(608e) Collateral Fitness Effects of Mutations

Mutations act as a driving force of evolution by providing the genetic variation upon which selective pressures act. A mutation to a protein can result in positive, negative, or neutral effects on fitness, with fitness being defined as a measure of an organism’s ability to survive and reproduce. Fitness effects can derive from the altered ability of a protein to fulfill it physiological function, which we refer to as primary fitness effects. Surprisingly, a protein’s functional significance plays only a minor role in determining protein evolutionary rates. Here, we reveal the importance of collateral fitness effects of mutations, which we define as effects that do not result from changes to the protein’s ability to perform its physiological function. We constructed a library of all single-codon missense mutations in the Escherichia coli TEM-1 antibiotic resistance gene. We used deep mutational scanning to comprehensively measure the collateral fitness effects of these mutations during growth competition experiments in the absence of antibiotic. We found that over 42% of all missense mutations were deleterious to growth rate in the absence of antibiotic. Deleterious mutations caused improper post-translational processing, incorrect disulfide-bond formation, protein aggregation and changes in gene expression. Collateral fitness effects have implications for directed evolution, as variants with improved specific activity but deleterious collateral fitness effects may be lost during selection. High-throughput in vivo screening experiments that seek to quantify the effects of mutations on protein function might be misled by not considering the impact of collateral fitness effects. By identifying the frequency, magnitude, and mechanisms of collateral fitness effects, we can understand their role in genetic selection and the shaping of protein evolutionary pathways.