title: Fitness cost and interference of Arm/Rmt aminoglycoside resistance with the RsmF housekeeping methyltransferases.
abstract: Arm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants of Escherichia coli, corresponding to the genotypes rsmF(+), ΔrsmF, rsmF(+) rmtC(+), and ΔrsmF rmtC(+). When analyzed for the antimicrobial resistance pattern, the ΔrsmF bacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility in E. coli. Competition experiments between the isogenic E. coli strains showed that, contrary to expectation, acquisition of rmtC does not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.
authors.affiliation: Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain.
authors.forename: Belen
authors.initials: B
authors.lastname: Gutierrez
authors.forename: Jose A
authors.initials: JA
authors.lastname: Escudero
authors.forename: Alvaro
authors.initials: A
authors.lastname: San Millan
authors.forename: Laura
authors.initials: L
authors.lastname: Hidalgo
authors.forename: Laura
authors.initials: L
authors.lastname: Carrilero
authors.forename: Cristina M
authors.initials: CM
authors.lastname: Ovejero
authors.forename: Alfonso
authors.initials: A
authors.lastname: Santos-Lopez
authors.forename: Daniel
authors.initials: D
authors.lastname: Thomas-Lopez
authors.forename: Bruno
authors.initials: B
authors.lastname: Gonzalez-Zorn
mesh_terms: D000617:Aminoglycosides / Q000737:chemistry / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D056084:Genetic Fitness; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism; D012475:Salmonella / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D007527:Isoenzymes; D012336:RNA, Ribosomal, 16S; D008780:Methyltransferases; D003545:Cysteine; D005998:Glycine