RNA modification enzymes in the manifestation of methyltransferases play a major role in antibiotic resistance in bacteria. The ribosome modifications are almost exclusively methylation of either the 2'-O-ribose position or various positions on the bases. A wealth of information on antibiotic resistance caused by methylation of rRNA has been revealed during the last ten years. Modifications at eight 23S rRNA nucleotides (G748, A1067, C1920, A2058, G2470, U2479, A2503 and G2535) on the large ribosomal subunit have so far been revealed as antibiotic resistance determinants. In addition, the absence of intrinsic methylation can also lead to reduced antibiotic susceptibility. Antibiotics typically bind to regions of functional importance and sterically block binding of interacting molecules and/or hinder structural rearrangements needed for ribosome activity. The RNA methyltransferases all act at RNA placed at or near the binding site of the antibiotic to which they confer resistance. The structures of about half of the methyltransferases associated with antibiotic resistance and known to act on 23S rRNA have been determined, but there is at present only limited information on the interaction of the methyltransferases with their RNA targets and the timing of the methylation reactions during ribosome assembly.

Introduction

Antibiotic resistance is a phenomenon of crucial importance in the treatment of diseases caused by pathogenic microorganisms. In general, antibiotic binding to a target interferes with or inhibits an essential cellular pathway or process. Bacteria have evolved various strategies to evade the cytotoxic effects of antibiotics. One of these strategies is alteration of the antibiotic binding site by enzymatic modification. The ribosome is a major site of antibiotic action in the bacterial cell and is targeted by a large and chemically diverse group of antibiotics (reviewed in refs. 1, 2). A number of these antibiotics have important applications in human and veterinary medicine in the treatment of bacterial infections. The antibiotic binding sites are clustered at functional centers of the ribosome, such as the decoding center on the 30S subunit, the peptidyltransferase center (PTC), GTPase center and peptide exit tunnel on the 50S subunit and the subunit interface spanning both subunits on the 70S ribosome (Fig. 1). Since the year 2000, high resolution structures of the Thermus thermophilus, Deinococcus radiodurans and Haloarcula marismotui ribosomal subunits bound to antibiotics have made significant contributions in localizing drug-binding sites and understanding antibiotic resistance (reviewed in refs. 1, 2). Upon binding, the drugs interfere with the positioning and movement of substrates, products and ribosomal components that are essential for protein synthesis.

Figure 1

Models of the large ribosomal subunit and the assembled ribosome showing the sites of methylations related to antibiotic resistance. The methyltransferases mentioned in the text and the antibiotic binding sites they target are indicated with arrows. The (more...)

Nature has evolved an effective and elegant way of preventing drug binding to the ribosome by simply adding methyl groups to rRNA at appropriate sites. Strangely, methylation is the only type of RNA modification found to date that provides acquired antibiotic resistance.³ A possible explanation for this is that these antibiotic resistance determinants evolved from other methyltransferases acting on rRNA performing so-called housekeeping modifications. These modification enzymes act on rRNA clustered at or close to the A, P and E sites, the ribosomal subunit interface and at other functionally important sites (reviewed in ref. 4), including those also targeted by antibiotics. These housekeeping modifications are believed to fine tune the function of the translational apparatus in various ways⁴ but their exact roles are not well described. In fact, the distinction between housekeeping modifications and modifications participating in antibiotic resistance is not completely clear and there seems to be some functional overlap. For example, RlmA^II is a housekeeping methyltransferase in Gram-positive bacteria, but it also contributes significantly to tylosin resistance when combined with the ErmN monomethylase as described below.

The absence of modifications is also known to confer antibiotic resistance. Some classic examples are the lack of the Ksg methyltransferase that causes kasugamycin resistance due to lack of methylation of 16S rRNA and the lack of methylation at U2584 (E. coli numbering) in Haloarcula marismortui 23S rRNA that causes sparsomycin resistance.^5,6 A recent discovery of intrinsic resistance concerns the Ψ at position 2504 in E. coli 23S rRNA, where inactivation of the rluC gene confers significant resistance to clindamycin, linezolid and tiamulin.⁷ None of the other eight intrinsic rRNA modifying enzymes targeting the PTC in E. coli seem to affect antibiotic susceptibility.⁷

Ribosomal RNA methyltransferases causing antibiotic resistance were discovered approximately 50 years ago. Shortly after the introduction of erythromycin into therapy in the 1950's, resistance to the drug was observed in bacterial pathogens (reviewed in ref. 8). The erythromycin-resistant strains were not only cross resistant to all other macrolides, but also to the chemically unrelated lincosamide and streptogramin B drugs and thus exhibited the MLS_Bresistance phenotype. The resistance was conferred by expression of the ErmC RNA methyltransferase that causes N-6 dimethylation of adenine⁹ and acts at position 2058 in 23S rRNA.¹⁰ Since then many more erm methyltransferase genes have been identified and the Erm family of methyltransferases, that mediate mono- or dimethylation of A2058, now consists of approximately 40 different classes of methylases (http://faculty.washington.edu/marilynr/).^11,12 The erm genes are now found all over the world and in many different kinds of bacteria. Other methyltransferases have been identified and there are now eight known classes of RNA methyltransferases that act on 23S rRNA and are related to antibiotic resistance (Table 1).

Table 1

23S Ribosomal RNA methyltransferases associated with antibiotic resistance in bacteria.

