be able to proceed in a colicin-led manner.
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