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 indigenous methyltransferase RlmN and its evolutionary-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. RlmN and Cfr are members of the Radical SAM superfamily, and contain the characteristic cysteine rich CX3CX2C motif. We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. S-adenosylmethionine (SAM) is both the methyl donor and the source of a 5′-deoxyadenosyl radical, which activates the substrate for methylation. Detailed analyses of the rRNA requirements show that the enzymes can utilize protein-free 23S rRNA as a substrate, but not the fully-assembled large ribosomal subunit, suggesting that the methylations take place during the assembly of the ribosome. The key recognition elements in the 23S rRNA are helices 90–92 and the adjacent single stranded RNA that encompasses A2503. To our knowledge, this study represents the first in vitro description of a methyl transfer catalyzed by a member of Radical SAM superfamily, and it expands the catalytic repertoire of this diverse enzyme class. Furthermore, by providing information on both the timing of methylation and its substrate requirements, our findings have important implications for the functional consequences of Cfr-mediated modification of rRNA in acquisition of antibiotic resistance.

Staphylococci are increasingly aggressive human pathogens suggesting that active evolution is spreading novel virulence and resistance phenotypes. Large staphylococcal plasmids commonly carry antibiotic resistances and virulence loci, but relatively few have been completely sequenced. We determined the plasmid content of 280 staphylococci isolated in diverse geographical regions from the 1940s to the 2000s and found that 79% of strains carried at least one large plasmid >20 kb and that 75% of these large plasmids were 20–30 kb. Using restriction fragment length polymorphism (RFLP) analysis, we grouped 43% of all large plasmids into three major families, showing remarkably conserved intercontinental spread of multiresistant staphylococcal plasmids over seven decades. In total, we sequenced 93 complete and 57 partial staphylococcal plasmids ranging in size from 1.3 kb to 64.9 kb, tripling the number of complete sequences for staphylococcal plasmids >20 kb in the NCBI RefSeq database. These plasmids typically carried multiple antimicrobial and metal resistances and virulence genes, transposases and recombinases. Remarkably, plasmids within each of the three main families were >98% identical, apart from insertions and deletions, despite being isolated from strains decades apart and on different continents. This suggests enormous selective pressure has optimized the content of certain plasmids despite their large size and complex organization.

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. Since many mutations could inactivate the rlmN gene, our findings unveil a potential mechanism for future linezolid resistance in clinical strains.