Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria

The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity

Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics

Structural basis for the methylation of G1405 in 16s rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: A diversity of active sites in m7G methyltransferases

10.1093/nar/gkq122Nucleic Acids Research38124120-4132NARH

Tobramycin at subinhibitory concentration inhibits the RhlI/R quorum sensing system in a <it>Pseudomonas aeruginosa </it>environmental isolate

<p>Abstract</p> <p>Background</p> <p>Antibiotics are not only small molecules with therapeutic activity in killing or inhibiting microbial growth, but can also act as signaling molecules affecting gene expression in bacterial communities. A few studies have demonstrated the effect of tobramycin as a signal molecule on gene expression at the transcriptional level and its effect on bacterial physiology and virulence. These have shown that subinhibitory concentrations (SICs) of tobramycin induce biofilm formation and enhance the capabilities of <it>P. aeruginosa </it>to colonize specific environments.</p> <p>Methods</p> <p>Environmental <it>P. aeruginosa </it>strain PUPa3 was grown in the presence of different concentrations of tobramycin and it was determined at which highest concentration SIC, growth, total protein levels and translation efficiency were not affected. At SIC it was then established if phenotypes related to cell-cell signaling known as quorum sensing were altered.</p> <p>Results</p> <p>In this study it was determined whether tobramycin sensing/response at SICs was affecting the two independent AHL QS systems in an environmental <it>P. aeruginosa </it>strain. It is reasonable to assume that <it>P. aeruginosa </it>encounters tobramycin in nature since it is produced by niche mate <it>Streptomyces tenebrarius</it>. It was established that SICs of tobramycin inhibited the RhlI/R system by reducing levels of C4-HSL production. This effect was not due to a decrease of <it>rhlI </it>transcription and required tobramycin-ribosome interaction.</p> <p>Conclusions</p> <p>Tobramycin signaling in <it>P. aeruginosa </it>occurs and different strains can have a different response. Understanding the tobramycin response by an environmental <it>P. aeruginosa </it>will highlight possible inter-species signalling taking place in nature and can possible also have important implications in the mode of utilization for human use of this very important antibiotic.</p