Fitness Cost and Interference of Arm/Rmt Aminoglycoside Resistance with the RsmF Housekeeping Methyltransferases

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Escherichia coli</jats:named-content>, corresponding to the genotypes<jats:italic>rsmF</jats:italic><jats:sup>+</jats:sup>, Δ<jats:italic>rsmF</jats:italic>,<jats:italic>rsmF</jats:italic><jats:sup>+</jats:sup><jats:italic>rmtC</jats:italic><jats:sup>+</jats:sup>, and Δ<jats:italic>rsmF rmtC</jats:italic><jats:sup>+</jats:sup>. When analyzed for the antimicrobial resistance pattern, the Δ<jats:italic>rsmF</jats:italic>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<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">E. coli</jats:named-content>. Competition experiments between the isogenic<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">E. coli</jats:named-content>strains showed that, contrary to expectation, acquisition of<jats:italic>rmtC</jats:italic>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.</jats:p>

Aminoglycoside resistance 16S rRNA methyltransferases block endogenous methylation, affect translation efficiency and fitness of the host

“…As opposed to our findings, it was reported that acquisition of rmtC, which like ArmA methylates G4015, does not entail a fitness cost for the host (Gutierrez et al 2012). In this study, RmtC was shown to block endogenous methylation at C1407 by RsmF in E. coli, but unexpectedly, an rsmF knockout was found to display slower growth and reduced fitness in competition with the parental strain (Gutierrez et al 2012).…”

“…Competition experiments should explore the entire growth cycle, i.e., the lag phase, the exponential, and the stationary phases on several cycles. As reported in Gutierrez et al (2012), 20 generations were reached after the fourth transfer performed every 24 h; this would correspond to an estimated, rather unusual, 4-h doubling time for E. coli. In addition, the proportion of resistant to susceptible strains was determined on 100 colonies by replica plating or PCR (Gutierrez et al 2012), whereas we surveyed 500-1000 colonies by replica plating on selective media.…”

“…In this study, RmtC was shown to block endogenous methylation at C1407 by RsmF in E. coli, but unexpectedly, an rsmF knockout was found to display slower growth and reduced fitness in competition with the parental strain (Gutierrez et al 2012). …”

“…Different experimental conditions could account for these contradictory results. In Gutierrez et al (2012), growth rates were measured every hour over a 12-h period as opposed to every 3 min at the beginning of the exponential phase (Foucault et al 2009). Competition experiments should explore the entire growth cycle, i.e., the lag phase, the exponential, and the stationary phases on several cycles.…”

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

“…The network of long-range interactions provides the basis for the action of the antibiotic kasugamycin, which binds in the P site and requires dimethylation of m 6 2 A1519 for its function 15 . In the A site of the decoding centre, the aminoglycoside class of antibiotics directly binds to a monomethylated residue, m 5 C1407 in 16S rRNA, which is needed for optimum drug activity 16 (Extended Data Fig. 5c).…”