The mobilization and spread of RNA methyltransferases causing antibiotic resistance in the microbial community is disturbing. Our knowledge regarding the various methyltransferases is highly variable with respect to their effects on normal growth, expression, detailed mechanisms of action, target interactions and dissemination mechanisms. In this chapter we focus on RNA methyltransferases that act on 23S rRNA in the 50S ribosomal subunit and confer antibiotic resistance by acting on 16S rRNA in the 30S ribosomal subunit are described in chapter by Conn, Savic and Macmaster in this volume.

The Cfr Methyltransferase Targets A2503 at the Peptidyl Transferase Center

The PTC on the large ribosomal subunit is composed mainly of the central loop of domain V of 23S rRNA and contains binding sites for various antibiotics of clinical and veterinary importance (Fig. 1). It is the site where amino acids are added to the nascent peptide chain and therefore an efficient place for drugs to block protein synthesis. For the same reason, the PTC is the target for alterations that prevent drug binding and thereby cause antibiotic resistance. The PTC is located in an RNA-lined cleft where there are no ribosomal proteins directly at the surface.¹³ Although the resistance mechanisms established so far at this site are mostly RNA mutations, a newly discovered methylation at the PTC mediated by the Cfr methyltransferase¹⁴ confers combined resistance to five different classes of antibiotics that bind to the PTC.¹⁵

The cfr gene was originally discovered on a multiresistance plasmid isolated during a surveillance study of florfenicol resistance in Staphylococcus spp. isolates of animal origin¹⁶ and shown to encode a methyltransferase that methylates nucleotide A2503 of 23S rRNA.¹⁴ In E. coli and S. aureus there is a natural m²A methylation at A2503 mediated by methyltransferases encoded by the yfgB (rlmN) and NWMN_1128 genes, respectively.¹⁷ The lack of the natural methylation at A2503 confers a slight increase in susceptibility to tiamulin, hygromycin A, sparsomycin and linezolid¹⁷ but we consider this methylation to be a housekeeping modification rather than a genuine antibiotic resistance determinant. Cfr adds an additional methyl group at A2503 and also, at least in the case of E. coli, prevents the natural 2'-O-ribose methylation at position C2498.¹⁴ The cfr and yfgB genes have 32% sequence identity and encode proteins containing a cysteine-rich motif that is characteristic of the radical SAM enzymes,^17,18 a superfamily whose members catalyze a diverse set of reactions including unusual methylations, isomerizations, sulfur insertions and anaerobic oxidations. A very recent study showed that Cfr adds a methyl group at the C-8 position of A2503 (Fig. 2),¹⁹ unveiling a hitherto unknown naturally occurring RNA modification that probably requires a radical-based reaction mechanism to attack position 8 of adenosine.

Figure 2

The Cfr methyltransferase confers PhLOPSa resistance by methylating A2503 at the C8 position. The panels are described from left to right. A schematic bacterial cell is depicted with a translating ribosome. The structure of the bacterial 50S ribosomal (more...)

Expression of cfr in a S. aureus laboratory strain and an E. coli strain with altered membrane permeability showed that the Cfr methyltransferase confers resistance to five different classes of relevant antimicrobial agents. The phenotype was named PhLOPS_Afor resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins and Streptogramin Aantibiotics.¹⁵ Superposition of the bound structures of chloramphenicol, clindamycin, tiamulin and dalfopristin from X-ray studies clearly shows that the drugs bind at overlapping, but nonidentical sites neighbouring A2503 (Fig. 2).¹⁵ The oxazolidinone binding site has been localized to the same region through crosslinking studies²⁰ and this has been confirmed in recent crystal structures of linezolid bound to the H. marismortui and D. radiodurans 50S subunits.²² The resistance is substantial and is likely the result of steric hindrance from a minor perturbation of the structure at or around A2503 caused by the methylation. It has also been shown recently that Cfr alone confers significant resistance to selected 16-membered ring macrolide drugs such as josamycin and spiramycin, but not to other macrolides such as tylosin.²³

As very few new antimicrobial agents are appearing on the market, the finding that one methyltransferase confers resistance to five different classes of relevant antimicrobial agents is alarming. This mechanism of resistance functions in both Gram-positive and Gram-negative organisms,¹⁵ and the cfr gene has been found on transposons and in different geographical locations, suggesting that it can be disseminated within the microbial community.^24,25 The cfr gene has recently been reported in Staphylococcus spp. isolates from humans, including a linezolid-resistant S. aureus hospital strain from Colombia with a chromosomally-encoded cfr gene.²⁶ In this strain, the ermB (described in the next section) and cfr genes are organized into one operon, where expression of the two methyltransferases confers resistance to essentially all clinically useful antibiotics that target the large ribosomal subunit.^23,26 Furthermore, the cfr gene has also been found on plasmids isolated from linezolid-resistant S. aureus and S. epidermis clinical isolates from the United States.²⁷

