Identification of essential residues in the Erm(B) rRNA methyltransferase of Clostridium perfringens.
Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein from Clostridium perfringens with the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried the erm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in the erm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of the C. perfringens Erm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.
Farrow
Kylie A
KA
Bacterial Pathogenesis Research Group, Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia.
Lyras
Dena
D
Polekhina
Galina
G
Koutsis
Katerina
K
Parker
Michael W
MW
Rood
Julian I
JI
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003016:Clostridium perfringens / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004917:Erythromycin / Q000494:pharmacology; D015151:Immunoblotting; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D017422:Sequence Analysis, DNA
D000900:Anti-Bacterial Agents; D004917:Erythromycin; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase