Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Savic, M. Articles by Conn, G. L. Search for Related Content PubMed PubMed Citation Articles by Savic, M. Articles by Conn, G. L.

 Previous Article  |  Next Article 

Journal of Bacteriology, September 2008, p. 5855-5861, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00076-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Critical Residues for Cofactor Binding and Catalytic Activity in the Aminoglycoside Resistance Methyltransferase Sgm ^,

Miloje Savic,¹ Tatjana Ilic-Tomic,² Rachel Macmaster,¹ Branka Vasiljevic,² and Graeme L. Conn^1*

Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom,¹ Institute of Molecular Genetics and Genetic Engineering (IMGGE), P.O. Box 23, 11010 Belgrade, Serbia²

Received 15 January 2008/ Accepted 20 June 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K_D (binding constant) of 17.6 µM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antibiotic resistance by rRNA methylation is a frequently used mechanism among macrolide and aminoglycoside antibiotic-producing actinomycete strains (8). Members of the FmrO protein family, named after fortimicin A, the resistance methyltransferase (MT) from Micromonospora olivasterospora (25), methylate 16S rRNA and thus protect bacteria against the action of aminoglycoside antibiotics. Like the majority of MT enzymes, they are dependent upon the methyl donor S-adenosyl-L-methionine (SAM) as a cofactor.

Two 16S rRNA modifications are most commonly employed by aminoglycoside-producing actinomycetes, the guanine-N7 methylation of G1405 (m⁷N G1405) and the adenine-N1 methylation of A1408 (m¹N A1408) (4, 8). To date, m¹N A1408 MTs were observed only among aminoglycoside producers, except for one instance of plasmid-mediated A1408 NpmA MT found in a clinically isolated Escherichia coli isolate that confers pan-aminoglycoside resistance among pathogenic bacteria (33). In contrast, a significantly increased spread of m⁷N G1405 MTs among various gram-negative pathogens from both clinical and veterinary isolates has been observed recently (9).

Around 130 members of the SAM-dependent MT family have been classified (EC 2.1.1.X) based on their substrate specificity (small molecule, lipid, protein, or nucleic acid) and on the atom targeted for methylation. Despite sharing almost no sequence similarity with other enzymes, actinomycete MTs are remote relatives of the RNA MT subfamily of SAM-dependent MTs. However, the limited overall sequence similarity and "domain swapping" make inference about the structure of the FmrO family very difficult compared to other SAM-dependent MT families. Structural studies indicate that the SAM-dependent MTs represent a large structurally conserved superfamily, where the profound structural conservation is not reflected in a corresponding sequence conservation. As an alternative to using the extent of sequence conservation at analogous positions, MTs can be grouped based on the relative linear order of functional regions. Broadly speaking, SAM-dependent MTs comprise the following three functional domains: SAM binding, substrate recognition and binding, and catalysis of methyl transfer. The MTs differ in the relative linear order of these regions as defined by the presence of a conserved sequence (11). However, identification of putative key residues forming these conserved motifs may provide a platform to begin structure-function analyses of the actinomycete MT enzymes.

A sisomicin-gentamicin aminoglycoside resistance MT gene, the sgm gene from the actinomycete "Micromonospora zionensis," prevents self-intoxication by a mechanism that involves methylation of the intact 30S ribosomal subunit (20). Although the precise site of methylation has not been directly determined, it has been inferred to be the same site as that of another family member, KgmB, which methylates G1405 in 16S rRNA (18; T. Ilic-Tomic, I. Moric, G. L. Conn, and B. Vasiljevic, unpublished data). To date, no further biochemical characterization or structure-function analysis of Sgm or any other FmrO family member (Fig. 1A) has been conducted. Here, we have used amino acid conservation within resistance MTs across 13 aminoglycoside-producing actinomycete strains to direct a mutagenesis strategy to characterize the SAM cofactor binding and catalytic properties of Sgm using both in vivo and in vitro assays of enzyme function.

