Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis*

Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N6-monomethyltransferases or N6,N6-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLSB) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLSB and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin.

Tuberculosis is caused by infection with bacteria belonging to the Mycobacterium tuberculosis complex (MTC) 4 and is responsible for 2 million deaths per annum worldwide with almost one-third of the world's population harboring asymptomatic infections (1). Treatment for tuberculosis often requires extended courses with several antibiotics, and with resistant strains becoming more prevalent (2), drug therapy is not always successful. In the absence of new antimycobacterial drugs, a better understanding of the resistance mechanisms to existing drugs is desirable.
Members of the MTC include Mycobacterium africanum, Mycobacterium microti, Mycobacterium bovis, and M. tuberculosis, all of which are intrinsically resistant to macrolide antibiotics (3)(4)(5). Although this is due in part to the imperviousness of the mycobacterial cell wall (6), the recently discovered resistance determinant, erm (37) (formerly ermMT), also contributes to the lack of macrolide susceptibility in MTC species (7). The erm (37) determinant is a truncated homologue of the erm genes found in a diverse range of pathogenic and drug-producing bacteria (8). Members of the erm family of genes all encode methyltransferases that specifically methylate the N 6 position of nucleotide A2058 in 23 S rRNA (Escherichia coli numbering) but differ as to whether they monomethylate or dimethylate this nucleotide (9,10). Erm monomethyltransferases are found predominantly in drug-producing actinomycetes species and confer the MLS B type I phenotype with high resistance to lincosamides, low to moderate resistance to macrolide and streptogramin B antibiotics (10,11), but no resistance to the latest generation of macrolides, the ketolides (12). Erm dimethyltransferases confer the MLS B type II phenotype with high resistance to all macrolide, lincosamide, and streptogramin B antibiotics (8,10) including ketolides (12). Type II MLS B resistance with dimethylation of the rRNA is the more common mechanism in bacterial pathogens.
The erm (37) gene is, however, atypical as it confers resistance that falls between the type I and type II categories. Expression of the M. tuberculosis erm (37) gene in the non-tuberculous mycobacterium, Mycobacterium smegmatis, confers a pattern similar to type I resistance (7), whereas authentic MTC hosts are resistant to the ketolide antibiotic telithromycin (3,4) which is more indicative of type II resistance. In this study, we first established that erm (37) is indeed responsible for telithromycin resistance in MTC strains. This was achieved using an attenuated MTC strain, BCG-Pasteur, utilized in production of the bacillus Calmette-Guérin vaccine; the BCG-Pasteur strain has lost the RD2 chromosome region containing erm (37) (13) and is susceptible to telithromycin (4). Complementation of BCG-Pasteur with recombinant erm(37) restores telithromycin resistance to the level observed in virulent MTC strains and in a BCG strain that still has an intact RD2 region (BCG-Moreau). Introduction of the dimethyltransferase gene erm(E) conferred BCG-Pasteur with even higher telithromycin resistance, whereas the monomethyltransferase erm(O) conferred no telithromycin resistance. Paradoxically (but consistent with a previous report (7)), expression of recombinant erm (37) in M. smegmatis conferred the same phenotype as erm(O), with no significant resistance to telithromycin. A molecular explanation is needed to understand the disparate erm (37) phenotypes in the different mycobacterial strains.
We employed matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) tandem mass spectrometry to define precisely the locations and number of methyl groups added by Erm (37) to the rRNAs in its authentic MTC host as well as to the rRNA of recombinant M. smegmatis strains. In its MTC host, Erm(37) initially monomethylates nucleotide A2058 but then proceeds to modify one or both of the neighboring nucleotides to add up to three methyl groups (at A2057, A2058, and A2059, Fig. 1). This unique configuration of methyl groups explains the resistance exhibited by the MTC strains, which is distinct from both the type I and type II MLS B patterns. In M. smegmatis, Erm(37) monomethylates A2058 but adds extra methyl groups only sparingly at A2057 or A2059 and produces an overall methylation pattern that is consistent with the type I MLS B phenotype.

