INVESTIGATING THE ROLE OF NOVEL ADAPTATIONS TO tRNA-ACTING ENZYMES IN OPPORTUNISTIC PATHOGENIC BACTERIA

Abstract

The decoding of mRNA into protein is the final step of the central dogma proposed by Francis Crick. The fidelity of this step relies on intermediate enzymes to ensure that the adapter molecule tRNA will interact with the correct mRNA trinucleotide and incorporate the proper amino acid during protein synthesis. Adaptations to these enzymes expand and restrict their functionality in the cell, and our continued investigation informs how life evolves in response to external stimuli. Herein, we investigate two tRNA-acting enzymes from opportunistic pathogenic bacteria. The Mycoplasma penetrans genome contains an unusually large metS gene. Further analysis indicated the gene product was a fusion protein with an appended aminotransferase to the N-terminus of the MetRS enzyme. We show that this N-terminal appended domain functions as an aminotransferase in vitro and can provide the synthetase domain with the Met substrate. In the bacterium Burkholderia cenocepacia, we identified mutations to the tRNA modifying enzyme TilS and its substrate tRNAIle2. These mutant strains could exit lag phase earlier than the wild-type ancestor, generating a fitness benefit in a nutrient depleted environment. These mutations led to an impaired functionality for the TilS enzyme, and as a consequence a reduced pool of modified tRNAIle2 which is responsible for decoding the underutilized AUA codon in these bacteria. Characterization of this enzyme with TilS orthologs suggests that bacteria may tune TilS catalytic efficiency based on the translational needs for this decoding event. Additionally, comparison of the mutations observed in BcTilS with these orthologs suggests that the positions are more important for B. cenocepacia. Finally, investigation into the cellular impact of these mutations in B. cenocepacia revealed that while translation may be reduced it seemed to be less than predicted. Our focus has primarily been on the mutations to the TilS enzyme, though we have done some investigation into the selected tRNAIle2 mutants. It seems these mutations provide a separate and as-yet undefined benefit, as the tRNA mutants were indistinguishable from wild-type tRNAIle2 in vitro.