Andrea González-González, Shaun M. Hug, Alejandra Rodríguez-Verdugo, Jagdish Suresh Patel, Brandon S. Gaut

Modifications to transcriptional regulators play a major role in adaptation. Here we compared the effects of multiple beneficial mutations within and between Escherichia coli rpoB, the gene encoding the RNA polymerase β subunit, and rho, which encodes a transcriptional terminator. These two genes have harbored adaptive mutations in numerous E. coli evolution experiments but particularly in our previous large-scale thermal stress experiment, where the two genes characterized alternative adaptive pathways. To compare the effects of beneficial mutations, we engineered four advantageous mutations into each of the two genes and measured their effects on fitness, growth, gene expression and transcriptional termination at 42.2°C. Among the eight mutations, two rho mutations had no detectable effect on relative fitness, suggesting they were beneficial only in the context of epistatic interactions. The remaining six mutations had an average relative fitness benefit of ∼20%. The rpoB mutations affected the expression of ∼1700 genes; rho mutations affected the expression of fewer genes but most (83%) were a subset of those altered by rpoB mutants. Across the eight mutants, relative fitness correlated with the degree to which a mutation restored gene expression back to the unstressed, 37.0°C state. The beneficial mutations in the two genes did not have identical effects on fitness, growth or gene expression, but they caused parallel phenotypic effects on gene expression and genome-wide transcriptional termination.