Purifying selection enduringly acts on the sequence evolution of highly expressed proteins in Escherichia coli.

The evolutionary speed of a protein sequence is constrained by its expression level, with highly expressed proteins evolving relatively slowly. This negative correlation between expression levels and evolutionary rates (known as the E-R anticorrelation) has already been widely observed in past macroevolution between species from bacteria to animals. However, it remains unclear whether this seemingly general law also governs recent evolution, including past and de novo, within a species. However, the advent of genomic sequencing and high-throughput phenotyping, particularly for bacteria, has revealed fundamental gaps between the 2 evolutionary processes and has provided empirical data opposing the possible underlying mechanisms which are widely believed. These conflicts raise questions about the generalization of the E-R anticorrelation and the relevance of plausible mechanisms. To explore the ubiquitous impact of expression levels on molecular evolution and test the relevance of the possible underlying mechanisms, we analyzed the genome sequences of 99 strains of Escherichia coli for evolution within species in nature. We also analyzed genomic mutations accumulated under laboratory conditions as a model of de novo evolution within species. Here, we show that E-R anticorrelation is significant in both past and de novo evolution within species in E. coli. Our data also confirmed ongoing purifying selection on highly expressed genes. Ongoing selection included codon-level purifying selection, supporting the relevance of the underlying mechanisms. However, the impact of codon-level purifying selection on the constraints in evolution within species might be smaller than previously expected from evolution between species.