Adaptive selection on N-terminal codon bias in horizontally transferred dihydrofolate reductase genes intensifies at sub-MIC trimethoprim regime

Horizontal gene transfer (HGT) is common in bacteria. Although almost any gene can be subject to HGT, the spread of antibiotic resistance-conferring genes is central to bacterial adaptation. Codon usage compatibility between foreign and recipient genomes was shown to increase the chances of successful HGT events. Conversely, functional analysis of horizontally transferred antibiotic resistance genes concluded that codon usage of foreign genes does not limit their functional integration within the host. To understand the mechanistic reason for the inconsistency between these findings, we used an experimental system in which the variability in codon bias of horizontally transferred antibiotic resistant genes was directly linked to organismal fitness of the host. Specifically, we replaced the chromosomal folA coding sequence in E. coli encoding dihydrofolate reductase (DHFR), an essential enzyme that constitutes a target for the antibiotic trimethoprim (TMP), with the original and codon-usage-optimized orthologous folA genes. When we used sub-minimal inhibitory concentrations (MIC) of TMP, the contribution of sequence composition was pronounced, reaching 3-8-fold difference in sensitivity to TMP. Also, we created synonymous libraries of orthologous DHFR’s. Each library was subjected to laboratory-based evolution in a range of TMP concentrations. We found that up to 40% of variability in fitness effects can be explained by the thermodynamic stability of mRNA secondary structure that controls translation initiation. Contribution of codon usage bias to translation elongation explains up to 12% of variability in observed fitness effects. Our findings support the notion that translation initiation constitute a rate limiting step for protein synthesis.