EXPERIMENTAL PROCEDURES
Bacterial Strains, Plasmids, and Growth Conditions-Bacterial strains and plasmids used in this study are listed in TABLE ONE. Mycobacteria were grown at 37°C in Middlebrook 7H9 (Difco) liquid broth containing 0.2% (v/v) glycerol and on solid Middlebrook 7H10 medium (Difco) containing 0.5% (v/v) glycerol. Kanamycin (Sigma) was added at 15 g/ml to maintain the plasmids in the mycobacterial strains. Erythromycin (Sigma) was added at 20 g/ml to cultures of the BCG-Moreau strain to stimulate expression of erm (37) prior to rRNA analyses. Minimal inhibitory concentrations of the ketolide antibiotic telithromycin (Sanofi-Aventis) for MTC strains were determined at pH 7.3 using the BACTEC radiometric method (BD Biosciences) as reported previously (14).
Preparation of Ribosomes and RNA Purification-M. bovis BCG and M. smegmatis strains (TABLE ONE) were grown to late log phase and harvested by centrifugation. Cells were washed twice by being suspended in 100 ml of buffer A (10 mM Tris-Cl, pH 7.2, 4 mM MgCl 2 , 10 mM NH 4 Cl, 100 mM KCl) and pelleted by centrifugation before being resuspended in 10 ml of buffer A and lysed using a French press (M. bovis) or by sonication (M. smegmatis). Cell debris was removed by centrifugation at 30,000 ϫ g for 20 min at 4°C; ribosomes were pelleted by centrifugation at 100,000 ϫ g for 2 h at 4°C and were redissolved in 200 l of cold buffer A. Ribosomal proteins were removed by phenol/chloroform extraction, and the rRNA was redissolved in 30 l of H 2 O.
MALDI Mass Spectrometry Analysis of rRNAs-The 23 S rRNA region around A2058 in M. bovis and M. smegmatis was isolated using a hybridization method. Briefly, 33 pmol of rRNA was hybridized to 330 pmol of a 53-mer oligodeoxynucleotide complementary to the nucleotide sequence G2035-G2087 (Fig. 1), which is identical in the MTC and M. smegmatis 23 S rRNAs. Unprotected RNA sequences were digested away with nucleases (15). The RNA-DNA hybrid was phenol-extracted and recovered by ethanol precipitation. The protected rRNA fragment was released on a denaturing gel, excised, and extracted (15).
The rRNA fragment was digested with RNase A or RNase T1 (Sigma) to produce oligonucleotides of a size suitable for MALDI mass spectrometry analysis. Samples of rRNA (2.5 pmol in 1 l) were mixed with 0.5 l of 3-hydroxypicolinic acid (0.5 M in 50% acetonitrile), 2.0 l of RNase (0.5 g/l), and 1.5 l of H 2 O and digested for 2.5 h at 37°C. Digested fragments were purified through Poros 50R3 columns (Applied Biosystems). Short oligonucleotides were eluted with 10 mM triethylammonium acetate/6% acetonitrile (pH 7.0), larger ones with 10 mM triethylammonium acetate/25% acetonitrile. The oligonucleotides were analyzed using a Voyager STR MALDI mass spectrometer (Applied Biosystems) in reflector time-of-flight mode detecting positive ions (15)(16)(17). Spectra were smoothed using the Proteometrics Inc. "m/z" software program. Tandem mass spectrometry was carried out on a MicroMass MALDI quadrupole-TOF Ultima mass spectrometer in positive ion mode. The window for parent ion selection was set to 2 m/z units, and collision energy was 70 eV. Spectra were smoothed using the MassLynx software supplied by the manufacturer.
Primer Extension-A 5Ј-32 P-labeled deoxynucleotide primer (5Ј-GTAGTGAAGG-TCCCGGGG) complementary to the C2063-C2080 region of Mycobacterium 23 S rRNA was extended with 1 unit of reverse transcriptase (Life Sciences) using a mixture of 1 mM dTTP, dCTP, 5 mM ddGTP, and 1.5 pmol rRNA as template. Extension products were run on denaturing gels (12). (37) plays an important role in resistance to MLS B antibiotics (7) as well as to the ketolide antibiotic telithromycin (TABLE TWO). M. tuberculosis, M. africanum, M. microti, M. bovis, and the BCG-Moreau strain all possess an active chromosomal copy of erm (37) and exhibit relatively high resistance to telithromycin. The BCG-Pasteur strain differs essentially only in its lack of the erm(37) determinant and its susceptibility to telithromycin. Complementation of BCG-Pasteur with a plasmid-encoded copy of erm (37) restores the telithromycin-resistant phenotype. Expression of the monomethyltransferase erm(O) confers no telithromycin resistance in BCG-Pasteur, whereas high resistance is conferred by the dimethyltransferase erm(E) (TABLE TWO).

