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

ABSTRACT: 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 blaTEM genes also deviate in numerous synonymous mutations, which are not well understood. blaTEM-3 (2be), blaTEM-33 (2br), and blaTEM-109 (2ber) were studied in comparison to blaTEM-1blaTEM-33 was chosen for more detailed studies because it deviates from blaTEM-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 blaTEM-1 and blaTEM-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 blaTEM-1 and blaTEM-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 blaTEM-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.

SUBMITTER: Faheem M

PROVIDER: S-EPMC8315918 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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

Faheem Mohammad M Zhang Charles J CJ Morris Monica N MN Pleiss Juergen J Oelschlaeger Peter P

Antimicrobial agents and chemotherapy 20210518 6

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 blaTEM genes also deviate in numerous synonymous mutations, which are not well understood. blaTEM-3 (2be), blaTEM-33 (2br), and bla<sub ...[more]

PMID: 33820762

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Project description:β-lactamases are enzymes produced by bacteria to hydrolyze β-lactam antibiotics as a common mechanism of resistance. Evolution in such enzymes has been rendering a wide variety of antibiotics impotent, therefore posing a major threat. Clinical and in vitro studies of evolution in TEM-1 β-lactamase have revealed a large number of single point mutations that are responsible for driving resistance to antibiotics and/or inhibitors. The distal locations of these mutations from the active sites suggest that these allosterically modulate the antibiotic resistance. We investigated the effects of resistance driver mutations on the conformational dynamics of the enzyme to provide insights about the mechanism of their long-distance interactions. Through all-atom molecular dynamics (MD) simulations, we obtained the dynamic flexibility profiles of the variants and compared those with that of the wild type TEM-1. While the mutational sites in the variants did not have any direct van der Waals interactions with the active site position S70 and E166, we observed a change in the flexibility of these sites, which play a very critical role in hydrolysis. Such long distance dynamic interactions were further confirmed by dynamic coupling index (DCI) analysis as the sites involved in resistance driving mutations exhibited high dynamic coupling with the active sites. A more exhaustive dynamic analysis, using a selection pressure for ampicillin and cefotaxime resistance on all possible types of substitutions in the amino acid sequence of TEM-1, further demonstrated the observed mechanism. Mutational positions that play a crucial role for the emergence of resistance to new antibiotics exhibited high dynamic coupling with the active site irrespective of their locations. These dynamically coupled positions were neither particularly rigid nor particularly flexible, making them more evolvable positions. Nature utilizes these sites to modulate the dynamics of the catalytic sites instead of mutating the highly rigid positions around the catalytic site.

Dataset Information

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

Publications

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

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