View larger version (40K): [in this window] [in a new window]

FIG. 1. FmrO MT family sequence analysis, amino acid conservation, and homology modeling. (A) Phylogenetic tree of MTs from aminoglycoside-producing actinomycete strains. Numbers at the nodes indicate the levels of bootstrap support based on neighbor-joining analyses of 1,000 resampled data sets (27). The scale bar represents 0.1 amino acid substitutions per position. (B) LOGOS representation (6, 29) of the FmrO MT family sequence alignment used to construct the phylogenetic tree in panel A. Asterisks denote the conserved, putative key residues for cofactor binding (red), the catalysis of methyl group transfer (blue), and target site recognition (green). The position of one additional mutation (N179A) made as a control in the analysis of SAM binding is marked with an arrow. (C) ConSurf model of the Sgm protein with the clusters of key residues identified in panel B highlighted. The color code bar above the model indicates amino acid conservation from variable residues (light blue) to highly conserved residues (purple). (D) Cartoon representation of the Sgm homology model with amino acids targeted for site-directed mutagenesis within the proposed SAM binding pocket (red) and putative catalytic residues (blue) shown in stick representation. Additional amino acid changes (discussed in the text) are the mutation at a variable site near the SAM pocket (N179A; magenta) and two spontaneous mutations identified during selection (cyan). The figure was prepared using PyMOL (http://www.pymol.org).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Targeted site-directed mutagenesis of Sgm. Conserved amino acids were identified by alignment of 13 MT sequences from aminoglycoside-producing actinomycete strains using ClustalW (32) and further analysis using MUSCLE (10). Conserved residues were plotted on an Sgm homology model (Fig. 1 and see supplemental material) using the ConSurf algorithm (21) and amino acids (Table 1) selected for mutation to alanine by QuikChange II site-directed mutagenesis (Stratagene). The presence of each target mutation and the absence of further mutations or indels in either the coding sequence or promoter were confirmed by automated DNA sequencing. All mutant Sgm proteins were expressed in E. coli isolates in a soluble form at sufficient levels to allow extraction and purification for in vitro analysis.

View this table: [in this window] [in a new window]

TABLE 1. Growth of E. coli transformants containing wild-type and mutant Sgm-encoding plasmids in liquid culture with gentamicin and in vitro SAM binding affinities

Determination of aminoglycoside MICs. Gentamicin and kanamycin MICs were measured in triplicate in liquid culture for E. coli strains DH5 and BL21(DE3) harboring plasmid-encoded wild-type and mutant Sgm proteins. Initial cultures were grown from a single colony in LB medium supplemented with 100 µg/ml ampicillin to an optical density at 600 nm (OD₆₀₀) of 0.7 to 0.8. A total of 100 µl of this culture was used to inoculate 10 ml of LB medium with 100 µg/ml of ampicillin and various concentrations of gentamicin or kanamycin in the range of 0 to 1,500 µg/ml. These cultures were grown at 37°C for a further 16 h, at which time a final OD₆₀₀ measurement was taken and the MIC was determined as the minimal concentration of gentamicin or kanamycin that prevents bacterial growth. Bacteria lacking a plasmid or those transformed with an empty vector were used as negative controls. Measurements were made with and without 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) induction of protein expression with no effect on the MIC observed.

Sgm protein expression and purification. N-terminal His₆-tagged wild-type and mutant Sgm proteins were expressed in E. coli BL21(DE3) as previously described (19). Protein expression was induced with 1 mM IPTG at an OD₆₀₀ of 0.6 to 0.8, and growth continued for 3 to 4 h at 37°C. Cell pellets were resuspended and incubated for 1 h in lysis buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM MgCl₂, 10% glycerol, and 10 mM imidazole) containing 1 mg/ml lysozyme, protease inhibitor cocktail (Sigma), and 0.1 mM phenylmethylsulfonyl fluoride before being fully broken by sonication. Sgm proteins were purified by metal ion affinity chromatography under native conditions (Ni²⁺-nitrilotriacetic acid agarose; Qiagen) in a batch, followed by cation-exchange and gel filtration chromatography using HiPrep 16/10 SP FF and HiPrep 26/60 Sephacryl S-100 columns (GE Healthcare) on an ÄKTApurifier system. Cation-exchange chromatography was performed in 20 mM HEPES, pH 7.5, 10% glycerol buffer, eluting Sgm by using a linear gradient of 1 M NaCl in the same buffer. Gel filtration chromatography was performed using 20 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM MgCl₂, 10% glycerol, 5 mM 2-mercaptoethanol. Purified proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualization with Coomassie blue staining. The identity of wild-type Sgm was confirmed by mass spectrometry following in-gel trypsin digestion of the excised sodium dodecyl sulfate-polyacrylamide gel electrophoresis band.