Resistance and erm(37)-Microbiological studies on members of the M. tuberculosis complex (TABLE ONE) indicate that erm
A2058 Methylation in MTC-The exact function of Erm(37) was determined by comparing the rRNA modification patterns in the BCG- Pasteur and -Moreau strains. The masses of rRNA oligonucleotides containing nucleotide A2058 were measured by MALDI mass spectrometry. Digestion of rRNA with the nucleotide-specific RNases A and T1 yields fragments of predictable and suitable sizes, although many fragments have similar or identical masses and often produce a spectrum that is difficult to interpret. Here, we obtained an unambiguous rRNA analysis by preselecting the mycobacterial 23 S sequence from G2035 to G2087 (Fig. 1). Digestion of this sequence gives rise to oligonucleotides containing A2058 (an RNase A octamer of m/z 2659.4 or an RNase T1 pentamer of m/z 1680.3) that form unique and clearly defined peaks in the mass spectra. erm (37) Expression-When the BCG-Pasteur and -Moreau strains were grown without erythromycin, the RNase T1 fragment AAAAGp formed a single peak at 1680.3 m/z (Fig. 2), showing that there had been no methylation at A2058. Growth of the BCG-Moreau strain with erythromycin results in loss of the m/z 1680.3 peak and the appearance of new peaks at m/z 1694.3, 1708.3, and 1722.3 (Fig. 2), corresponding to the addition of one, two, and three methyl groups. This methylation pattern was verified by RNase A digestion of the rRNAs, producing a fragment containing A2058 (GAAAAGACp) that runs at m/z 2659.4 and increases to m/z 2673.4, 2687.4, and 2701.4 when cells are incubated with erythromycin (data not shown). Methylation of the rRNA and the ability to grow in erythromycin were dependent on erm(37) expression. The BCG-Pasteur strain did not grow in the presence of erythromycin at 20 g/ml.
rRNA Target of Erm(37)-The sites of Erm(37) methylation in the rRNA were pinpointed by analyzing the 1694.3, 1708.3, and 1722.3 m/z fragments using MALDI quadrupole-TOF tandem mass spectrometry. In this approach, the individual RNA ions are selected and then fragmented to reveal the mass of each nucleotide (Fig. 3A). The 14-Da   increase in the m/z of the 1694.3 ion was shown unambiguously to reside in nucleotide A2058 (Fig. 3B) and corresponds to the addition of a single methyl group here. The 1708.3 m/z ion with two methyl groups was analyzed in a similar manner (Fig. 3C) and was a mixture of products, which collectively were methylated at A2057, A2058, and A2059. This pattern is adequately explained by the presence of two products, one being methylated at A2057 and A2058 and the second being methylated at A2058 and A2059. In theory, a product with single methyl groups at A2057 and A2059 is also possible but is extremely unlikely as no intermediate methylated only at A2057 or A2059 was observed in the 1694.3 m/z ion. Another formal possibility, that one of the adenosines was dimethylated, was ruled out in parallel experiments using reverse transcriptase primer extension (Fig. 4). The largest AAAAGp ion at 1722.3 m/z was unambiguously identified by tandem mass spectrometry to be a unique product with one methyl group on each of the three adenosines at 2057, 2058, and 2059 (Fig. 3D).
erm (37) Expression in M. smegmatis-The erm (37) gene from M. tuberculosis was inserted after a constitutive promoter in a plasmid and expressed in M. smegmatis (TABLE ONE). Similar constructions were made with erm(O) and erm(E), which encode well characterized A2058 mono-and dimethyltransferases, respectively. M. smegmatis possesses its own chromosome-encoded methyltransferase gene erm (38), expression of which requires induction by erythromycin or similar compounds (18 -20). The erm (38) gene was held silent under the growth conditions used here, and this is evident from the lack of A2058 methylation in the control strain containing the empty plasmid (Fig. 5B). The rRNA from the M. smegmatis strain expressing erm(O) acquired a single methyl group (Fig. 5C), and further analysis by tandem mass spectrometry (not shown) demonstrated unambiguously that A2058 was stoichiometrically monomethylated and that no methylation occurred at either of the neighboring nucleotides. The rRNA analyses from the strain expressing erm(E) were also unequivocal and revealed specific and stoichiometric dimethylation at A2058 (Fig. 5D).
