engleski

Mutations in the A site of 16S rRNA reveal recognition site of aminoglycoside resistance methyltransferase Sgm on the bacterial ribosome

Sgm (Sisomicin-gentamicin methylase) is a methyltransferase found in Micromonospora zionensis that produces the antibiotic G-52 (6-N-methyl-sisomicin). Sgm modifies nucleotide G1405 in the A site of 16S rRNA, conferring high-level resistance to aminoglycosides because of the inability of the drugs to bind to their target site in the 30 S ribosomal subunit. Sgm belongs to the Arm (aminoglycoside resistance methyltransferase) family, whose members were recently found to be spreading by horizontal transfer in growing number of clinical strains, which poses a serious threat for the successful treatment of severe bacterial infections. E. coli cells constitutively transcribe their ribosomal RNAs from seven rrn operons. In this work, we used a specialized E. coli system, in which all rrn operons were inactivated, and ribosomal RNA was transcribed from a vector-based rrn operon. We mutated nucleotides in the part of the operon corresponding to the A site of 16S rRNA, and monitored the impact of these mutations on the essential ribosomal functions. We determined generation time and investigated the ability of these cells to grow in the presence of various concentrations of aminoglycoside kanamycin. We then introduced actively expressing methyltransferase Sgm into these cells and monitored the impact of the mutations on the methyltransferase activity. This highly specialized experimental system enabled us to monitor the effect of the mutations introduced in the A site of 16S rRNA on the interaction between aminoglycoside resistance methyltransferase Sgm and bacterial ribosome. These findings are leading towards the defining of the recognition motif of methyltransferase Sgm on the bacterial ribosome, which will help to construct effective inhibitors of the members of Arm family of enzymes, thus fighting aminoglycoside resistance.

16S rRNA; A site; ribosome; mutations; aminoglycoside resistance; methyltransferase Sgm

nije evidentirano