RNA Methyltransferases Acting on Nucleotides in the Peptide Exit Tunnel

The nascent peptides synthesized in the PTC progress into a tunnel that runs through the 50S ribosomal subunit (Fig. 1). The upper part of the tunnel adjoins the PTC region and contains antibiotic binding sites, as reviewed by,¹ where the drugs interfere with the passage of the nascent peptide and thereby inhibit protein synthesis. The macrolides and streptogramin B antibiotics bind here and share resistance mechanisms based on mutations and RNA methylations. In particular, 23S rRNA nucleotide A2058 is positioned at the tunnel entrance and plays an important role in antibiotic binding. As mentioned in the introduction, the ErmC methyltransferase dimethylates A2058 of 23S rRNA¹⁰ causing a macrolide-lincosamide-streptogramin B resistance phenotype named MLS_B. Similar erm methylase genes have been identified in a wide range of Gram-positive and Gram-negative bacteria with the transposon-borne erm (B), erm (F) and erm (A) genes, as well as the plasmid-borne erm (C) gene having the broadest host range.¹¹ The high incidence of resistance is probably caused by the extensive use of macrolides for the treatment of bacterial infections in humans and animals and by their nontherapeutic use as growth promoters in livestock. The acquired resistance is not especially detrimental to bacteria and can persist for a long time, which in turn, promotes its spread. Not surprisingly, mutations at A2058 in 23S rRNA can cause the same resistance as the Erm methylations (reviewed in ref. 27).

The erm genes are either inducible or constitutively expressed in a number of different bacteria causing either a full MLS_Bresistance or resistance to a subset of these antibiotics depending on the degree of mono- and dimethylation of A2058. While Erm dimethylases cause high-level resistance to all MLS_Bantibiotics, the Erm monomethylases acting at A2058 confers high-level resistance to lincosamides but low to medium levels of resistance to macrolides and streptogramin B drugs.^29,30

Resistance methyltransferases can act synergistically as first illustrated by the A2058 monomethylase ErmN and the G748 methylase RlmA^II that together confer resistance to tylosin, whereas neither of the enzymes alone confers tylosin resistance.³¹ RlmA^II transfers a single methyl group to the N1 position of G748. This nucleotide is located in the loop of helix 35 in domain II of 23S rRNA and is situated farther down the peptide exit tunnel relative to A2058. The large macrolide tylosin contacts both the G748 and the A2058 nucleotides. The interaction at G748 may enable tylosin to establish contact to monomethylated A2058 by induced fit, whereas methylation at G748 prevents this weak but important interaction (reviewed in ref. 3). The substrate for RlmA^II and its RNA contact points have been defined^32,33 and for several groups of Gram-positive bacteria it appears to be a housekeeping modification. Also, RlmA^II is very similar to RlmA^I, a housekeeping methyltransferase that acts at the nearby nucleotide G745.

An NMR structure of ErmAM³⁴ (class B) and X-ray structures of ErmC'³⁵ (class C) as well as ErmC' complexed with its cofactor S-adenosylmethionine (SAM)³⁶ have been reported (listed in Table 1). The structures of the Erm methyltransferases are organized into two domains and are very similar, despite sequence diversity. Their SAM binding domains resemble that of DNA methyltransferases and other methylases binding adenosine-based cofactors in their catalytic sites. The RNA target recognised by Erm methyltransferases has been studied in detail^37,38 And references herein and show that both structural features and sequences in the immediate vicinity of A2058 are important for methylation. Although no structures of Erm-RNA complexes have been determined, a model of ErmC' with a minimal RNA target has been proposed based on alanine-scanning mutagenesis to identify protein-RNA interactions.³⁹ A similar mutagenesis study of amino acids in the catalytic site revealed that few of the amino acids in the site are essential for function.⁴⁰

The Tsr Methyltransferase Targets Nucleotide A1067 at the GTPase Center

The GTPase Center on the 50S subunit (Fig. 1) is implicated in GTP hydrolysis in connection with the function of translation factors. The thiopeptide antibiotics thiostrepton and micrococcin bind to a highly conserved ribonucleoprotein domain at the GTPase center composed of ribosomal protein L11 and part of domain II of 23S ribosomal RNA (position 1051-1108)⁴¹ and a recent paper has documented the interaction between L11 and thiostrepton.⁴² Resistance in the thiostrepton producer Streptomyces azureus is mediated by the Tsr methyltransferase that modifies the 2'-O-ribose position of nucleotide A1067 and this methylation also confers resistance to micrococcin (Table 1).⁴³ Tsr methylation of the 1051-1108 RNA fragment is inhibited by the binding of thiostrepton and also L11, suggesting that this methylation occurs before L11 binding during ribosome assembly.^44,45 Mutagenesis of the 1051-1108 RNA fragment has shown that nucleotides A1056, U1061, U1066, A1067, G1068, A1069 and A1070 of 23S rRNA are required for Tsr methylation.⁴⁴ An assay based on inhibition of the Tsr methylation has been developed to probe binding of thiopeptide antibiotics and novel compounds to the 1055-1081 RNA fragment.⁴⁵

A forthcoming crystal structure of Tsr confirms that it is a member of the SpoU family of methyltransferases that exist as homodimers.⁴⁶ Each Tsr monomer is composed of two structural domains, including a C-terminal domain common to SpoU methyltransferases and a N-terminal target specificity domain, which are connected by a long flexible linker. Molecular docking experiments suggest that a single RNA is bound by the Tsr homodimer, where the N-terminal domains of both the catalytic monomer bound to the methyl-group donating SAM molecule and the other noncatalytic monomer are necessary for substrate recognition.⁴⁶ These studies further indicate that Tsr interacts with 23S rRNA nucleotides 1055-1081 and that the interactions are primarily electrostatic in nature.

