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    Replaying the evolution of FMET : experimental evidence that translation initiation in bacteria was invaded by a selfish genetic element.

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    Thesis embargoed until 11-04-2017 (15.10Mb)
    Author
    Rickerby, Alannah M.
    Date
    2016
    Permanent Link
    http://hdl.handle.net/10092/12825
    Thesis Discipline
    Cellular and Molecular Biology
    Degree Grantor
    University of Canterbury
    Degree Level
    Masters
    Degree Name
    Master of Science

    The core machinery for protein synthesis is universal to cellular life. However, idiosyncrasies exist that differentiate the process of translation across the three domains (Archaea, Bacteria, Eukarya). One such example is found in bacteria, mitochondria and chloroplasts, where a formyl group is added to methionine prior to initiation of translation. Intriguingly, this formyl group is removed from the nascent polypeptide by peptide deformylase before protein production is complete, and appears to have no clear function. Despite this, formylation is essential to bacterial translation: interrupting formylation is deleterious. Such a well-conserved, and apparently deleterious, process would be expected to play an important role in bacterial translation initiation. Previous work in our group has indicated that formylation, and the removal of the formyl group by deformylation, likely evolved from an ancient, plasmid transmitted, toxin antitoxin system capable of post-segregational killing. These systems work by addicting cells to their presence; the toxin is more stable than the antitoxin, so if the gene pair is interrupted, the toxin is able to exert its lethal effects. A line devoid of the def-fmt gene pair was generated, and evolved in the absence of the genes for 1,500 generations. Despite suffering a large decrease in fitness, wildtype growth rates were observed after 1,500 generations, showing that bacteria are capable of wildtype growth in the absence of formylation. Further to this, when def-fmt is reintroduced on a plasmid, a PSK phenotype was observed, with cell death occurring upon interruption of the plasmid. We have now investigated the initial evolution of formylation in bacteria by reintroducing the def-fmt gene pair into a line evolved in its absence for 1,500 generations. A further 3,000-generation evolution experiment was performed with these lines. Our results indicate that an immediate reassertion of addiction has occurred. Once we reintroduce these genes into the genome, they immediately become addictive once again. We have been unable to knock-out these genes, and they also appear to out-compete cells which are not performing formylation. Whole genome sequencing has revealed a number of parallel compensatory mutations across the evolved lines, as well as a number of reversions of mutations previously observed in lines evolved in the absence of def-fmt. These results ultimately indicate that formylation evolved as a selfish genetic element, invading bacteria and persisting through addiction as opposed to any functional advantage.

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    • Science: Theses and Dissertations [3299]
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