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

The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site

Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally crowded stretch of the rRNA sequence. Here, we show that the Sgm methyltransferase confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides by introducing the 16S rRNA modification m7G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m4Cm1402 and m5C1407. Modification at m5C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance, is less able to interfere with RsmF methylation on the 30S subunit. The two methylations at 16S rRNA nucleotide m4Cm1402 are unaffected by both the wild-type and the mutant versions of Sgm. The data indicate that interplay between resistance methyltransferases and the cell's own indigenous methyltransferases can play an important role in determining resistance levels

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