Compensatory evolution facilitates loss of prfB autoregulation in Pseudomonas fluorescens SBW25.

Understanding why some traits are maintained whereas others are repeatedly lost is a central question in evolutionary biology. Here, we address this question through the evolutionary dynamics of autoregulation of prfB, which encodes peptide-chain release factor 2 (RF2), a factor in bacterial translation termination. RF2 recognizes UGA and UAA stop codons and catalyzes polypeptide release. In many species, prfB contains an internal UGA stop codon that causes premature termination by RF2. Full RF2 synthesis depends on a + 1 programed ribosomal frameshifting (PRF) event at this stop codon, which occurs more frequently when RF2 levels are low, resulting in autoregulation of prfB expression. While widespread, this mechanism has been lost repeatedly across bacteria. We combined phylogenetics, experimental evolution, and molecular genetics to investigate the evolutionary forces underlying this loss. Phylogenetically informed analyses revealed no significant correlation between autoregulation and UGA stop codon usage, and autoregulation elimination in Pseudomonas fluorescens SBW25 had no detectable fitness effect. However, engineered mutations that reduced frameshifting at the prfB autoregulatory site caused fitness defects that were compensated by two classes of mutation: mutations affecting ribosome-associated proteins (RsmA, RsmH, RplI), and single-nucleotide deletions in prfB that adjusted the reading frame to bypass the internal stop codon, eliminating autoregulation. These results suggest that loss of prfB autoregulation can be facilitated by compensatory mutations when frameshifting at the prfB autoregulatory site is compromised and RF2 production is insufficient. Our findings illustrate how compensatory evolution can favor trait loss when the fitness benefit of losing the trait outweighs its cost.