Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA

Abstract

The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients.

Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence

“…This complete loss of HisA activity is in contrast to what is observed when random mutations (without any demand for acquiring TrpF activity) are introduced into HisA. Under such conditions, HisA appears relatively robust and very few single mutations abolish its activity ( Lundin et al, 2018 ). Thus, the loss of HisA activity is associated with the gain of TrpF activity and not a consequence of mutation accumulation per se .…”

“…Additionally, as shown previously, by expressing HisA from a constitutive promoter, at levels where the growth rate of cells is limited by the rate of histidine or tryptophan synthesis, allowed the use of bacterial growth rates as a proxy of the total cellular HisA or TrpF activities. Throughout the text, activity designates the product of intracellular enzyme level and enzyme specific activity ( Lundin et al, 2018 ). Thus, mutations that improve total cellular TrpF activity lead to faster growth in medium lacking tryptophan and mutations that reduce the total cellular HisA activity cause slower growth in medium lacking histidine, although we do not know if the relationship between activity and growth rate is linear or non-linear.…”

Collateral fitness effects of mutations

“…For essential genes, small-effect mutations on the protein's specific activity or abundance may be neutral on the level of fitness due to the non-linear mapping of protein physicochemical properties on organismal fitness (20,23,24) (Fig. 6CD).…”

“…Fitness effects were interpreted as arising solely from mutational effects on protein specific activity/abundance and how these physiochemical traits map onto the level of organismal fitness. While primary fitness effects may overshadow collateral fitness effects for mutations with a large effect on protein specific activity or abundance, intrinsic buffering mechanisms minimize the fitness effects of mutations with small effects on specific activity/abundance (20,23,24). For example, most enzymes are well above the threshold in activity that the cell requires.…”

“…Studies that present organismal fitness effects of random mutations in proteins (e.g. (16,(19)(20)(21)(22)) have not delineated…”