Expression of erm (37) in M. smegmatis gave rise to more complicated MALDI spectra (Fig. 5E) with signals corresponding to a small quantity of unmethylated rRNA (m/z 2659.4), a major product with one methyl group (m/z 2673.4), and a minor product with two methyl groups (m/z 2687.4). The monomethylated rRNA fragment was shown by tandem mass spectrometry to be methylated solely on A2058. Analysis of the peak with two methyl groups at m/z 2687.4 showed it to be identical to FIGURE 3. Tandem mass spectrometry analysis of the A2058 region. A, fragmentation of the RNA backbone results in a, b, c, and d ions from the 5Ј-end, and w, x, y, and z ions from the 3Ј-end (40). In the instrumentation setup used here, the y ions predominate (41), and only these are indicated on the AAAAGp sequence. The location of the methyl groups can be seen from the shift in the y ions peaks. Loss of an unmodified adenosine nucleotide corresponds to a difference of 329 Da, whereas a singly methylated adenosine is 343 Da. B, in the monomethylated peak, the mass differences between the parent ion (1694.3 m/z), the y 4 ion (1365.3 m/z), the y 3 ion (1022.2 m/z), and the y 2 ion (693.1 m/z) clearly show that the methyl group is located on the second adenosine (A2058). C, the 1708.3 m/z peak is most likely composed of two ion species (see text), one with single methyl groups at A2057 and A2058 (open arrowheads) and the other with methyl groups at A2058 and A2059 (filled arrowheads). D, the 1722.3 m/z ion was unambiguously assigned and has one methyl group on each of the nucleotides A2057, A2058, and A2059. The sites of methylation were additionally verified by other ions present in lower abundance; one such series of ions from m/z 542.1 to 1571.3 (y ions missing the guanine base) is indicated.  (37). Primer extension is stopped either by N 6 ,N 6 -dimethylation at nucleotide A2058 or by incorporation of ddGTP at nucleotide C2055. N 6 -monomethylation at adenosines does not stop reverse transcription. The 23 S rRNA template for the dideoxy sequencing reactions (CUAG) was from the BCG-Pasteur strain. NOVEMBER 25, 2005 • VOLUME 280 • NUMBER 47 the corresponding peak from BCG-Moreau cells containing a mixture of rRNA with single methyl groups on A2057 and A2058 and rRNA with single methyl groups on A2058 and A2059. No ion at m/z 2687.4 (potentially with three methyl groups) could be detected following erm (37) expression in M. smegmatis. All data were corroborated by mass spectrometry analysis following RNase T1 digestion (not shown). Reverse transcriptase analyses of the rRNAs (Fig. 4) confirmed the lack of any N 6 ,N 6 -dimethylation by Erm (37) or Erm(O) and showed that Erm(E) catalyzed N 6 ,N 6 -dimethylation exclusively at A2058 in M. smegmatis 23S rRNA.

DISCUSSION
The Erm(37) methyltransferase is highly conserved in the MTC members M. africanum, M. microti, M. bovis, and M. tuberculosis and is 100% identical in the latter two species (21)(22)(23), in keeping with their extremely close phylogeny (24). Two strains of MTC were used to deduce the function of erm (37). The two M. bovis BCG vaccine strains, Pasteur and Moreau, originate from an isogenic source but have diverged over the last few decades such that erm (37) has been lost in the Pasteur strain but retained in the Moreau strain. The Moreau strain, like the other MTC strains, is resistant to MLS B antibiotics (7) and to the ketolide antibiotic telithromycin (TABLE TWO). The Pasteur strain shows no such resistance; however, the MTC resistance phenotype is reestablished in the Pasteur strain when it is complemented with a plasmid-encoded copy of erm (37). The MLS B ketolide resistance conferred by Erm (37) is unusual, as it falls between the type I and type II patterns (4,7). We show here that this phenotype is linked with the unique manner in which Erm(37) methylates the mycobacterial 23 S rRNA.
Expression of the chromosome-coded erm (37) gene in MTC strains requires pre-incubation with an antibiotic such as erythromycin. Expression of erm (37) results in methylation of the 23 S rRNA, and this is seen as a series of shifts in the AAAAGp ion signal, corresponding to the addition of one, two, and three methyl groups (Fig. 2). The singly methylated AAAAGp ion was fragmented and analyzed by tandem mass spectrometry revealing that the methyl group was attached exclusively to A2058. Thus, the first reaction carried out by Erm(37) is to monomethylate specifically at A2058.
The methyltransferase then adds a second methyl group at A2057 or A2059 (the m/z 1708.3 ion, Fig. 3C) before proceeding to add a third methyl group (m/z 1722.3 ion) such that the rRNA becomes equipped with a single methyl group on each of the three nucleotides, A2057, A2058, and A2059. Despite its extended span of activity, Erm(37) acts as a monomethyltransferase at each of the nucleotides it modifies.
