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Fitness and epistatic interactions among mutations to less-preferred synonymous codons in an essential gene of Escherichia coli

Hauber, David J.
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1289235057

Abstract Details

2010, PhD, University of Cincinnati, Arts and Sciences: Biological Sciences.
The accumulation of very slightly deleterious mutations (VSDMs) in organisms with large genomes and small populations, as are found in many species, theoretically can lead to multiple lethal equivalents over time. One of the resolutions to this paradox is the action of negative, synergistic epistasis among the VSDMs. As a model to study epistasis as a mechanism to prevent the excessive genetic load from this accumulation, I used the codon bias found in highly transcribed genes of Escherichia coli. Synonymous, silent mutations, i.e., those nucleotide mutations of protein genes that code for the same amino acid, were originally thought to be neutral or nearly neutral, imparting little or no selective advantage or disadvantage to the organism; however, silent sites have been shown to have effects on the expression of some genes in some organisms. No one, however, has yet measured or demonstrated a direct impact on fitness for any silent polymorphisms in the native genome of an organism. In this work, I inserted mutations to 5 less-preferred synonymous codon (LSPCs) into the ribosomal protein gene rplQ of Escherichia coli strain K-12 (substrain MG1655) at codon positions L38, K40, K42, L44, and V47 by means of allele replacement with a temperature-sensitive plasmid and measured a relative fitness under rapid growth conditions in competition experiments with the wild type. In order to distinguish between two populations in competition experiments, however, I first developed a substrain of K-12 incapable of catabolizing arabinose by inserting a stop codon and a 2-basepair deletion in the araA gene by the same temperature-sensitive plasmid method and demonstrated its neutrality to approximately 2x10-3. The rplQ allele with the 5 LPSCs was placed within this Ara- marked strain. Three clones, independently developed from separate plasmid excisions, demonstrated a relative fitness of approximately 0.81 under the high growth competition experiment conditions. I also reverted the mutant rplQ strain back to the wild type and demonstrated its recovery in fitness. Finally, I inserted each of the 5 rplQ mutations singly into the bacterial chromosome and measured their relative fitnesses under the same competition conditions to determine if negative, synergistic epistasis is occurring. The relative fitnesses measured for the strains with the single, less-preferred synonymous mutations at the specific sites are as follows: 0.977 for L38, 1.006 for K40, 0.973 for K42, 1.004 for L44, and 0.974 for V47. The codon changes were identical for the two leucine codon positions (CUG to CUA) and for the two lysine positions (AAA to AAG), but the relative fitnesses were significantly different for the same codon in two nearby positions. The fitness expected from these 5 synonymous mutations together was 0.935, calculated by a multiplicative formula, and 0.934, calculated by an additive formula. The fact that the measured relative fitness of the strain bearing the rplQ allele with all 5 of these synonymous mutations was 0.81 and, therefore, substantially lower than estimated fitness values calculated on the basis of either formula is indicative of negative, synergistic epistasis operating among LPSCs.
Ronald Debry, PhD (Committee Chair)
Brian Kinkle, PhD (Committee Member)
Kenneth Petren, PhD (Committee Member)
Dennis Grogan, PhD (Committee Member)
Patrick Limbach, PhD (Committee Member)
147 p.
Biology
synonymous mutation; epistasis; fitness; codon bias; allele replacement; experimental evolution

Recommended Citations

Citations

  • Hauber, D. J. (2010). Fitness and epistatic interactions among mutations to less-preferred synonymous codons in an essential gene of Escherichia coli [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1289235057

    APA Style (7th edition)

  • Hauber, David. Fitness and epistatic interactions among mutations to less-preferred synonymous codons in an essential gene of Escherichia coli. 2010. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1289235057.

    MLA Style (8th edition)

  • Hauber, David. "Fitness and epistatic interactions among mutations to less-preferred synonymous codons in an essential gene of Escherichia coli." Doctoral dissertation, University of Cincinnati, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1289235057

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Cincinnati and OhioLINK.