Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria

X-ray crystal structures were determined of the broad-spectrum aminoglycoside-resistance A1408 16S rRNA methyltransferases KamB and NpmA, from the aminoglycoside-producer Streptoalloteichus tenebrarius and human pathogenic Escherichia coli, respectively. Consistent with their common function, both are Class I methyltransferases with additional highly conserved structural motifs that embellish the core SAM-binding fold. In overall structure, the A1408 rRNA methyltransferase were found to be most similar to a second family of Class I methyltransferases of distinct substrate specificity (m⁷G46 tRNA). Critical residues for A1408 rRNA methyltransferase activity were experimentally defined using protein mutagenesis and bacterial growth assays with kanamycin. Essential residues for SAM coenzyme binding and an extended protein surface that likely interacts with the 30S ribosomal subunit were thus revealed. The structures also suggest potential mechanisms of A1408 target nucleotide selection and positioning. We propose that a dynamic extended loop structure that is positioned adjacent to both the bound SAM and a functionally critical structural motif may mediate concerted conformational changes in rRNA and protein that underpin the specificity of target selection and activation of methyltransferase activity. These new structures provide important new insights that may provide a starting point for strategies to inhibit these emerging causes of pathogenic bacterial resistance to aminoglycosides.     

Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics

The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles.     

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

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.     

Functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization

Methylation of the bacterial small ribosomal subunit (16S) rRNA on the N1 position of A1408 confers exceptionally high-level resistance to a broad spectrum of aminoglycoside antibiotics. Here, we present a detailed structural and functional analysis of the Catenulisporales acidiphilia 16S rRNA (m¹A1408) methyltransferase (‘CacKam’). The apo CacKam structure closely resembles other m¹A1408 methyltransferases within its conserved SAM-binding fold but the region linking core β strands 6 and 7 (the ‘β6/7 linker’) has a unique, extended structure that partially occludes the putative 16S rRNA binding surface, and sequesters the conserved and functionally critical W203 outside of the CacKam active site. Substitution of conserved residues in the SAM binding pocket reveals a functional dichotomy in the 16S rRNA (m¹A1408) methyltransferase family, with two apparently distinct molecular mechanisms coupling cosubstrate/ substrate binding to catalytic activity. Our results additionally suggest that CacKam exploits the W203-mediated remodeling of the β6/7 linker as a novel mechanism to control 30S substrate recognition and enzymatic turnover.     

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

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 m⁷G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m⁴Cm1402 and m⁵C1407. Modification at m⁵C1407 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 m⁴Cm1402 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.     

Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit

NpmA, a methyltransferase that confers resistance to aminoglycosides was identified in an Escherichia coli clinical isolate. It belongs to the kanamycin–apramycin methyltransferase (Kam) family and specifically methylates the 16S rRNA at the N1 position of A1408. We determined the structures of apo-NpmA and its complexes with S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.4, 2.7 and 1.68 Å, respectively. We generated a number of NpmA variants with alanine substitutions and studied their ability to bind the cofactor, to methylate A1408 in the 30S subunit, and to confer resistance to kanamycin in vivo. Residues D30, W107 and W197 were found to be essential. We have also analyzed the interactions between NpmA and the 30S subunit by footprinting experiments and computational docking. Helices 24, 42 and 44 were found to be the main NpmA-binding site. Both experimental and theoretical analyses suggest that NpmA flips out the target nucleotide A1408 to carry out the methylation. NpmA is plasmid-encoded and can be transferred between pathogenic bacteria; therefore it poses a threat to the successful use of aminoglycosides in clinical practice. The results presented here will assist in the development of specific NpmA inhibitors that could restore the potential of aminoglycoside antibiotics.     

Mapping of the second tetracycline binding site on the ribosomal small subunit of E.coli

Tetracycline blocks stable binding of aminoacyl-tRNA to the bacterial ribosomal A-site. Various tetracycline binding sites have been identified in crystals of the 30S ribosomal small subunit of Thermus thermophilus. Here we describe a direct photo- affinity modification of the ribosomal small subunits of Escherichia coli with 7-[³H]-tetracycline. To select for specific interactions, an excess of the 30S subunits over tetracycline has been used. Primer extension analysis of the 16S rRNA revealed two sites of the modifications: C936 and C948. Considering available data on tetracycline interactions with the prokaryotic 30S subunits, including the presented data (E.coli), X-ray data (T.thermophilus) and genetic data (Helicobacter pylori, E.coli), a second high affinity tetracycline binding site is proposed within the 3′-major domain of the 16S rRNA, in addition to the A-site related tetracycline binding site.     

YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

Methylation at the 5-position of cytosine [m⁵C (5-methylcytidine)] occurs at three RNA nucleotides in Escherichia coli. All these modifications are at highly conserved nucleotides in the rRNAs, and each is catalyzed by its own m⁵C methyltransferase enzyme. Two of the enzymes, RsmB and RsmF, are already known and methylate 16S rRNA at nucleotides C967 and C1407, respectively. Here, we report the identity of the third E. coli m⁵C methyltransferase. Analysis of rRNAs by matrix-assisted laser desorption/ionization mass spectrometry showed that inactivation of the yccW gene leads to loss of m⁵C methylation at nucleotide 1962 in E. coli 23S rRNA. This methylation is restored by complementing the knockout strain with a plasmid-encoded copy of the yccW gene. Purified recombinant YccW protein retains its specificity for C1962 in vitro and methylates naked 23S rRNA isolated from the yccW knockout strain. However, YccW does not methylate assembled 50S subunits, and this is somewhat surprising as the published crystal structures show nucleotide C1962 to be fully accessible at the subunit interface. YccW-directed methylation at nucleotide C1962 is conserved in bacteria, and loss of this methylation in E. coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m⁵C methyltransferases RsmB and RsmF and is in fact more similar to known m⁵U (5-methyluridine) RNA methyltransferases. In keeping with the previously proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI.     

RNA Sequence Determinants for Aminoglycoside Binding to an A-site rRNA Model Oligonucleotide

The codon-anticodon interaction on the ribosome occurs in the A site of the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosomal RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spectroscopy were used to characterize the interaction between the aminoglycoside antibiotic paromomycin and a small model oligonucle otide that mimics the A site ofEscherichia coli16 S ribosomal RNA. Upon chemical modification, the RNA oligonucleotide exhibits an accessibility pattern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The anti biotic binding site forms an asymmetric internal loop, caused by non-canonical base-pairs. Nucleotides that are important for binding of paromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407·G1494 base-pair, and A·U base-pair at positions 1410/1490, and nucleotides A1408, A1493 and U1495. The asymmetry of the internal loop, which requires the presence of a nucleotide in position 1492, is also crucial for antibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA result in weaker binding of paromomycin to the oligonucleotide. Oligonucleotides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific action of aminoglycosides.     

Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases

Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 Å resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m⁷G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.     

Identification of novel methyltransferases,Bmt5 and Bmt6, responsible for the m3U methylations of 25S rRNA in Saccharomyces cerevisiae

RNA contains various chemical modifications that expand its otherwise limited repertoire to mediate complex processes like translation and gene regulation. 25S rRNA of the large subunit of ribosome contains eight base methylations. Except for the methylation of uridine residues, methyltransferases for all other known base methylations have been recently identified. Here we report the identification of BMT5 (YIL096C) and BMT6 (YLR063W), two previously uncharacterized genes, to be responsible for m³U2634 and m³U2843 methylation of the 25S rRNA, respectively. These genes were identified by RP-HPLC screening of all deletion mutants of putative RNA methyltransferases and were confirmed by gene complementation and phenotypic characterization. Both proteins belong to Rossmann-fold–like methyltransferases and the point mutations in the S-adenosyl-l-methionine binding pocket abolish the methylation reaction. Bmt5 localizes in the nucleolus, whereas Bmt6 is localized predominantly in the cytoplasm. Furthermore, we showed that 25S rRNA of yeast does not contain any m⁵U residues as previously predicted. With Bmt5 and Bmt6, all base methyltransferases of the 25S rRNA have been identified. This will facilitate the analyses of the significance of these modifications in ribosome function and cellular physiology.     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

Yeast Rrp8p,a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA

Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2′-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m¹A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m¹A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.     

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.     

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6′)-Ii that encodes a 6′-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m⁵C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m⁵C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m⁵C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes'' respective specificities for nucleotides C1404 and C1407.     

Identification of pseudouridine methyltransferase in Escherichia coli

In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem–loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m³Ψ) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Ψ1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m³Ψ1915 is the only methylated pseudouridine in bacteria described to date.