The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site

Abstract

Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally crowded stretch of the rRNA sequence. Here, we show that the Sgm methyltransferase confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides by introducing the 16S rRNA modification m(7)G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m(4)Cm1402 and m(5)C1407. Modification at m(5)C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance, is less able to interfere with RsmF methylation on the 30S subunit. The two methylations at 16S rRNA nucleotide m(4)Cm1402 are unaffected by both the wild-type and the mutant versions of Sgm. The data indicate that interplay between resistance methyltransferases and the cell's own indigenous methyltransferases can play an important role in determining resistance levels.

FIGURE 1.

Representation of the 16S rRNA secondary structure showing the sequence in helix 44 (boxed) that was isolated for analysis by mass spectrometry. The sites of post-transcriptional housekeeping modification in this E. coli rRNA region are indicated, and the methyltransferases that introduce these modifications are given (where known); the site of the m⁷G1405 modification added by the Sgm resistance methyltransferase is also shown.

FIGURE 2.

Gel autoradiogram of primer extension through the G1405 region of 16S rRNA. The rRNAs were from E. coli cells without sgm (lanes 1,2) or from cells expressing wild-type sgm (lanes 3,4); samples were either untreated (lanes 1,3) or were treated with NaBH₄/aniline prior to primer extension. In lane 4, rRNA scission stops reverse transcriptase immediately 3′ to the site of Sgm methylation at G1405.

FIGURE 3.

MALDI-MS spectra of RNase T1 oligonucleotides from the E. coli 16S rRNA sequence C1378–G1432. (A) Spectrum of rRNA from cells without the sgm gene. The theoretical monoisotopic masses of the RNase digestion products with 3′-end cyclic phosphates (>p) are shown in the box. Fragments smaller than trinucleotides are not shown. The theoretical and empirical masses (given above the peaks) match to within 0.2 Da. (B) The corresponding spectrum from Sgm⁺ cells showing that the fragments at m/z 1289 and 3179 are missing and remain combined in the longer sequence at m/z 4467 and 4481. (Inset) Enlargement of this spectral region; the multiple tops in these fragments reflect the natural distribution of ¹²C and ¹³C isotopes. (C) The rRNA spectrum from Sgm-G135A cells; this differs from the Sgm⁺ rRNA in the relative amplitudes of the m/z 4467 and 4481 peaks, and also peaks at m/z 1289 and 3179 are evident.