Role of Synonymous Mutations in the Evolution of TEM β-Lactamase Genes

Nonsynonymous mutations are well documented in TEM β-lactamases. The resulting amino acid changes often alter the conferred phenotype from broad spectrum (2b) conferred by TEM-1 to extended spectrum (2be), inhibitor resistant (2br), or both extended spectrum and inhibitor resistant (2ber). The encoding bla_TEM genes also deviate in numerous synonymous mutations, which are not well understood. bla_TEM-3 (2be), bla_TEM-33 (2br), and bla_TEM-109 (2ber) were studied in comparison to bla_TEM-1bla_TEM-33 was chosen for more detailed studies because it deviates from bla_TEM-1 by a single nonsynonymous mutation and three additional synonymous mutations. Genes encoding the enzymes with only nonsynonymous or all (including synonymous) mutations plus all permutations between bla_TEM-1 and bla_TEM-33 were expressed in Escherichia coli cells. In disc diffusion assays, genes encoding TEM-3, TEM-33, and TEM-109 with all synonymous mutations resulted in higher resistance levels than genes without synonymous mutations. Disc diffusion assays with the 16 genes carrying all possible nucleotide change combinations between bla_TEM-1 and bla_TEM-33 indicated different susceptibilities for different variants. Nucleotide BLAST searches did not identify genes without synonymous mutations but did identify some without nonsynonymous mutations. Energies of possible secondary mRNA structures calculated with mfold are generally higher with synonymous mutations, suggesting that their role could be to destabilize the mRNA and facilitate its unfolding for efficient translation. In summary, our data indicate that transition from bla_TEM-1 to other variant genes by simply acquiring the nonsynonymous mutations is not favored. Instead, synonymous mutations seem to support the transition to other variant genes with nonsynonymous mutations leading to different phenotypes.