Three Different RNA Methyltransferases That Confer Orthosomycin Resistance

The best-known orthosomycin compounds are evernimicin that was developed for use in humans but never made it to the market and avilamycin that is used as a growth promoter. The orthosomycin antibiotics target a site on the 50S ribosomal subunit that includes protein L16 and 23S rRNA helices 89 and 91 and is close to both the elbow of tRNA positioned in the A-site⁴⁷ (Fig. 1) and the initiation factor 2 binding site.⁴⁸ Mutations in ribosomal protein L16 and in domain V of 23S rRNA (at positions 2469-72, 2479-80, 2535-6) confer resistance to orthosomycins (summarised in ref. 46). The EmtA methyltransferase acting at 23S rRNA position G2470 was found in Enterococcus faecium from animal sources on the basis of its resistance to the growth promoter avilamycin and it also confers resistance to evernimicin (Table 1).⁴⁹ It was shown that purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain⁴⁹ indicating that the entire 50S subunit can act as a substrate for this methyltransferase. Soon after this discovery, four genes in the natural producer of avilamycin, the actinomycetes Streptomyces viridochromogenes Tü57, were found to be resistance determinants.⁵⁰ Two of the genes are similar to an ATP binding cassette transporter system that probably exports avilamycin across the cell membrane, whereas the other two genes, aviRa and aviRb, encode rRNA methyltransferases. The modifications were later identified as a 2'-O-ribose methylation at U2479 conferred by AviRb and a base methylation at G2535 conferred by AviRa (Table 1).⁵¹ Both of these sites are in the same RNA region on the 50S subunit as all the resistance mutations. AviRb confers a higher level of resistance than AviRa, but both are necessary for self-protection in the avilamycin producer and they are thought to act together like the tylosin resistance methyltransferases described above. The crystal structure of the AviRa methyltransferase has been determined by X-ray diffraction at 1.5 Å resolution.⁵² Its overall fold is similar to that of most methyltransferases, but it contains two additional helices as a specific feature. Guided by the target, the enzyme was docked to the cognate ribosomal surface, where it fit well into a deep cleft without contacting any ribosomal proteins. A putative-binding site for the SAM cofactor was derived from homologous structures, but because the transferred methyl group is in a pocket beneath the enzyme surface, the targeted guanine base has to flip out for methylation.⁵²

The TlyA Methyltransferase Targets Nucleotides on Intersubunit Bridge B2A at the Ribosomal Subunit Interface

The ribosomal subunits are held together through a network of intermolecular contacts called intersubunit bridges. The intersubunit cavity is spanned by tRNAs, with anticodons base-paired with mRNA codons bound on the small subunit and 3'-CCA ends carrying amino acids positioned in the peptidyl transferase cavity on the large subunit. Intersubunit bridge B2a is conserved throughout evolution and is necessary for the association of ribosomal subunits and binding of tRNAs.⁵³ It is located at the center of the subunit interface between the ribosomal A and P sites and is composed entirely of rRNA nucleotides from helix 44 of 16S rRNA and helix 69 of 23S rRNA.⁵⁴ The modified nucleotides in helix 69 have recently been shown to have a collective significance in Saccharomyces cerevisiae, in that blocking three to five modifications impairs growth and strongly affects ribosome function.⁵⁵ Intersubunit bridge B2a is targeted by the cyclic peptide antibiotics capreomycin and viomycin that are unique in that they recognize the assembled ribosome by interacting with nucleotides on both subunits.⁵⁶ Capreomycin is an important drug in the treatment of multidrug-resistant Mycobacterium tuberculosis that is resistant to the first-line antibiotics isoniazid and rifampicin. Isolation and characterization of several capreomycin-resistant M. tuberculosis and M. smegmatis mutants revealed that these strains contained mutations that inactivated the tlyA gene.⁵⁷ Viomycin and capreomycin inhibit translation by trapping an intermediate state in ribosomal subunit movement and thereby impede tRNA movement.^56,58

The tlyA gene has recently been shown to encode a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and C1920 in helix 69 of 23S rRNA (Table 1).⁵⁶ The inactivation of the TlyA methyltransferase and the resulting lack of these methylations confers capreomycin resistance in M. tuberculosis isolates.⁵⁶ Thus, TlyA belongs to the group of methyltransferases that function as housekeeping enzymes, where the lack rather than presence of methylation confers an antibiotic resistance phenotype (Table 1). The lack of natural homologs of tlyA in many bacteria is correlated with decreased susceptibility to capreomycin and viomycin relative to mycobacteria. In support of this, expression of plasmid-encoded tlyA in E. coli results in 2'-O-methylation at the above nucleotides and increased susceptibility to capreomycin and viomycin.⁵⁶ Sequence analysis of the capreomycin biosynthetic gene cluster from the capreomycin-producing bacterium Saccharothrix mutabilis subsp. capreolus has identified an additional capreomycin resistance gene called cmnU.⁵⁹ CmnU is a homolog of 16S rRNA methyltransferases that confer resistance to the aminoglycosides kanamycin and apramycin by methylating the N1 position of nucleotide A1408 and is further described in chapter by Conn, Savic and Macmaster in this volume.

