The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase.

The ErmE methyltransferase confers resistance to MLS antibiotics by specifically dimethylating adenine 2058 (A2058, Escherichia coli numbering) in bacterial 23S rRNA. To define nucleotides in the rRNA that are part of the motif recognized by ErmE, we investigated both in vivo and in vitro the effects of mutations around position A2058 on methylation. Mutagenizing A2058 (to G or U) completely abolishes methylation of 23S rRNA by ErmE. No methylation occurred at other sites in the rRNA, demonstrating the fidelity of ErmE for A2058. Breaking the neighboring G2057-C2611 Watson-Crick base pair by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058. Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformation of this region in 23S rRNA was probed with chemical reagents and it was shown that the A2057 and U2611 mutations alone and in combination alter the reactivity of A2058 and adjacent bases. However, mutagenizing position G-->A2032 in an adjacent loop, which has been implicated to interact with A2058, alters neither the ErmE methylation at A2058 nor the accessibility of this region to the chemical reagents. The data indicate that a less-exposed conformation at A2058 leads to reduction in methylation by ErmE. Nucleotide G2057 and its interaction with C2611 maintain the conformation at A2058, and are thus important in forming the structural motif that is recognized by the ErmE methyltransferase.     

Core sequence in the RNA motif recognized by the ErmE methyltransferase revealed by relaxing the fidelity of the enzyme for its target.

Under physiological conditions, the ErmE methyltransferase specifically modifies a single adenosine within ribosomal RNA (rRNA), and thereby confers resistance to multiple antibiotics. The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated efficiently, and with equally high fidelity, in rRNAs from phylogenetically diverse bacteria. However, the fidelity of ErmE is reduced when magnesium is removed, and over twenty new sites of ErmE methylation appear in E. coli 16S and 23S rRNAs. These sites show widely different degrees of reactivity to ErmE. The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA. This suggests that methylation at the new sites results from changes in the RNA substrate rather than the methyltransferase. Chemical probing confirms that the rRNA structure opens upon magnesium depletion, exposing potential new interaction sites to the enzyme. The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclusively at adenosines (underlined); these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are slight preferences for the nucleotides shown in lower case). This consensus is believed to represent the core of the motif that Erm methyltransferases recognize at their canonical A2058 site. The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recognition.     

Substrate requirements for ErmC' methyltransferase activity.

ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.     

A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin-resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.     

Site of action of a ribosomal RNA methylase responsible for resistance to erythromycin and other antibiotics

The enzyme which confers resistance to erythromycin in the producing organism Streptomyces erythraeus dimethylates a single adenine residue in Bacillus stearothermophilus 23 S rRNA. This corresponds to residue Ade 2058 in Escherichia coli 23 S RNA. The methylase responsible for resistance to macrolides, lincomycin, and streptogramin B-related antibiotics in Staphylococcus aureus also acts at this site.     

Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition

Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m⁶ ₂A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity–binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.     

Functional interactions within 23S rRNA involving the peptidyltransferase center.

A molecular genetic approach has been employed to investigate functional interactions within 23S rRNA. Each of the three base substitutions at guanine 2032 has been made. The 2032A mutation confers resistance to the antibiotics chloramphenicol and clindamycin, which interact with the 23S rRNA peptidyltransferase loop. All three base substitutions at position 2032 produce an erythromycin-hypersensitive phenotype. The 2032 substitutions were compared with and combined with a 12-bp deletion mutation in domain II and point mutations at positions 2057 and 2058 in the peptidyltransferase region of domain V that also confer antibiotic resistance. Both the domain II deletion and the 2057A mutation relieve the hypersensitive effect of the 2032A mutation, producing an erythromycin-resistant phenotype; in addition, the combination of the 2032A and 2057A mutations confers a higher level of chloramphenicol resistance than either mutation alone. 23S rRNAs containing mutations at position 2058 that confer clindamycin and erythromycin resistance become deleterious to cell growth when combined with the 2032A mutation and, additionally, confer hypersensitivity to erythromycin and sensitivity to clindamycin and chloramphenicol. Introduction of the domain II deletion into these double-mutation constructs gives rise to erythromycin resistance. The results are interpreted as indicating that position 2032 interacts with the peptidyltransferase loop and that there is a functional connection between domains II and V.     

Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA.

We have used chemical modification to examine the conformation of 23 S rRNA in Escherichia coli ribosomes bearing erythromycin resistance mutations in ribosomal proteins L22 and L4. Changes in reactivity to chemical probes were observed at several nucleotide positions scattered throughout 23 S rRNA. The L4 mutation affects the reactivity of G799 and U1255 in domain II and that of A2572 in domain V. The L22 mutation influences modification in domain II at positions m5U747, G748, and A1268, as well as at A1614 in domain III and G2351 in domain V. The reactivity of A789 is weakly enhanced by both the L22 and L4 mutations. None of these nucleotide positions has previously been associated with macrolide antibiotic resistance. Interestingly, neither of the ribosomal protein mutations produces any detectable effects at or within the vicinity of A2058 in domain V, the site most frequently shown to confer macrolide resistance when altered by methylation or mutation. Thus, while L22 and L4 bind primarily to domain I of 23 S rRNA, erythromycin resistance mutations in these ribosomal proteins perturb the conformation of residues in domains II, III and V and affect the action of antibiotics known to interact with nucleotide residues in the peptidyl transferase center of domain V. These results support the hypothesis that ribosomal proteins interact with rRNA at multiple sites to establish its functionally active three-dimensional structure, and suggest that these antibiotic resistance mutations act by perturbing the conformation of rRNA.     

Characteristics of a 50S ribosomal subunit precursor particle as a substrate for ermE methyltransferase activity and erythromycin binding in Staphylococcus aureus.

Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. A model for 50S ribosomal subunit formation suggests that the precursor particle which accumulates in erythromycin treated cells is the target for methyl transferase activity. Hybridization experiments identified the presence of 23S rRNA in the 50S precursor particle. The protein content of the 50S precursor particle was analyzed by MALDI-TOF mass spectrophotometry. These studies have identified 23 of 36 50S ribosomal proteins in the precursor. Methyltransferase assays demonstrated that the 50S precursor particle was a substrate for ermE methyltransferase. Competition experiments indicated that the enzyme could displace erythromycin from the 50S precursor particle and that the methyltransferase had a higher association constant for the precursor particle compared to that of erythromycin. Inhibition experiments showed that macrolide, lincosamide and streptogramin B compounds bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. These studies shed light on the interaction of ermE methyltransferase and erythromycin in this clinically important pathogen.     

Resistance to macrolides and lincosamides in Streptomyces lividans and to aminoglycosides in Micromonospora purpurea.

Ribosomal (r) resistance to gentamicin in clones containing DNA from the producing organism Micromonospora purpurea is determined by grmA, and not by kgmA as originally reported. The kgmA gene originated in Streptomyces tenebrarius and is identical to kgmB. Both grmA and kgm encode enzymes that methylate single specific sites within 16S rRNA, although the site of action of the grmA product has not yet been determined. In either case, the methylated nucleoside is 7-methyl G. Inducible resistance to lincomycin (Ln) and macrolides in Streptomyces lividans TK21 results from expression of two genes: lrm, encoding an rRNA methyltransferase and mgt, encoding a glycosyl transferase (MGT), that specifically inactivates macrolides. The lrm product monomethylates residue A2058 within 23S rRNA (Escherichia coli numbering scheme) and confers high-level resistance to Ln with much lower levels of resistance to macrolides. Substrates for MGT, which utilises UDP-glucose as cofactor, include macrolides with 12-, 14-, 15- or 16-atom cyclic polyketide lactones (as in methymycin, erythromycin, azithromycin or tylosin, respectively) although spiramycin and carbomycin are not apparently modified. The enzyme is specific for the 2'-OH group of saccharide moieties attached to C5 of the 16-atom lactone ring (corresponding to C5 or C3 in 14- or 12-atom lactones, respectively). The lrm and mgt genes have been cloned and sequenced. The deduced lrm product is a 26-kDa protein, similar to other rRNA methyltransferases, such as the carB, tlrA and ermE products, whereas the mgt product (deduced to be 42 kDa) resembles a glycosyl transferase from barley.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resistance to macrolides, lincosamides and streptogramin type B antibiotics due to a mutation in an rRNA operon of Streptomyces ambofaciens.

