Compensatory evolution facilitates loss of prfB autoregulation in Pseudomonas fluorescens SBW25

Abstract

Understanding why some traits are maintained whereas others are repeatedly lost is a central question in evolutionary biology. Here, we address this problem through an analysis of the evolutionary dynamics of autoregulation of the prfB gene, which encodes peptide-chain release factor 2 (RF2), a key factor in bacterial translation termination. RF2 recognizes UGA and UAA stop codons and catalyzes the release of the completed polypeptide. In many species, prfB contains an internal UGA stop codon, triggering premature translation termination by RF2 itself. Complete RF2 translation depends on a +1 programmed ribosomal frameshifting (PRF) event on the internal stop codon, which occurs more frequently when RF2 levels are low, resulting in autoregulation of prfB expression. While widespread, this autoregulatory mechanism has been lost in multiple bacterial lineages. We combined phylogenetics, experimental evolution and molecular genetics to investigate the evolutionary forces behind this loss. We found no significant correlation between PRF loss and stop codon usage using phylogenetically informed analyses, and PRF disruption in Pseudomonas fluorescens SBW25 had no detectable fitness effect. However, engineered mutations that reduced frameshifting caused fitness defects, which were compensated by two classes of mutation: (i) mutations that impair specific ribosomal proteins, and (ii) single-nucleotide deletions in prfB that adjust the reading frame and bypass the internal stop codon. These results suggest that compensatory mutations facilitate the loss of prfB autoregulation under RF2-limiting conditions. We discuss three potential scenarios that could account for this process.