Conclusions and Future Prospects

The methylation of specific rRNA nucleotides by methyltransferase enzymes can efficiently prevent the binding of protein synthesis inhibitors to their target sites on the ribosome and thereby lead to antibiotic resistance. It should be noted that the methylation of specific rRNA nucleotides can also confer antibiotic sensitivity, as observed in cases where the lack of methylation by housekeeping methyltransferases confers antibiotic resistance. Our understanding of the detailed architecture of antibiotic binding sites has increased rapidly in recent years due to the elucidation of high-resolution structures of antibiotic-ribosome complexes. However, knowledge on the interaction between these sites and the corresponding methyltransferases is still very limited. The structures of several enzymes have been published and it can be expected, for at least some of the enzymes, that the details of their interactions with target RNA will be revealed in the coming years. The fact that the target sites of some of the enzymes are not accessible in mature ribosomes implies that the methylation reactions occur before or during ribosome assembly. Almost all methyltransferases described to date that are associated with changes in antibiotic susceptibility are specific for a single methylation site. The exception to this is the dual specificity exhibited by the TlyA methyltransferase described above that methylates two nucleotides on intersubunit bridge B2a, including one 16S and one 23S rRNA nucleotide. Combined resistance to chemically unrelated classes of drugs is observed in cases where methylations are present at overlapping drug binding sites, as with those mediated by the Cfr and Erm methyltransferases.

The RNA methyltransferase genes providing drug resistance have probably spread from organisms that produce antibiotics and therefore needed them to protect themselves. These methyltransferase genes are often associated with extrachromosomal elements that are acquired from other bacteria in the environment.⁶¹ Thus, the localization of methyltransferase genes on mobile DNA elements such as plasmids, transposons and integrons is important for estimating their dissemination potential via horizontal gene transfer. Methyltransferase genes can also be clustered into resistance units controlled by a common promoter as in the mlr operon on the chromosome of methicillin-resistant S. aureus strain CM05 that contains the cfr and erm (B) genes.²³ The extensive human use of some antibiotics has probably increased the selection of such events. In general, a deeper understanding of the mechanisms through which resistance determinants including methyltransferase genes are transferred in both agricultural settings and the human community is important in preventing the spread of antibiotic resistance.⁶¹

Several approaches have been used in attempting to circumvent antibiotic resistance mediated by methyltransferases. One approach is to design new antibiotics that are not affected by known methylations at the target site. For example, drug companies have tried to circumvent the Erm-mediated resistance by developing semisynthetic macrolide antibiotics, such as the ketolide telithromycin, that make additional interactions with the ribosome⁶² and thereby possess improved resistance properties.⁶³ Other approaches have been aimed at inhibiting methyltransferases with small molecules and peptides. In one study a group of triazine-containing compounds were shown to inhibit Erm-mediated methylation in the low micromolar range.⁶⁴ However, the structures of enzyme-inhibitor complexes revealed that these inhibitors bind in the SAM binding site. As this site is highly conserved, it is likely that these inhibitors would be nonselective and target multiple methyltransferases. A recent investigation used the crystal structure of the ErmC methyltransferase for structure-based virtual screening to find lead compounds that bind to the unique RNA binding site.⁶⁵

A number of new methyltransferases that confer antibiotic resistance have been described in recent years and it is likely that additional modifying enzymes will be found in the future. It is becoming increasingly apparent that soil bacteria are a reservoir of resistance determinants that can be mobilized and spread to other microorganisms, including pathogenic bacteria.⁶⁶ A recent study described a high frequency of antibiotic resistance in a diverse collection of soil bacteria, where resistance was observed to every major antibiotic class, regardless of antibiotic origin.⁶⁷ In support of this, a recent paper has shown that a phylogenetically diverse set of bacteria isolated from soil, where some are closely related to human pathogens, can subsist on antibiotics as a sole carbon source.⁶⁸ Investigation of this antibiotic resistome could identify new methyltransferase genes and also foreshadow clinically relevant antibiotic resistance mechanisms of the future.

Acknowledgements

We thank Jacob Poehlsgaard for figures and Graeme L. Conn for sharing results prior to publication.

References

1.

Poehlsgaard J, Douthwaite S. The bacterial ribosome as a target for antibiotics. Nat Rev Microbiol. 2005;3(11):870–881. [PubMed: 16261170]

2.

Long KS, Vester B. Antibiotic resistance mechanisms, with an emphasis on those related to the ribosome. Chapter 2.5.7. In: EcoSal-Escherichia Coli and Salmonella: Cellular and Molecular Biology. Washington, DC:ASM Press. [PubMed: 26443725]

3.

Douthwaite S, Fourmy D, Yoshizawa S. Nucleotide methylations in rRNA that confer resistance to ribosome-targeting antibiotics. In: Topics in Current Genetics, Fine-Tuning of RNA Functions by Modification and Editing. Berlin, Heidelberg: Springer-Verlag. 2005;12:285–307.

4.

Chow CS, Lamichhane TH, Mahto SK. Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications. ACS Chemical Biology. 2007;2(9):610–619. [PMC free article: PMC2535799] [PubMed: 17894445]

5.

Lazaro E, Rodriguez-Fonseca C, Porse B. et al. A sparsomycin-resistant mutant of Halobacterium salinarium lacks a modification at nucleotide U2603 in the peptidyl transferase centre of 23 S rRNA [published erratum appears in J Mol Biol 1996; 264(4):839] J Mol Biol. 1996;261(2):231–238. [PubMed: 8757290]

6.

Hansen MA, Kirpekar F, Ritterbusch W. et al. Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria. RNA. 2002;8(2):202–213. [PMC free article: PMC1370243] [PubMed: 11911366]

7.