Streptomyces ambofaciens produces spiramycin, a macrolide antibiotic and expresses an inducible resistance to macrolides, lincosamides and streptogramin B antibiotics (MLS). From a mutant of S.ambofaciens exhibiting a constitutive MLS resistance phenotype a resistance determinant was cloned on a low copy number vector (pIJ61) through its expression in Streptomyces lividans. Further characterization has shown that this determinant corresponded to a mutant rRNA operon with a mutation in the 23S rRNA gene. In different organisms, mutations leading to MLS resistance have been located at a position corresponding to the adenine 2058 of Escherichia coli 23S rRNA. In the 23S rRNA from S.ambofaciens a similar position for the mutation has been postulated and DNA sequencing of this region has shown an adenine to guanine transition at a position corresponding to 2058. S.ambofaciens possesses four rRNA operons which we have cloned. In Streptomyces, contrary to other bacteria, a mutation in one among several rRNA operons confers a selectable MLS resistance phenotype. Possible reasons for this difference are discussed.     

Mutation from guanine to adenine in 25S rRNA at the position equivalent to E. coli A2058 does not confer erythromycin sensitivity in Sacchromyces cerevisae

The macrolide erythromycin binds to the large subunit of the prokaryotic ribosome near the peptidyltransferase center (PTC) and inhibits elongation of new peptide chains beyond a few amino acids. Nucleotides A2058 and A2059 (E. coli numbering) in 23S rRNA play a crucial role in the binding of erythromycin, and mutation of nucleotide A2058 confers erythromycin resistance in both gram-positive and gram-negative bacteria. There are high levels of sequence and structural similarity in the PTC of prokaryotic and eukaryotic ribosomes. However, eukaryotic ribosomes are resistant to erythromycin and the presence of a G at the position equivalent to E. coli nucleotide A2058 is believed to be the reason. To test this hypothesis, we introduced a G to A mutation at this position of the yeast Saccharomyces cerevisiae 25S rRNA and analyzed sensitivity toward erythromycin. Neither growth studies nor erythromycin binding assays on mutated yeast ribosomes indicated any erythromycin sensitivity in mutated yeast strains. These results suggest that the identity of nucleotide 2058 is not the only determinant responsible for the difference in erythromycin sensitivity between yeast and prokaryotes.     

ermSF, a ribosomal RNA adenine N6-methyltransferase gene from Streptomyces fradiae, confers MLS (macrolide-lincosamide-streptogramin B) resistance to E. coli when it is expressed.

The Erm family of methyltransferases confers the MLS antibiotic resistance to pathogenic microorganism through the mono- or dimethylation of a single adenine residue in 23S rRNA, which is known as the target site for modification. One of the erm genes, ermSF was cloned from Streptomyces fradiae NRRL 2702 by PCR and overexpressed in E. coli BL21(DE3) as both a soluble protein and insoluble aggregate (inclusion body) using the T7 promoter driven expression vector, pET23b. Even though most of the overexpressed protein existed as an inclusion body, E. coli cells showed resistance to erythromycin. The lowering of incubation temperature from 37 degrees C to 22 degrees C facilitated the purification of the protein by increasing the fraction of soluble protein. The soluble protein was purified using immobilized metal ion (Ni2+) affinity chromatography in a one-step manner to the apparent homogeneity. The 23S rRNA of E. coli was found to be a good substrate for the purified ErmSF.     

