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Functional characterization of aminoglycoside resistance methyltransferase Sgm and its interaction with ribosomal RNA

High-level aminoglycoside resistance in bacteria is conferred by Arm and Kam family of enzymes. Sgm methyltransferase, a member of Arm family of enzymes, is originally found in Micromonospora zionensis, a natural producer of aminoglycoside antibiotic sisomicin. Recently, members of this family have been found in many clinical strains. The available data on Sgm structure, function and mechanism of action are still very limited. In order to fight this fast spreading and dangerous mode of bacterial action against aminoglycosides, more information about these enzymes is needed. Recently we modelled the Sgm structure and analyzed sequence– structure-function relationships by limited proteolysis and mutagenesis. In this work we defined the position of the Sgm methylation, as well as the substrate requirements of the enzyme. We investigated how the presence of the Sgm enzyme affects the exponential growth of E. coli by growing cells in different media at different temperatures. In competition growth assays we monitored the competition of the cells carrying the Sgm methyltransferase with the cells lacking the enzyme. 30S subunits were shown to be the substrate for Sgm MTase, while entire ribosome or naked RNA could not be methylated. Both primer extension analysis and MALDI-TOF MS confirmed that the methylation occurs at G1405 in 16S rRNA. E. coli cells expressing the Sgm grow slightly faster compared with the non expressing strain. Our results set the basis for current kinetic studies of the reaction mechanism and recognition elements in 16S rRNA, which will help to find the specific inhibitor of Arm MTases and thus fight the aminoglycoside resistance.

aminoglycoside resistance; 16S rRNA methylation; Sgm methyltransferase; MALDI-TOF MS

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