CD spectroscopy. Proteins (6 to 15 µM) were dialyzed into a solution containing 20 mM sodium phosphate buffer, pH 7.5, and 150 mM NaF. Circular dichroism (CD) spectra were recorded on a Jasco J-810 spectropolarimeter equipped with a Peltier element temperature controller, using a 0.01-cm light path quartz cell (Hellma). Each protein CD spectrum represents the average of 10 accumulations recorded between 190- and 260-nm wavelengths, with 0.2-nm resolution, 0.5-nm bandwidth, 4-s response time, 100-millidegree sensitivity, and a scan speed of 10 nm/min. Sgm unfolding experiments were collected over the temperature range of 5 to 85°C in increments of 5°C. Background reference spectra were recorded before and after sample spectrum recording.

The background-corrected spectrum of the wild-type Sgm at 20°C was analyzed using the K2D (16) and SELCON3 (30) algorithms via Dichroweb (http://www.cryst.bbk.ac.uk/cdweb/html/home.html). For a comparison to the experimental estimates of Sgm secondary structure content, the predicted secondary structure was also calculated from the Sgm protein sequence using the method of Garnier et al. (15) and Jpred (7) and from the Sgm homology model with the program STRIDE (13).

In vitro MT activity assays. Methylation reaction mixtures contained 10 mM HEPES-KOH, pH 7.5, 10 mM MgCl₂, 50 mM NH₄Cl, 5 mM 2-mercaptoethanol, 1 mCi/ml [³H]SAM (15 Ci/mM; Amersham-Pharmacia), 500 units/ml RNasin (Ambion), 20 pmol 30S ribosomal subunits, and 5 µg of Sgm proteins (or the equivalent volume of cell extract from cells with an empty vector as the negative control). Incubation was carried out at 35°C for 40 min with aliquots removed at 0, 10, 20, 30, and 40 min. The reaction was stopped by adding ice-cold 5% trichloroacetic acid. The samples were allowed to precipitate at 0°C for 10 min and were collected on GF/C filters (Whatman). The filters were suspended in Ultima Gold F scintillation fluid (Perkin Elmer), and the incorporated radioactivity was measured using a scintillation counter (1219 RackBeta liquid scintillation counter; LKB Wallac). Each assay was performed at least twice using independent preparations of protein.

ITC. The thermodynamics of Sgm-SAM interactions were measured using a VP-ITC microcalorimeter (MicroCal) with Sgm (20 to 70 µM) in the sample cell and SAM (1 to 2 mM) in the injection syringe. Wild-type, G135A, D156A, N179A, and D182A Sgm proteins were dialyzed exhaustively into solution containing 20 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM MgCl₂, and 10% glycerol, and SAM was prepared using the final buffer from dialysis. K199A, E205A, R236A, and E267A Sgm proteins were found to precipitate during dialysis and were therefore purified by gel filtration chromatography in the isothermal titration calorimetry (ITC) buffer, concentrated, and used directly in titration experiments. In these cases, SAM solutions were prepared using the final buffer flowthrough from the concentration device. Titration experiments were performed at 20°C and involved a single 2-µl injection, followed by 25 to 60 4-µl injections of SAM with 360-s spacing. Titration curves were fit by a nonlinear least-squares method in Origin software (MicroCal) using a model for one or two binding sites. A model with one binding site was found to give the optimal fit and was used to extract the K_D (binding constant) and N (stoichiometry) of binding for all proteins except K199A, E205A, and E267A Sgm, where reasonable fits could only be obtained with fixed stoichiometry (N = 1).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Identification of putative key residues for cofactor binding and methyl group transfer. Conserved residues in Sgm were identified based on a sequence alignment of all complete annotated open reading frames of FmrO family members and mapped onto an Sgm homology model using ConSurf (21) (Fig. 1). Within the C-terminal domain, a conserved cluster of residues was identified that might form a pocket for binding SAM cofactor. Putative key residues for methyl group transfer (and/or target site recognition) were also identified in the C-terminal domain. Finally, two further clusters of conserved basic/charged residues were identified within the N-terminal domain that we hypothesize are important for target site recognition and binding (Fig. 1B and C). To test these functional predictions, we chose to focus on residues identified as potentially being involved in cofactor binding or methyl group transfer (see Discussion for comments on the likely complexity of target recognition). Each of these residues (Table 1) was mutated to alanine by site-directed mutagenesis, and Sgm mutants were initially selected by streaking colonies in parallel on LB agar containing either ampicillin or gentamicin. A lack of growth on gentamicin was observed for all targeted mutants, indicating a defect in MT activity. During this process, a further two spontaneous mutants defective in growth on LB agar containing gentamicin were identified (Table 1). Curiously, however, these mutants could support growth in liquid medium at levels similar to the wild-type protein and were therefore not analyzed further.