Expression of the macrolide-lincosamide-streptogramin-B-resistance methylase gene, ermE, from Streptomyces erythraeus in Escherichia coli results in N6-monomethylation and N6,N6-dimethylation of ribosomal RNA

The ermE gene was cloned from Streptomyces erythraeus into Escherichia coli on a series of plasmids. When transcribed from the lac promoter, ermE conferred high-level resistance to erythromycin and other macrolide-lincosamide-streptogramin-B (MLS) antibiotics. A methylase activity capable of N6-mono- and N6,N6-dimethylation of adenine residues in E. coli rRNA was detected in extracts of MLS-resistant cells. In addition, rRNA extracted from MLS-resistant E. coli contained N6-mono- and N6,N6-dimethylated adenine residues.     

A 50S ribosomal subunit precursor particle is a substrate for the ErmC methyltransferase in Staphylococcus aureus cells

Macrolide antibiotics like erythromycin can induce the synthesis of a specific 23S rRNA methyltransferase which confers resistance to cells containing the erm gene. Erythromycin inhibits both protein synthesis and the formation of 50S subunits in bacterial cells. We have tested the idea that the 50S precursor particle that accumulates in antibiotic-treated Staphylococcus aureus cells is a substrate for the methyltransferase enzyme. Pulse-chase labeling studies were conducted to examine the rates of ribosomal subunit formation in control and erythromycin-induced cells. Erythromycin binding to 50S subunits was examined under the same conditions. The rate of 50S subunit formation was reduced for up to 30 min after antibiotic addition, and erythromycin binding was substantial at this time. A nuclease protection assay was used to examine the methylation of adenine 2085 in 23S rRNA after induction. A methyl-labeled protected RNA sequence was found to appear in cells 30 min after induction. This protected sequence was found in both 50S subunits and in a subunit precursor particle sedimenting at about 30S in sucrose gradients. 23S rRNA isolated from 50S subunits of cells could be labeled by a ribosome-associated methlytransferase activity, with (3)H-S-adenosylmethionine as a substrate. 50S subunits were not a substrate for the enzyme, but the 30S gradient region from erythromycin-treated cells contained a substrate for this activity. These findings are consistent with a model that suggests that antibiotic inhibition of 50S formation leads to the accumulation of a precursor whose 23S rRNA becomes methylated by the induced enzyme. The methylated rRNA will preclude erythromycin binding; thus, assembly of the particle and translation become insensitive to the inhibitory effects of the drug.     

The macrolide-ketolide antibiotic binding site is formed by structures in domains II and V of 23S ribosomal RNA

The macrolide antibiotic erythromycin interacts with bacterial 23S ribosomal RNA (rRNA) making contacts that are limited to hairpin 35 in domain II of the rRNA and to the peptidyl transferase loop in domain V. These two regions are probably folded close together in the 23S rRNA tertiary structure and form a binding pocket for macrolides and other drug types. Erythromycin has been derivatized by replacing the L-cladinose moiety at position 3 by a keto group (forming the ketolide antibiotics) and by an alkyl-aryl extension at positions 11/12 of the lactone ring. All the drugs footprint identically within the peptidyl transferase loop, giving protection against chemical modification at A2058, A2059 and G2505, and enhancing the accessibility of A2062. However, the ketolide derivatives bind to ribosomes with widely varying affinities compared with erythromycin. This variation correlates with differences in the hairpin 35 footprints. Erythromycin enhances the modification at position A752. Removal of cladinose lowers drug binding 70-fold, with concomitant loss of the A752 footprint. However, the 11/12 extension strengthens binding 10-fold, and position A752 becomes protected. These findings indicate how drug derivatization can improve the inhibition of bacteria that have macrolide resistance conferred by changes in the peptidyl transferase loop.     

