Local genic base composition impacts protein production and cellular fitness

The maintenance of a G + C content that is higher than the mutational input to a genome provides support for the view that selection serves to increase G + C contents in bacteria. Recent experimental evidence from Escherichia coli demonstrated that selection for increasing G + C content operates at the level of translation, but the precise mechanism by which this occurs is unknown. To determine the substrate of selection, we asked whether selection on G + C content acts across all sites within a gene or is confined to particular genic regions or nucleotide positions. We systematically altered the G + C contents of the GFP gene and assayed its effects on the fitness of strains harboring each variant. Fitness differences were attributable to the base compositional variation in the terminal portion of the gene, suggesting a connection to the folding of a specific protein feature. Variants containing sequence features that are thought to result in rapid translation, such as low G + C content and high levels of codon adaptation, displayed highly reduced growth rates. Taken together, our results show that purifying selection acting against A and T mutations most likely results from their tendency to increase the rate of translation, which can perturb the dynamics of protein folding.