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

Abstract

In Gram-negative bacteria, acquired 16S rRNA methyltransferases ArmA and NpmA confer high-level resistance to all clinically useful aminoglycosides by modifying, respectively, G1405 and A1408 in the A-site. These enzymes must coexist with several endogenous methyltransferases that are essential for fine-tuning of the decoding center, such as RsmH and RsmI in Escherichia coli, which methylate C1402 and RsmF C1407. The resistance methyltransferases have a contrasting distribution--ArmA has spread worldwide, whereas a single clinical isolate producing NpmA has been reported. The rate of dissemination of resistance depends on the fitness cost associated with its expression. We have compared ArmA and NpmA in isogenic Escherichia coli harboring the corresponding structural genes and their inactive point mutants cloned under the control of their native constitutive promoter in the stable plasmid pGB2. Growth rate determination and competition experiments showed that ArmA had a fitness cost due to methylation of G1405, whereas NpmA conferred only a slight disadvantage to the host due to production of the enzyme. MALDI MS indicated that ArmA impeded one of the methylations at C1402 by RsmI, and not at C1407 as previously proposed, whereas NpmA blocked the activity of RsmF at C1407. A dual luciferase assay showed that methylation at G1405 and A1408 and lack of methylation at C1407 affect translation accuracy. These results indicate that resistance methyltransferases impair endogenous methylation with different consequences on cell fitness.

Keywords: 16S rRNA methyltransferase; aminoglycoside resistance; fitness; translation.

FIGURE 1.

Schematic representation of 16S rRNA helix 44 (positions 1378–1432) secondary structure isolated for mass spectrometry analysis. The sites of post-transcriptional endogenous (RsmH, RsmI, RsmF, and RsmE) and exogenous (ArmA and NpmA) methylation are indicated.

FIGURE 2.

Competition experiments between susceptible and resistant strains. Competition was carried out in LB broth at 37°C. MM294 harboring (A) pGB2 and pGB2ΩarmA or pGB2ΩarmA* and (B) pGB2ΩnpmA were mixed at a 1:1 ratio at an initial inoculum of 10³ CFU and transferred every 12 h (corresponding to about 20 generations) in fresh medium for up to six passages. The competition index (CI) was calculated as the CFU ratio of the resistant and susceptible strains (R/S) at time (t₁) divided by the same R/S at time (t₀), and the selection coefficient s was then calculated as the slope of the following linear regression model s= ln(CI)/[t × ln(2)], where t is the number of generations. Values are the mean ± SE of at least three independent experiments.

FIGURE 3.

MALDI MS analysis of ArmA methylation in 16S rRNA fragment C1378–G1432. (A) Spectrum of RNase T1 rRNA from E. coli MM294/pGB2. The empirical m/z values are given above the peaks and match the theoretical masses (see below) to within 0.1 Da. (B) Spectrum from E. coli MM294/pGB2ΩarmA. Fragments at m/z 1305 and 3195 are missing and fused in the longer fragment at m/z 4483. (Inset) Enlargement of the spectrum around m/z 4480. Spectrum of RNase T1 rRNA from (C) E. coli MM294▵rsmF/pGB2 and (D) E. coli MM294▵rsmF/pGB2ΩarmA. Fragments at m/z 1305 and 3181 are missing and are fused in the longer fragment at m/z 4469. (Inset) Enlargement of the spectrum around m/z 4460. (E) MALDI tandem MS of RNase A rRNA from E. coli MM294/pGB2ΩarmA. The fragment at m/z 986 corresponding to GCCp fragment (positions 1401–1403) was selected and subjected to further fragmentation. Peaks corresponding to mCCp (Z₂), Cp (W₁), and GmC (c₂) are indicated. These ions confirm that C1402 was monomethylated. (F) Theoretical masses of the monoisotopic E. coli RNase T1 fragments with 3′-linear phosphate (p). Only fragments that are tetranucleotides and larger are shown.

FIGURE 4.

MALDI MS analysis of NpmA methylation in 16S rRNA fragment C1378–G1432. (A) Spectrum of RNase T1 rRNA from E. coli MM294/pGB2ΩnpmA. The empirical m/z values are given above the peaks and match the theoretical masses (see below) to within 0.1 Da. (Inset) Higher mass region of rRNA showing the monomethylated C1406–G1415 fragment. (B) MALDI tandem MS of RNase A nucleotides from E. coli MM294/pGB2ΩnpmA. The CACp fragment (positions 1407–1409) at m/z 970 was selected and subjected to further fragmentation. Peaks corresponding to C (d₁), CmA (a₂), and mACp (Y₂) are indicated. (C) Theoretical masses of the monoisotopic E. coli RNase T1 fragments with 3′-linear phosphate (p). Only fragments that are tetranucleotides and larger are shown.

FIGURE 5.

Translational accuracy in E. coli MM294 in the presence of ArmA, ArmA*, or NpmA or in the absence of RsmF. Strain MM294 harboring pGB2 (white), pGB2ΩarmA (strips), pGB2ΩarmA* (gray), or pGB2ΩnpmA (dotted) was transformed with plasmids encoding Renilla luciferase (RLuc) starting with the AUG codon and Firefly luciferase (FLuc) starting with codon AUG or AUU on a single transcript. Translation initiation efficiency was measured by dividing the chemiluminescence of FLuc by that of RLuc and normalizing the ratio to that of MM294/pGB2 (relative F/R value). To measure the efficiency of +1 reading frame maintenance and UGA readthrough in the presence of ArmA or NpmA, fusion constructs containing the RLuc and FLuc open reading frames separated by short windows containing a stop codon (UGA) or a frameshift site (+1) were used. The efficiency of translation initiation at AUG and the accuracy of UGA readthrough in the absence of RsmF (black) were also determined. In this case, the ratio FLuc/RLuc was normalized to the ratio FLuc/RLuc obtained for E. coli MM294. The values are the means of three independent experiments carried out in quintuplates with error deviations. (*) Significant difference (P< 0.01) of the mean value.