The ribosome is essential for production the of all proteins in the cell, thus a large portion of antibiotics work by inhibiting the function of the bacterial ribosome. Since these antibiotic binding pockets are primarily composed of ribosomal RNA (rRNA), a common resistance mechanism involves acquisition of rRNA-modifying enzymes. The enzyme Cfr is one such example. Cfr methylates rRNA at adenosine 2503 within the peptidyl transferase center (PTC) and bacteria that acquire the cfr gene become resistant to 8 classes of PTC-targeting antibiotics. However, our understanding of the principles that govern Cfr-mediated resistance are lacking, including how the extent of Cfr methylation influence phenotypic resistance. To address this question, we used directed evolution to generate Cfr variants that confer enhanced antibiotic resistance in E. coli. We used MALDI-TOF MS to assess the percentage of rRNA methylated by Cfr in vivo, and surprisingly observe a non-linear correlation between resistance and methylation. These Cfr variants accomplish enhanced RNA methylation through a novel mechanism, namely by enhancing translation of the cfr gene through early-codon mutations. Interestingly, these translation-enhancing codons can be found upstream of cfr genes that occur in nature, suggesting that this mechanism may be exploited by bacteria to enhance Cfr expression under antibiotic stress.

Investigating antibiotic resistance of a ribosomal-RNA methylating enzyme through directed evolution

ACS Fall 2020 Virtual Meeting

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