engleski

Methyltransferases responsible for resistance to aminoglycoside antibiotics can methylate immature small ribosomal subunits

Methyltransferases play important role in ribosome biogenesis and decorate rRNA with methyl groups during different stages of ribosome subunits maturation. However, rRNA methyltransferases can also have critical importance for antibiotic resistance, and one the emerging mechanisms of resistance to aminoglycoside antibiotics is the methylation of 16S rRNA in the decoding center of small ribosomal subunit. 16S rRNA methyltransferases conferring resistance to aminoglycoside antibiotics can be found in antibiotic producers, as well as resistant pathogenic strains found in clinical isolates, and represent a major health concern in the fight against aminoglycoside antibiotic resistance. Enzymatic assays have shown that 16S rRNA methyltransferases responsible for resistance to aminoglycoside antibiotics modify 16S rRNA only in fully assembled 30S ribosomal subunits. Recent crystallographic data have shown that these methyltransferases dock and recognize four disparate 16S rRNA helices brought into proximity by 30S assembly, revealing structural basis for their requirement for assembled 30S subunits. However, whether aminoglycoside- resistance methyltransferases act upon fully assembled mature 30S subunits, or late-stage ribosome biogenesis 30S intermediates, remained an open question. Therefore, we have analyzed in vitro and in vivo propensity of aminoglycoside-resistance methyltransferases to methylate immature 30S precursors. Immature 30S subunits were isolated from ΔrsgA and ΔrimM knock-out strains, which accumulate immature 30S precursors. The isolated immature 30S were subjected to in vitro methylation and the modifications analyzed by primer extension analysis. The results have shown that both m1A1408 and m7G1405 methyltransferases were capable of 16S rRNA methylation incorporated in immature 30S precursors. The results were confirmed in vivo, by analysis of methylation of accumulated 30S ribosome biogenesis intermediates in knock-out strains. Direct interaction between methyltransferases and different maturation stage 30S subunits was observed by biophysical methods such as microscale thermophoresis and pull-experiments. Taken all together, our results suggest that methylation of small ribosomal subunit decoding center can occur during late stages of ribosome biogenesis, and not only on mature 30S subunits as previously thought.

methyltransferase ; small ribosomal subunit ; aminoglycoside antibiotic

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