Toh SM, Mankin AS. An indigenous posttranscriptional modification in the ribosomal peptidyl transferase center confers resistance to an array of protein synthesis inhibitors. J Mol Biol. 2008;380(4):593–597. [PMC free article: PMC5367387] [PubMed: 18554609]

8.

Leclercq R, Courvalin P. Bacterial resistance to macrolide, lincosamide and streptogramin antibiotics by target modification. Antimicrob Agents Chemother. 1991;35(7):1267–1272. [PMC free article: PMC245156] [PubMed: 1929280]

9.

Lai CJ, Weisblum B. Altered methylation of ribosomal RNA in an erythromycin-resistant strain of Staphylococcus aureus. Proc Natl Acad Sci USA. 1971;68(4):856–860. [PMC free article: PMC389059] [PubMed: 5279527]

10.

Skinner R, Cundliffe E, Schmidt FJ. Site of action of a ribosomal RNA methylase responsible for resistance to erythromycin and other antibiotics J-Biol-Chem 1983258(20):12702–12706. issn: 10021-19258. [PubMed: 6195156]

11.

Roberts MC, Sutcliffe J, Courvalin P. et al. Nomenclature for macrolide and macrolide-lincosamide- streptogramin B resistance determinants. Antimicrob Agents Chemother. 1999;43(12):2823–2830. [PMC free article: PMC89572] [PubMed: 10582867]

12.

Roberts MC. Update on macrolide-lincosamide-streptogramin, ketolide and oxazolidinone resistance genes. FEMS Microbiol Lett. 2008;282(2):147–159. [PubMed: 18399991]

13.

Nissen P, Hansen J, Ban N. et al. The structural basis of ribosome activity in peptide bond synthesis. Science. 2000;289(5481):920–930. [PubMed: 10937990]

14.

Kehrenberg C, Schwarz S, Jacobsen L. et al. A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503. Mol Microbiol. 2005;57(4):1064–1073. [PubMed: 16091044]

15.

Long KS, Poehlsgaard J, Kehrenberg C. et al. The Cfr rRNA methyltransferase confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A antibiotics. Antimicrob Agents Chemother. 2006;50(7):2500–2505. [PMC free article: PMC1489768] [PubMed: 16801432]

16.

Schwarz S, Werckenthin C, Kehrenberg C. Identification of a plasmid-borne chloramphenicol-florfenicol resistance gene in Staphylococcus sciuri. Antimicrob Agents Chemother. 2000;44(9):2530–2533. [PMC free article: PMC90098] [PubMed: 10952608]

17.

Toh SM, Xiong L, Bae T. et al. The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA. RNA. 2008;14(1):98–106. [PMC free article: PMC2151032] [PubMed: 18025251]

18.

Sofia HJ, Chen G, Hetzler BG. et al. Radical SAM a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Res. 2001;29(5):1097–1106. [PMC free article: PMC29726] [PubMed: 11222759]

19.

Giessing AMB, Jensen SS, Rasmussen A. et al. Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistence in bacteria. RNA. 2009;15(2):327–336. [PMC free article: PMC2648713] [PubMed: 19144912]

20.

Leach KL, Swaney SM, Colca JR. et al. The Site of Action of oxazolidinone Antibiotics in Living bacteria and in Human Mitochondria. Molecular Cell. 2007;26:393–402. [PubMed: 17499045]

21.

Ippolito JA, Kanyo ZF, Wang D. et al. Crystal structure of the oxazolidinone antibiotic linezolid Bound to the 50S Ribosomal Subunit. J Med Chem. 2008;51:3353–3356. [PubMed: 18494460]

22.

Wilson DN, Schluenzen F, Harms JM. et al. The oxazolidinone antibiotics perturb the ribosomal peptidyl transferase center and effect tRNA positioning. Proc Natl Acad Sci USA. 2008;105(36):13339–13344. [PMC free article: PMC2533191] [PubMed: 18757750]

23.

Smith LK, Mankin AS. Transcriptional and translational control of the mlr operon, which confers resistance to seven classes of protein synthesis inhibitors. Antimicrob Agents Chemother. 2008;52(5):1703–1712. [PMC free article: PMC2346656] [PubMed: 18299405]

24.

Kehrenberg C, Schwarz S. Distribution of florfenicol resistance genes fexA and cfr among chloramphenicol-resistant Staphylococcus isolates. Antimicrob Agents Chemother. 2006;50(4):1156–1163. [PMC free article: PMC1426988] [PubMed: 16569824]

25.

Kehrenberg C, Aarestrup FM, Schwarz S. IS21-558 insertion sequences are involved in the mobility of the multiresistance gene cfr. Antimicrob Agents Chemother. 2007;51(2):483–487. [PMC free article: PMC1797725] [PubMed: 17145796]

26.

Toh SM, Xiong L, Arias CA. et al. Acquisition of a natural resistance gene renders a clinical strain of methicilinresistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid. Molecular Microbiology. 2007;64(6):1506–1514. [PMC free article: PMC2711439] [PubMed: 17555436]

27.

Mendes RE, Deshpande LM, Castanheira M. et al. First report of cfr-mediated resistance to linezolid in human staphylococcal clinical isolates recovered in the United States. Antimicrob Agents Chemother. 2008;52(6):2244–2246. [PMC free article: PMC2415768] [PubMed: 18391032]

28.

Vester B, Douthwaite S. Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob Agents Chemother. 2001;45(1):1–12. [PMC free article: PMC90232] [PubMed: 11120937]

29.