Analysis of Sgm protein expression and folding. Wild-type Sgm and mutant proteins were successfully expressed and purified according to procedures previously established for the wild-type enzyme (19). CD spectroscopy was first used to assess the secondary structure and thermostability of the wild-type Sgm protein. CD spectra can be deconvoluted, by comparison to large data sets of protein CD spectra, in order to obtain an experimental estimate of the protein secondary structure content (16, 30). The Sgm CD spectrum (Fig. 2) was thus used to determine the protein secondary structure content using Dichroweb, giving an estimated -helical content of 40 to 46%. The values obtained agree well with both the predicted secondary structure from the Sgm amino acid sequence and the homology model structure (Table 2). The CD spectrum obtained for Sgm therefore indicates the protein is folded and that it has a structure consistent with our model.

View larger version (17K): [in this window] [in a new window]

FIG. 2. CD spectroscopy analysis of Sgm. CD spectrum of purified wild-type Sgm collected at 20°C and used for analysis of protein secondary structure content via Dichroweb (Table 2). deg., degrees.

View this table: [in this window] [in a new window]

TABLE 2. Prediction and experimental estimation of Sgm secondary structure

Sgm thermostability was examined by collecting CD spectra over the range of 5 to 85°C in 5°C increments. Sgm is fully stable up to 45 to 50°C, after which point the spectra become progressively more like those of an unstructured random coil (data not shown). No further change is observed beyond 65°C, where all secondary structure appears to be lost. The temperature-induced melting of -helical secondary structures, monitored at 220 nm, showed an increase in molar ellipticity beyond 45°C, indicating a rapid disorder in the molecular structure after this point.

Before beginning a detailed analysis of the function of mutant Sgm proteins, we wished to eliminate the possibility that any of the mutations introduced might cause major misfolding and/or degradation of Sgm. Each mutated protein was successfully expressed in E. coli BL21(DE3) and purified under conditions identical to those of the wild-type Sgm. CD spectra were recorded for each mutant protein (see Fig. S1 in the supplemental material). Although some small differences were observed, the CD spectrum for each mutant indicated it was a folded protein with a structure similar to that of the wild-type Sgm. None of the mutants displayed gross changes in the CD spectrum similar to those obtained in the thermally induced unfolding experiments with the wild-type protein.

Measurement of MIC in liquid culture. The function of wild-type and mutant Sgm proteins was assessed in vivo by determination of MICs in liquid cultures of E. coli transformants containing an sgm-carrying plasmid (Table 1). The wild-type Sgm conferred resistance to both of the 4,6-disubstituted 2-deoxystreptamines tested (gentamicin and kanamycin) with a MIC of >1,500 µg/ml. Each protein containing an alanine mutation at one target residue within the Sgm C-terminal domain was classified into one of the following two groups according to the observed phenotype: mutations that had no apparent phenotype and mutations that had a dramatic influence on cell survival in the presence of gentamicin (similar results were obtained with kanamycin). The mutation of all residues that were implicated in either cofactor binding or methyl group transfer resulted in a severely gentamicin-sensitive phenotype (Table 1). Only one mutation to alanine of a nonconserved amino acid (N179) near the proposed SAM binding pocket produced a protein with a wild-type phenotype (Table 1).

Methylation assay of 30S ribosomal subunits. The MT activity of wild-type and mutant Sgm proteins was assessed by an in vitro assay of 30S ribosomal subunit methylation with [³H]SAM cofactor (Fig. 3). The reaction resulted in the transfer of the tritiated methyl group to the 16S rRNA component of the 30S subunit isolated from the E. coli BL21(DE3) strain ("sensitive 30S"). In vivo-methylated 30S subunits, isolated from bacteria harboring a plasmid encoding wild-type Sgm ("resistant 30S") were not further in vitro methylated by Sgm (Fig. 3A). A further negative control, the addition of extract from E. coli BL21(DE3) without Sgm-encoding vector, provided a measure of the background incorporation for all experiments. The wild-type Sgm was able to efficiently methylate sensitive 30S subunits (Fig. 3A), but none of the mutant proteins showed activity above the background. For mutations targeted to both putative SAM binding (Fig. 3B) and catalytic (Fig. 3C) residues, the rate of tritiated methyl group incorporation was the same as that for the background (negative control).

