Transcriptional Effects of Adaptive Synonymous Mutations in Pseudomonas fluorescens

Abstract

Synonymous mutations have traditionally been thought to have no significant effect on fitness. However, a growing body of recent research has shown that this is not always the case. In an experimentally evolved population of Pseudomonas fluorescens grown in minimal glucose media, synonymous mutations arose in a glucose transport gene that resulted in beneficial fitness effects comparable to those of non-synonymous mutations. We found that the increase in fitness was a direct result of increased gene expression; however, the precise mechanism was unclear. Synonymous mutations have been shown to affect gene expression on transcriptional and translational levels through changes in mRNA secondary structure and codon usage.

Our study investigates the underlying mechanisms in which these evolved synonymous mutations lead to increased gene expression. In addition to the evolved mutations, we have a library of 42 strains with single synonymous mutations within the glucose transport gene and found a positive correlation between fitness and gene expression. To determine whether these mutations affect transcript levels, translational efficiency or a combination of both, we systematically incorporated transcriptional and translational fusions of a yellow fluorescent protein within the glucose transport operon. We found that the evolved mutations predominantly act on the level of transcription and have strong polar downstream effects. Additionally, through manipulation of the local genetic sequence, we investigated the specific molecular requirements necessary for the increased expression. We found that for one of our evolved synonymous mutants, mRNA secondary structure does not play an essential role, but we speculate that the mutation may strengthen a weak internal promoter sequence to confer its increased expression. Our study provides evidence of the adaptive mechanisms of beneficial synonymous mutations in an experimentally evolved setting.