Weisblum B. Erythromycin resistance by ribosome modification Antimicrob agents chemother 199539(3):577–585. issn: 0066-4804. [PMC free article: PMC162587] [PubMed: 7793855]

30.

Pernodet JL, Fish S, Blondelet Rouault MH. et al. The macrolide-lincosamide-streptogramin B resistance phenotypes characterized by using a specifically deleted, antibiotic-sensitive strain of Streptomyces lividans Antimicrob-Agents-Chemother 199640(3):581–585. issn: 0066-4804. [PMC free article: PMC163161] [PubMed: 8851574]

31.

Liu M, Douthwaite S. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy. Proc Natl Acad Sci USA. 2002;99(23):14658–14663. [PMC free article: PMC137475] [PubMed: 12417742]

32.

Douthwaite S, Jakobsen L, Yoshizawa S. et al. Interaction of the tylosin-resistance methyltransferase RlmA II at its rRNA target differs from the orthologue RlmA I. J Mol Biol. 2008;378(5):969–975. [PubMed: 18406425]

33.

Lebars I, Husson C, Yoshizawa S. et al. Recognition elements in rRNA for the tylosin resistance methyltransferase RlmA(II). J Mol Biol. 2007;372(2):525–534. [PubMed: 17673230]

34.

Yu L, Petros AM, Schnuchel A. et al. Solution structure of an rRNA methyltransferase (ErmAM) that confers macrolide-lincosamide-streptogramin antibiotic resistance. Nat Struct Biol. 1997;4(6):483–489. [PubMed: 9187657]

35.

Bussiere DE, Muchmore SW, Dealwis CG. et al. Crystal structure of ErmC', an rRNA methyltransferase which mediates antibiotic resistance in bacteria. Biochemistry. 1998;37(20):7103–7112. [PubMed: 9585521]

36.

Schluckebier G, Zhong P, Stewart KD. et al. The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism. J Mol Biol. 1999;289(2):277–291. [PubMed: 10366505]

37.

Nielsen AK, Douthwaite S, Vester B. Negative in vitro selection identifies the rRNA recognition motif for ErmE methyltransferase. RNA. 1999;5(8):1034–1041. [PMC free article: PMC1369827] [PubMed: 10445878]

38.

Villsen ID, Vester B, Douthwaite S. ErmE methyltransferase recognizes features of the primary and secondary structure in a motif within domain V of 23 S rRNA. J Mol Biol. 1999;286(2):365–374. [PubMed: 9973557]

39.

Maravic G, Bujnicki JM, Feder M. et al. Alanine-scanning mutagenesis of the predicted rRNA-binding domain of ErmC' redefines the substrate-binding site and suggests a model for protein-RNA interactions. Nucleic Acids Res. 2003;31(16):4941–4949. [PMC free article: PMC169915] [PubMed: 12907737]

40.

Maravic G, Feder M, Pongor S. et al. Mutational analysis defines the roles of conserved amino acid residues in the predicted catalytic pocket of the rRNA:m6A methyltransferase ErmC'. J Mol Biol. 2003;332(1):99–109. [PubMed: 12946350]

41.

Wimberly BT, Guymon R, McCutcheon JP. et al. A detailed view of a ribosomal active site: the structure of the L11-RNA complex. Cell. 1999;97(4):491–502. [PubMed: 10338213]

42.

Lee D, Walsh JD, Yu P. et al. The structure of free L11 and functional dynamics of L11 in free, L11-rRNA(58 nt) binary and L11-rRNA(58 nt)-thiostrepton ternary complexes. J Mol Biol. 2007;367(4):1007–1022. [PMC free article: PMC2045704] [PubMed: 17292917]

43.

Thompson J, Schmidt F, Cundliffe E. Site of action of a ribosomal RNA methylase conferring resistance to thiostrepton. J Biol Chem. 1982;257(14):7915–7917. [PubMed: 6806287]

44.

Bechthold A, Floss HG. Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 22-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA. Eur J Biochem. 1994;224(2):431–437. [PubMed: 7925357]

45.

Lentzen G, Klinck R, Matassova N. et al. Structural basis for contrasting activities of ribosome binding thiazole antibiotics. Chem Biol. 2003;10(8):769–778. [PubMed: 12954336]

46.

Dunstan MS, Hang PC, Honek JF. Structure of the thiostrepton-resistance methyltransferase and its interaction with cofactor and ribosomal RNA (Submitted). [PMC free article: PMC2719339] [PubMed: 19369248]

47.

Kofoed CB, Vester B. Interaction of avilamycin with ribosomes and resistance caused by mutations in 23S rRNA. Antimicrob Agents Chemother. 2002;46(11):3339–3342. [PMC free article: PMC128742] [PubMed: 12384333]

48.

Marzi S, Knight W, Brandi L. et al. Ribosomal localization of translation initiation factor IF2. RNA. 2003;9(8):958–969. [PMC free article: PMC1370462] [PubMed: 12869707]

49.

Mann PA, Xiong L, Mankin AS. et al. EmtA a rRNA methyltransferase conferring high-level evernimicin resistance. Mol Microbiol. 2001;41(6):1349–1356. [PubMed: 11580839]

50.

