The genetic environment of the cfr gene and the presence of other mechanisms account for the very high linezolid resistance of Staphylococcus epidermidis isolate 426-3147L.

The clinical Staphylococcus epidermidis isolate 426-3147L exhibits an unusually high resistance to linezolid that exceeds 256 μg/ml. The presence of the cfr gene, encoding the RNA methyltransferase targeting an rRNA nucleotide located in the linezolid binding site, accounts for a significant fraction of resistance. The association of cfr with a multicopy plasmid is one of the factors that contribute to its elevated expression.

Genetic environment and stability of cfr in methicillin-resistant Staphylococcus aureus CM05.

The Cfr methyltransferase confers resistance to many 50S ribosomal subunit-targeted antibiotics, including linezolid (LZD), via methylation of the 23S rRNA base A2503 in the peptidyl transferase center. Methicillin-resistant Staphylococcus aureus strain CM05 is the first clinical isolate documented to carry cfr. While cfr is typically plasmid borne, in CM05 it is located on the chromosome and is coexpressed with ermB as part of the mlr operon.

Low fitness cost of the multidrug resistance gene cfr.

The recently described rRNA methyltransferase Cfr that methylates the conserved 23S rRNA residue A2503, located in a functionally critical region of the ribosome, confers resistance to an array of ribosomal antibiotics, including linezolid. A number of reports of linezolid-resistant cfr-positive clinical strains indicate the possible rapid spread of this resistance mechanism. Since the rate of dissemination and the efficiency of maintenance of a resistance gene depend on the fitness cost associated with its acquisition, we investigated the fitness cost of cfr expression in a laboratory Staphylococcus aureus strain.

Inactivation of the indigenous methyltransferase RlmN in Staphylococcus aureus increases linezolid resistance.

The indigenous methyltransferase RlmN modifies A2503 in 23S rRNA. A recently described rlmN mutation in a clinical Staphylococcus aureus isolate decreases susceptibility to linezolid and was thought to increase the extent of A2503 modification. However, we show that the mutation in fact abolishes RlmN activity, resulting in a lack of A2503 modification.

RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA.

Posttranscriptional modifications of ribosomal RNA (rRNA) nucleotides are a common mechanism of modulating the ribosome's function and conferring bacterial resistance to ribosome-targeting antibiotics. One such modification is methylation of an adenosine nucleotide within the peptidyl transferase center of the ribosome mediated by the endogenous methyltransferase RlmN and its evolutionarily related resistance enzyme Cfr. These methyltransferases catalyze methyl transfer to aromatic carbon atoms of the adenosine within a complex 23S rRNA substrate to form the 2,8-dimethylated product.

Synthesis of a hemoglobin polymer containing antioxidant enzymes using complementary chemistry of maleimides and sulfhydryls.

To increase the overall size of hemoglobin (Hb), we developed a novel system of polymerization based on the complementary chemistry between sulfhydryls and maleimides. The maleimides were introduced onto the protein through N-(-maleimidobutyryloxy) succinimide, while the sulfhydryls were added using 2-iminothiolane hydrochloride (Trauts reagent). Resulting polymers showed SDS-PAGE bands with molecular weights as high as 96 kDa.