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.

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

“…3D), peaks at m/z 1305 and 3181 were missing and replaced by a longer fragment at m/z 4469 corresponding to the C1402-G1415 sequence but with only two methyl groups. The m 7 G1405 modification must be present in the peak at m/z 4469 to prevent its cleavage by RNase T1; taken together, these data indicate that it is one of the two rRNA methylations at C1402 that is missing rather than that at C1407 as previously suggested (Cubrilo et al 2009;Gutierrez et al 2012). …”

“…3B). The presence of the C1402-G1415 fragments resistant to cleavage by RNase T1 indicated that ArmA methylation is complete but that one of these endogenous modifications is no longer stoichiometric, as already shown for other G1405 methyltransferases (Cubrilo et al 2009;Gutierrez et al 2012). To discriminate between partial interference at position C1402 or C1407, pIP1204 was introduced into MM294ΔrsmF.…”

“…In MM294/ pGB2ΩarmA * , the rRNA fragment produced a spectrum pattern identical to that of MM294/pGB2. As already mentioned, in E. coli expression of m 7 G1405 resistance methyltransferases such as Sgm or RmtC was proposed to reduce endogenous RsmF methylation at C1407 (Cubrilo et al 2009;Gutierrez et al 2012). The MS spectrum of the fragment purified from MM294ΔrsmF showed peaks at m/z 1305 (containing m 4 Cm1402) and m/z 3181 (fragment U1406-G1415), the latter being 14 Da less than that from the rsmF + strain due to loss of C1407 methylation (Fig.…”

“…It was previously shown that methyltransferases Sgm and RmtC, which confer resistance by modification of G1405, impede RsmF methylation at C1407 (Cubrilo et al 2009;Gutierrez et al 2012). …”

Synthesis, antibacterial and cytotoxic activity evaluation of hydroxyurea derivatives

“…It is interesting, however, that compounds 5, 7 and 15 showed less evident antibacterial activity, either on the strain resistant to macrolide antibiotics or on the sensitive DH5a strain, while 10, 16 and 17 showed stronger antibacterial activity on the strain resistant to aminoglycoside antibiotics. Aminoglycoside resistant strain expressed 16S rRNA methyltransferase Sgm, a member of the Arm family of enzymes that methylate the N 7 position of residue G1405 within the decoding-or A-site in the small ribosomal subunit (23). This modification prevents 4,6-disubstituted 2-deoxystreptamine aminoglycosides from binding to the A-site, thus rendering them bacteria resistant (12).…”