Weitnauer G, Gaisser S, Trefzer A. et al. An ATP-binding cassette transporter and two rRNA methyltransferases are involved in resistance to avilamycin in the producer organism Streptomyces viridochromogenes Tu57. Antimicrob Agents Chemother. 2001;45(3):690–695. [PMC free article: PMC90357] [PubMed: 11181344]

51.

Treede I, Jakobsen L, Kirpekar F. et al. The avilamycin resistance determinants AviRa and AviRb methylate 23S rRNA at the guanosine 2535 base and the uridine 2479 ribose. Mol Microbiol. 2003;49(2):309–318. [PubMed: 12828631]

52.

Mosbacher TG, Bechthold A, Schulz GE. Crystal structure of the avilamycin resistance-conferring methyltransferase AviRa from Streptomyces viridochromogenes. J Mol Biol. 2003;329(1):147–157. [PubMed: 12742024]

53.

Ali IK, Lancaster L, Feinberg J. et al. Deletion of a conserved, central ribosomal intersubunit RNA bridge. Mol Cell. 2006;23(6):865–874. [PubMed: 16973438]

54.

Yusupov MM, Yusupova GZ, Baucom A. et al. Crystal structure of the ribosome at 5.5 A resolution. Science. 2001;292(5518):883–896. [PubMed: 11283358]

55.

Liang XH, Liu Q, Fournier MJ. rRNA modifications in an intersubunit bridge of the ribosome strongly affect both ribosome biogenesis and activity. Mol Cell. 2007;28(6):965–977. [PubMed: 18158895]

56.

Johansen SK, Maus CE, Plikaytis BB. et al. Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2'-O-methylations in 16S and 23S rRNAs. Mol Cell. 2006;23(2):173–182. [PubMed: 16857584]

57.

Maus CE, Plikaytis BB, Shinnick TM. Mutation of tlyA confers capreomycin resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2005;49(2):571–577. [PMC free article: PMC547314] [PubMed: 15673735]

58.

Ermolenko DN, Spiegel PC, Majumdar ZK. et al. The antibiotic viomycin traps the ribosome in an intermediate state of translocation. Nat Struct Mol Biol. 2007;14(6):493–497. [PubMed: 17515906]

59.

Felnagle EA, Rondon MR, Berti AD. et al. Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin. Appl Environ Microbiol. 2007;73(13):4162–4170. [PMC free article: PMC1932801] [PubMed: 17496129]

60.

Alekshun MN, Levy SB. Molecular mechanisms of antibacterial multidrug resistance. Cell. 2007;128(6):1937–1050. [PubMed: 17382878]

61.

Salyers A, Shoemaker NB. Reservoirs of antibiotic resistance genes. Anim Biotechnol. 2006;17(2):137–146. [PubMed: 17127525]

62.

Tu D, Blaha G, Moore PB. et al. Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance. Cell. 2005;121(2):257–270. [PubMed: 15851032]

63.

Farrell DJ, Morrissey I, Bakker S. et al. In vitro activities of telithromycin, linezolid and quinupristin- dalfopristin against Streptococcus pneumoniae with macrolide resistance due to ribosomal mutations. Antimicrob Agents Chemother. 2004;48(8):3169–3171. [PMC free article: PMC478535] [PubMed: 15273142]

64.

Hajduk PJ, Dinges J, Schkeryantz JM. et al. Novel inhibitors of Erm methyltransferases from NMR and parallel synthesis. J Med Chem. 1999;42(19):3852–3859. [PubMed: 10508434]

65.

Feder M, Purta E, Koscinski L. et al. Virtual screening and experimental verification to identify potential inhibitors of the ErmC methyltransferase responsible for bacterial resistance against macrolide antibiotics. Chem Med Chem. 2008;3(2):316–322. [PubMed: 18038381]

66.

Wright GD. The antibiotic resistome: the nexus of chemical and genetic diversity. Nat Rev Microbiol. 2007;5(3):175–186. [PubMed: 17277795]

67.

D'Costa VM, McGrann KM, Hughes DW. et al. Sampling the antibiotic resistome. Science. 2006;311(5759):374–377. [PubMed: 16424339]

68.

Dantas G, Sommer MO, Oluwasegun RD. et al. Bacteria subsisting on antibiotics. Science. 2008;320(5872):100–103. [PubMed: 18388292]

69.

Mosbacher TG, Bechthold A, Schulz GE. Structure and function of the antibiotic resistance-mediating methyltransferase AviRb from Streptomyces viridochromogenes. J Mol Biol. 2005;345(3):535–545. [PubMed: 15581897]

70.

Schuwirth BS, Borovinskaya MA, Hau CW. et al. Structures of the bacterial ribosome at 3.5 A resolution. Science. 2005;310(5749):827–834. [PubMed: 16272117]

71.

Selmer M, Dunham CM, Murphy FVt. et al. Structure of the 70S ribosome complexed with mRNA and tRNA. Science. 2006;313(5795):1935–1942. [PubMed: 16959973]

72.

Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics J Mol Graph 199614(1):33–38., 27-38. [PubMed: 8744570]

73.

Harms JM, Schlunzen F, Fucini P. et al. Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin. BMC Biol. 2004;2(1):4. [PMC free article: PMC400760] [PubMed: 15059283]

74.

Schlunzen F, Pyetan E, Fucini P. et al. Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin. Mol Microbiol. 2004;54(5):1287–1294. [PubMed: 15554968]

75.

Schlunzen F, Zarivach R, Harms J. et al. Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature. 2001;413(6858):814–821. [PubMed: 11677599]