engleski

Mutations in the ribosomal A site reveal a difference between ribosomal binding patterns among Arm methyltransferases

Aminoglycoside antibiotics act by binding to the A site 16S rRNA, thereby causing lethal errors in bacterial translation process. Natural producers of aminoglycosides use methyltransferases that modify target nucleotides in the A site of 16S rRNA and thus surpass the toxic effect of their own product. Enzymes that are involved in this process are members of Arm or Kam family of enzymes. Recently, members of these families of enzymes were found to be spreading by horizontal transfer in growing number of clinical strains, causing high-level aminoglycoside resistance. In this work we mutated nucleotides in the highly conserved A site of 16S rRNA and explored the ribosomal A site binding pattern of a Sgm methyltransferase from the natural producer of aminoglycoside and the Arm enzymes isolated from clinical pathogens, ArmA, RmtA, RmtB, RmtC and RmtD. We used a specialized E. coli system in which various 16S rRNA mutations were introduced in a strain carrying only one copy of 16S rRNA. Resulting cells harboured a uniform population of mutated ribosomes. We measured the susceptibility of these cells to the various concentrations of aminoglycoside kanamycin. We then introduced actively expressing Arm methyltransferases into these cells and monitored the impact of the mutations on the methyltransferase activity by determining minimal inhibitory concentration of kanamycin for these cells and analyzing the target nucleotide methylation with primer extension. Our results show that mutations in the A site of 16S rRNA affect the interaction between Arm methyltransferases and the bacterial ribosomes for Sgm methyltransferase. The enzymes from clinical strains apparently exhibit no sequence requirements for successful methylation, possibly as an evolutionary adaptation to modify rRNA in different bacterial species. We therefore strongly believe that it is of great importance to functionally characterize more members of the family in order to construct effective inhibitors of the members of the whole Arm family that would help to fight aminoglycoside resistance.

aminoglycoside resistance; methyltransferases; Arm; Kam; Ribosome; A site; mutagenesis

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