Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics

Abstract

The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles.

Analysis of gene expression from theWolbachiagenome of a filarial nematode supports both metabolic and defensive roles within the symbiosis

“…This suggests that S21 is not essential for ribosomal function but may have a regulatory role in protein biosynthesis. Two nonstructural components with ancillary roles in translation were also up-regulated in the gonad: polypeptide deformylase (Mazel et al 1994) and a predicted S-adenosyl-L-methionine-dependent methyltransferase (Pleshe et al 2005;Savic et al 2009). Additionally, evidence for a higher rate of DNA replication in this tissue relative to the soma was apparent in the increased expression of uracil-DNA glycosylase, a key enzyme of the base excision system (Holmquist 1998), and a nucleoid DNA-binding protein of the HU family, which exhibits peak expression during the exponential phase of growth in Escherichia coli (Luijsterburg et al 2006).…”

rRNA Methyltransferases and their Role in Resistance to Antibiotics

“…Closely related GrmA (gentamicin-resistance methyltransferase), found in the gentamicinproducing strain Micromonospora echinospora (formerly known as Micromonospora purpurea) (60) also catalyze the modification of G1405 at the N7 The KamA MTase from Streptomyces tenjimariensis acts at the N1 position of A1408 conferring resistance to kanamycin, tobramycin, sisomicin, and apramycin but not to gentamicin (22). The exact same site of the methylation is also confirmed for the KamB MTases from Streptoalloteichus tenebrarius (56) and KamC MTases from Saccharopolyspora hirsuta (62) ( Figure 1A). These MTases constitute the Kam family of producers' MTases.…”

“…Two distinct groups of 16S rRNA aminoglycoside resistance MTases, Kgm and Kam families, conferring resistance to overlapping sets of aminoglycosides, have been distinguished based upon their target nucleotides, G1405 or A1408, respectively (55,56). Methylation of these two nucleotides affects drug binding not only by steric hindrance but also by charge repulsion.…”

“…The KgmB (kanamycin-gentamicin methyltransferase) from Streptoalloteichus tenebrarius (57), formerly Streptomyces tenebrarius, a producer of nebramycin complex (58), and the Sgm (sisomicingentamicin methyltransferase) from Micromonospora zionensis, a producer of G-52 (59), modify the N7 position of G1405, in the A-site of 16S rRNA (22,56). Closely related GrmA (gentamicin-resistance methyltransferase), found in the gentamicinproducing strain Micromonospora echinospora (formerly known as Micromonospora purpurea) (60) also catalyze the modification of G1405 at the N7 The KamA MTase from Streptomyces tenjimariensis acts at the N1 position of A1408 conferring resistance to kanamycin, tobramycin, sisomicin, and apramycin but not to gentamicin (22).…”

“…Soon after circuitously proving that Sgm MTase shares the same site of modification with KgmB, we have experimentally determined that G1405 is the Sgm target nucleotide, as well as for GrmA from M. echinospora and Krm from Frankia sp. Ccl3 (56). Methylation sites have also been identified for functionally equivalent MTases from isolates of bacterial pathogens, as G1405 for ArmA and RmtB, and A1408 for NpmA (69,78,79).…”