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

The distribution of fitness effects (DFE) of mutations is a factor of fundamental importance in evolutionary biology. We determined the DFE of 510 mutants that each carried between 1 to 10 mutations (synonymous and non-synonymous) 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, non-synonymous 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 average, amplified the combined effect of non-synonymous mutations. (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 standard deviation of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients.