engleski

Site-directed mutagenesis of the aminoglycoside resistance methylase Sgm

Methylation is the most frequent posttranscriptional modification found in rRNA, exerted mostly by S-adenosyl-methionine dependent methylases. In addition to the essential set of enzymes, some antibiotic producing bacteria as well as growing number of antibiotic resistant strains contain different rRNA methylases, which protect their protein synthesis machinery from the destructive action of ribosomal antibiotics. The self-defense mechanism of natural producers of deoxystreptamine-containing aminoglycoside antibiotics is methylation of a specific guanine residue within the 16S rRNA. In addition, this is a relatively new mechanism of resistance found in members of clinically important Enterobacteriaceae family and in Pseudomonas aeruginosa and Serratia marcescens clinical strains. Methylation of 16S rRNA in aminoglycoside resistant bacteria is carried out by enzymes from the aminoglycoside resistance methylase (Agr) family, which are now found to be spreading among clinical strains by rapid horizontal transfer. Very little is known on the mechanism of action of Agr methylases, while the structural data virtually do not exist. We are therefore studying the molecular basis of this mechanism, in order to help to prevent it before it becomes omnipresent, especially among multiresistant pathogens. The aim of our study is to functionally characterise the methyltransferase Sgm. We cloned the sgm gene into pET-25b(+) vector and developed the assay for functional test in vivo. By site-directed mutagenesis we changed the evolutionary conserved amino acids into alanines. We transformed the susceptible E. coli cells with recombinant vector carrying mutant genes and determined the minimal inhibitory concentration for kanamycin. Comparison of the in vivo activity of mutant variants with the action of the wild type enzyme gave us the first insight into the localisation of the active site and cofactor S-adenosyl-methione binding site. Obtained results together with the additional kinetic analysis in vitro will enable us to propose a reaction mechanism and to investigate the possibilities of finding a specific inhibitor that would block aminoglycoside resistance based on ribosome methylation.

aminoglycosides; resistance; methyltransferase Sgm

nije evidentirano