In many bacteria that possess an erm gene, expression requires prior induction with low concentrations of erythromycin or a similar compound. Induction mechanisms are found in pathogenic and drug-producing bacteria (25) as well as in the saprophyte M. smegmatis (20) and generally function via translational attenuation at a leader sequence upstream of the erm cistron. The MTC erm (37) gene has no such leader sequence. Instead, erm(37) expression appears to be transcriptionally controlled as part of a multidrug resistance regulon (26). The regulon is activated by the protein WhiB7, which is expressed in response to several compounds including erythromycin. The intensity of the response increases with the concentration of these compounds (26).
The concentration of erythromycin used here to promote erm(37) expression results in a relatively minor proportion of trimethylated rRNA (Fig. 2). Our resistance data indicate that telithromycin also elicits WhiB7/Erm(37) expression. The higher concentrations of telithromycin used in the minimal inhibitory concentration experiments would be expected to stimulate Erm(37) production and consequently increase the proportion of trimethylated rRNA beyond that seen here with erythromycin. Multiple methylations are required at or adjacent to A2058 to confer telithromycin resistance; thus the relatively high level of resistance observed here (TABLE TWO) is consistent with a substantial proportion of the rRNA being trimethylated.
The trimethylation pattern created by Erm(37) on mycobacterial rRNA has not been seen in any other Erm/host systems studied previously. Also unusual is the 2057-2611-bp closing helix 73 (Fig. 1), which is A-U in mycobacterial 23 S rRNA as opposed to G-C in the majority of bacteria, including other Gram-positive bacteria that harbor an erm gene (27). It could thus be envisaged that the structure of mycobacterial rRNA might reduce the stringency of Erm methyltransferases for their target and might be responsible for the atypical methylation pattern. We ruled out this idea after expressing erm(O) and erm(E) from plasmids in the BCG-Pasteur strain, where the genes produced phenotypes fully consistent with erm mono-and dimethyltransferases, respectively. Furthermore, mass spectrometry analyses of the methylation patterns produced by Erm(O) and Erm(E) in M. smegmatis rRNA confirmed that both enzymes completely retain their fidelity and mono-and dimethylate exclusively at nucleotide A2058 (Fig. 5). The current evidence indicates that the lax stringency of the M. tuberculosis Erm(37) methyltransferase is an intrinsic property of its own structure.
This lax stringency is also evident, albeit to a lesser extent, when erm (37) is expressed in M. smegmatis. In M. smegmatis, the major Erm(37) product is monomethylated A2058 (Fig. 5) with minor amounts of methylation at A2057 or A2059; however, no product with three methyl groups was detected. The methylation pattern produced by Erm (37) in M. smegmatis thus fits well with the MLS B type I phenotype that has been reported (7).
Some general inferences about the interaction of Erm methyltransferases with their rRNA substrate can be drawn. NMR studies on Erm(B) (formerly ErmAM) (28) and crystals of Erm(C)Ј (29,30) show identical structural folds organized into distinct N-and C-terminal domains. The larger N-terminal domain contains the catalytic site adjacent to the binding site for the S-adenosylmethionine cofactor (30). Mutagenesis studies on Erm(B) (31) and Erm(C)Ј (32) fit well with the structures and indicate that the main substrate recognition motifs are within the positively charged cleft between the two domains (33). The Erm(37), Erm(E), and Erm(O) sequences can be modeled into the Erm(B)/Erm(C)Ј structures and show the same distribution of surface charges; any significant differences lie within the C-terminal domain. As Erm(37) seems to possess all of the structures necessary for cofactor binding, catalytic activity, and RNA recognition, the portions missing from the truncated C-terminal domain of Erm (37) probably function to stabilize methyltransferases on the rRNA substrate.
The configuration of methyl groups added by Erm (37) is unique and confers its MTC hosts with an atypical MLS B phenotype including resistance to the ketolide telithromycin. Although the semi-synthetic ketolide drugs are too recent an addition to the antimicrobial scene to be linked with selection for the unusual function of Erm(37), the ketolide binding site (34,35) overlaps that of several classes of naturally occurring compounds including the MLS B drugs (35)(36)(37)(38). It is thus highly feasible that the atypical methylation pattern of Erm(37) also provides a selective advantage against some of the naturally occurring antimicrobial compounds.