Evolution of New Genes and Functions

To answer major evolutionary questions, we need a better understanding of the effects of mutations on specific functions and organism fitness. The aim of this thesis was to elucidate how new functions evolve and potential trade-offs with the original function. Paper I identified a bimodal distribution of fitness effects of mutations in Salmonella enterica HisA protein. Most mutations negatively affected protein function but the effect was masked at high gene expression. Expression levels and the extent to which the studied protein limited growth were important in determining the fitness effects of mutations. No fitness prediction tool was satisfactorily alone but in combination predictions were improved. In Paper II, S. enterica HisA was evolved to acquire TrpF activity. Numerous pathways towards improved TrpF activity were examined and several improvement mechanisms were identified. Improved TrpF activity extensively reduced the original activity, generalist enzymes were rare and restoring original activity after an initial loss was difficult. Furthermore, expression levels had a major impact on the shape of the trade-off curve. In Paper III, adaptation during serial passage of Escherichia coli and S. enterica under laboratory conditions were examined. Adaptive mutations were identified in four different laboratory media and their fitness effects were determined. Little overlap in mutation spectra was found in the different media and species suggesting that adaptation was media-specific. Furthermore, media adaptation mutations reduced the accuracy of fitness assays and the use of pre-adapted strains improved the sensitivity of fitness assays 10-fold. Paper IV examined evolution of novel metabolic capabilities in S. enterica by analyzing growth on 124 non-native carbon sources. Growth was observed on 25 compounds and for five of these, the causative mutation was identified. Increased gene expression of cryptic genes was a major mechanism for acquiring the novel phenotypes. In conclusion, my results show that in most cases many types of mutations can improve a function and allow adaptive evolution but this often is associated with a trade-off and loss in other abilities. Increased gene expression was a major mechanism by which bacteria could compensate for loss of an activity as well as acquire new metabolic capabilities