Impact of synonymous mutations in the bla TEM-3 gene on gene expression and Escherichia coli fitness

Synonymous mutations (SMs) have a significant effect on the expression of the β-lactamase TEM-3 and the resistance phenotype conferred. Cell-free transcription did not reproduce this effect. The amount of mRNA obtained was identical for bla TEM-3 transcripts with or without SMs, and the transcripts were indistinguishable in terms of secondary structure and thermal de- and renaturation, as shown by circular dichroism experiments. In asymmetric competition assays, cells expressing the gene with SMs replaced cells expressing the gene without SMs after five days of passaging in a low concentration of ceftazidime. However, without antibiotic present, cells expressing the gene without SMs replaced cells expressing the gene with SMs. These experiments indicate that either post-transcriptional or in cellulo processes are responsible for the differences in expression levels and fitness. Possible mechanisms to be tested in future studies include epigenetic effects (different degrees of methylation) and the removal of the gene in the absence of β-lactam by recombination. Although the phagemid used in this study has the ampicillin resistance gene removed and replaced with a chloramphenicol resistance gene, the 5′-terminal portion of the bla TEM-1a gene is still present and might account for deletion by recombination. The question would then become why recombination is observed more in the phagemids with the gene without SMs. IMPORTANCE Most studies focusing on the evolution of antibiotic resistance focus on nonsynonymous mutations (NMs) in genes encoding proteins that cause resistance, such as β-lactamases. NMs result in amino acid changes that are often responsible for an extended substrate spectrum, insensitivity to inhibitors, or suppression of destabilizing effects introduced by other NMs. Although these mutations are very important, comparisons of natural sequences as well as directed evolution experiments have also identified synonymous mutations (SMs), but their impact is rarely studied. Here, we provide evidence that SMs in a β-lactamase gene alter the fitness levels conferred to Escherichia coli cells. Our results suggest that transcription efficiency is not affected, but that epigenetic factors could affect the stability of the gene inside the bacteria. Translation and protein folding efficiency could also not be ruled out thus far. Studying SMs will improve our understanding of how antibiotic resistance evolves and help us combat it.