Identification of Essential Residues in the Erm(B) rRNA Methyltransferase ofClostridium perfringens

Abstract

Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein from Clostridium perfringens with the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried the erm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in the erm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of the C. perfringens Erm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.Macrolide, lincosamide, and streptogramin B (MLS) antibiotics are a diverse group of antibacterial agents that are chemically distinct but that have similar modes of action. They act at the early stages of protein synthesis by blocking the growth of the nascent peptide chain (1), which then presumably causes the premature dissociation of the peptidyl-tRNA molecule from the ribosome (22). These antibiotics include erythromycin, clindamycin, and lincomycin and are active against various bacteria, including gram-positive cocci and rods and gramnegative cocci.Four mechanisms of bacterial resistance to MLS antibiotics have been detected; they involve enzymatic modification of the antibiotic, active efflux from the bacterial cell, mutation of the ribosomal target site, or, most commonly, enzyme-mediated chemical alteration of the rRNA target (18,19,33). The last mechanism is mediated by the synthesis of a 23S rRNA methyltransferase, which is responsible for the N 6 -dimethylation of a specific adenine residue in the 23S rRNA molecule (17).Methyltransferases are enzymes that methylate a wide variety of substrates; they use S-adenosyl-L-methionine (SAM) as the universal methyl donor and release S-adenosyl-L-homocysteine as the reaction product (4). Comparative analysis of over 40 DNA amino-methyltransferases (21) revealed the presence of nine conserved sequence motifs that are important in target sequence specificity, catalysis, and SAM binding (Fig. 1). Motif I is highly conserved and forms a secondary structure known as the G loop which binds to the methionine moiety of SAM.Motifs II and III are less conserved, with motif II containing a negatively charged amino acid that interacts with the ribose hydroxyls of SAM and a bulky hydrophobic side chain that makes contact with the...

The Expression of Antibiotic Resistance Methyltransferase Correlates with mRNA Stability Independently of Ribosome Stalling

“…An antibody against C. perfringens ErmB (52) was used to probe the expression level of ErmB. A catalytically inactive mutant of ErmB (Y103A, Y104 numbering in ErmC [65]) served as a control.…”

“…Total soluble proteins (40 μg/lane) were resolved using 4 to 20% TGX SDS-PAGE (Bio-Rad), and the proteins were transferred to a nitrocellulose membrane using a Trans-Blot Turbo system (Bio-Rad). A 1/1,000 dilution of anti-ErmB (kindly provided by J. Rood) (52) and a 1/10,000 dilution of anti-RNAP α (NeoClone) were used for immunoblotting. The intensity of immunoblot bands was quantitated by using ImageJ.…”

Identification and Antimicrobial Susceptibility Testing of Anaerobic Bacteria: Rubik’s Cube of Clinical Microbiology?

“…Several studies showed that macrolides (azithromycin, clarithromycin) are potent agents against some anaerobic bacteria, although their bacteriostatic effects hinder their usefulness in serious infections. Resistance to the macrolide-lincosamide-streptogramine-group of antimicrobials manifest uniformly (which can be attributed to various erm genes): due to the methylation of two specific adenine residues of the 23S rRNA, the effective binding of the drugs to ribosome is prevented [ 159 , 160 , 161 , 162 , 163 ].…”

Potential Target Site for Inhibitors in MLSB Antibiotic Resistance

“…Although motif X, one of the nine common SAM-dependent MTase functional motifs, motifs I to VIII and X observed in amino (N 4 -cytosine or N 6 -adenine) MTases [ 18 ] is well known to be located before motif I in Erm and KsgA/Dim1, its identity, especially the range of sequence encompassing motif X, is different depending on the report. It ranged from 18 amino acids [ 19 ], 6 amino acids [ 37 ] to 4 amino acids [ 17 ] (GQNF is the most appeared amino acids). Residue V21 in M .…”

“…In ErmC’ and ErmB, the motif X sequence is located near the active site and presumably, each amino acid might perform more than just one role. The exact location and range of motif X in Erm proteins are somewhat different depending on the reports [ 17 , 19 , 20 ]. However, in that motif, some consensus G(S/T)-Q-N(H)-F(L, Y) in which Q is highly conserved among almost all members of the families of Erm, and another sequence might be altered to amino acid(s) in parenthesis, could be identified with Erm families and some other slight variation in that consensus could be found with KsgA/Dim1 (see below).…”