A 50S Ribosomal Subunit Precursor Particle Is a Substrate for the ErmC Methyltransferase in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Cells

Macrolide antibiotics like erythromycin can induce the synthesis of a specific 23S rRNA methyltransferase which confers resistance to cells containing the erm gene. Erythromycin inhibits both protein synthesis and the formation of 50S subunits in bacterial cells. We have tested the idea that the 50S precursor particle that accumulates in antibiotic-treated Staphylococcus aureus cells is a substrate for the methyltransferase enzyme. Pulse-chase labeling studies were conducted to examine the rates of ribosomal subunit formation in control and erythromycin-induced cells. Erythromycin binding to 50S subunits was examined under the same conditions. The rate of 50S subunit formation was reduced for up to 30 min after antibiotic addition, and erythromycin binding was substantial at this time. A nuclease protection assay was used to examine the methylation of adenine 2085 in 23S rRNA after induction. A methyl-labeled protected RNA sequence was found to appear in cells 30 min after induction. This protected sequence was found in both 50S subunits and in a subunit precursor particle sedimenting at about 30S in sucrose gradients. 23S rRNA isolated from 50S subunits of cells could be labeled by a ribosome-associated methlytransferase activity, with ³H-S-adenosylmethionine as a substrate. 50S subunits were not a substrate for the enzyme, but the 30S gradient region from erythromycin-treated cells contained a substrate for this activity. These findings are consistent with a model that suggests that antibiotic inhibition of 50S formation leads to the accumulation of a precursor whose 23S rRNA becomes methylated by the induced enzyme. The methylated rRNA will preclude erythromycin binding; thus, assembly of the particle and translation become insensitive to the inhibitory effects of the drug. Received: 21 June 2002 / Accepted: 21 August 2002     

Purification and biochemical characterization of the ErmSF macrolide-lincosamide-streptogramin B resistance factor protein expressed as a hexahistidine-tagged protein in Escherichia coli

The erm proteins confer resistance to the MLS (macrolide-lincosamide-streptogramin B) antibiotics in various microorganisms, including pathogens, through dimethylation of a single adenine residue (A2085: Bacillus subtilis coordinate) of the 23S rRNA to reduce the affinity of antibiotics, thereby enabling the cells to escape from the antibiotics' action, and this mechanism is predominantly adopted by microorganisms resistant to MLS antibiotics. ErmSF methyltransferase is one of the four gene products synthesized by Streptomyces fradiae NRRL 2338 to be resistant to its autogenous antibiotic, tylosin. In order to have a convenient source for the purification of milligram amounts, we expressed ErmSF in Escherichia coli using a T7 promoter-driven expression vector system, pET 23b, and the protein was expressed with a carboxy-terminal addition of six histidine residues in order to facilitate purification. Expression at 22 degrees C reduced the formation of insoluble aggregate, inclusion body, and resulted in accumulation of soluble hexahistidine-ErmSF up to 30% of total cell protein after 18 h. Metal-chelation chromatography yielded 126 mg of hexahistidine-ErmSF per liter of culture with a purity slightly greater than 95%. To examine the function of ErmSF in vivo and in vitro, its activity in E. coli (antibiotic susceptibility assay) andin vitro methyltransferase activity using in vitro-produced B. subtilis domain V, 434-, 257-, and 243-nt RNAs were investigated. The ErmSF in E. coli conferred resistance to erythromycin, whereas E. coli harboring an empty vector, pET23b, was susceptible. The purified recombinant protein successfully methylated domain V of 23S rRNA, which is known to contain all of the substrate elements recognized and to be methylated by erm proteins. However, the truncated substrates were methylated with decreased efficiencies. Almost all of domain V was monomethylated with less than 0.2 pM S-[methyl-(3)H]adenosylmethionine concentration. The roles of three structurally divided regions of domain V in recognition and methylation by ErmSF are proposed through kinetic studies using RNA substrates, in which each region is deleted, under the monomethylation condition.