View larger version (21K): [in this window] [in a new window]

FIG. 3. In vitro methylation activities of wild-type and mutant Sgm proteins. (A) Comparison of wild-type Sgm methylation activity against sensitive 30S (S-30S; solid line) and resistant 30S (R-30S; dotted line) ribosomal subunit substrates. MT-free cell extract from E. coli lacking an Sgm-encoding plasmid was used as a negative control (EC cell extract; dashed line) in all methylation assays and indicates background levels of methylation activity. (B) Methylation activities against sensitive 30S ribosomal subunits of Sgm proteins mutated at a nonconserved residue near the SAM pocket (N179A; squares) and at putative key residues for cofactor binding, as follows: G135A (circles), D156A (triangles), and D182A (diamonds). (C) Methylation activities against sensitive 30S ribosomal subunits of Sgm proteins mutated at putative key residues for methyl group transfer, as follows: K199A (circles), E205A (triangles), R236A (squares), and E267A (diamonds). In panels B and C, the wild-type (solid line) and negative control (dashed line) data are the same as in panel A.

Measurement of SAM binding by ITC. To test whether the functional defect observed in the putative cofactor pocket mutants (G135A, D156A, and D182A) was indeed due to a deficiency in SAM binding, we used ITC to measure the thermodynamics of binding (Table 1; Fig. 4). The wild-type Sgm and a protein mutated at a variable residue near the proposed binding site (N179A) showed clear binding to SAM, with an affinity in the low micromolar range. The fits for both titrations produced reasonable stoichiometries, with N equal to 1.07 ± 0.7 and 0.89 ± 0.3 for the wild-type and N179A Sgm, respectively, indicating one SAM molecule bound per protein. Each of the proteins mutated at potential catalytic residues (K199A, E205A, R236A, and E267A) also retained SAM-binding affinity (Table 1). In contrast, none of the proteins mutated at putative key residues for cofactor binding (G135A, D156A, and D182A) showed detectable binding of SAM, confirming their role in creating the cofactor binding pocket.

View larger version (38K): [in this window] [in a new window]

FIG. 4. Isothermal titration calorimetric analysis of SAM binding to Sgm. (A) Titration of SAM (via syringe) into the wild-type Sgm protein (sample cell). The upper panel shows the baseline-corrected titration data, and the lower panel shows the binding isotherm fit to a model for a single SAM binding site. Equivalent titration data are shown for N179A (B), D182A (C), and R236A (D) mutant Sgm proteins.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Aminoglycoside antibiotics comprise a structurally varied family of polycationic bactericidal agents that interfere with protein translation by mimicking the conformational change in 16S rRNA induced by correct codon-anticodon pairing (24). Mutations or modifications, such as methylation, that perturb hydrogen bonding between aminoglycosides and target residues or that generate steric clashes within the A site result in a lowered affinity for the antibiotic for the A site and can lead to bacterial resistance.

To provide a structural platform for functional characterization of the sisomicin-gentamicin resistance MT Sgm, we employed a range of computational structure modeling and sequence analysis methods. The model shows a clear division of Sgm into two major structural domains separating the N- and C-terminal amino acids. The 164 C-terminal amino acids form a Rossmann-like MT fold, as expected for an MT enzyme, and the structure closely resembles a previous model of this domain constructed for a homologous MT from Streptomyces kanamyceticus (5). The complete Sgm protein model was experimentally evaluated by analysis of the protein CD spectrum, which showed good correspondence between predicted and observed secondary structure content. We next incorporated amino acid conservation across all FmrO family members by mapping these onto the homology model. An initial classification was made for three clusters of conserved residues as potential key residues for SAM binding, catalysis, or target recognition.

Amino acids G135, D156, and D182 were identified as potentially forming the SAM binding pocket and were mutated to alanine. All three mutations abrogated Sgm activity as measured in vivo by growth of E. coli in liquid media and by in vitro methylation assays of 30S substrate. To confirm the role of these residues in SAM cofactor binding, the affinity for each protein was assessed by ITC. The wild-type Sgm bound SAM with a dissociation constant, K_D, of 17.5 µM, and a similar value of 14.2 µM was obtained for the functional N179A mutant. This lies within the range of known affinities for SAM-dependent MTs (0.1 to 19 µM) (22, 23, 26, 31) and is comparable to that of another RNA MT, RsmC (SAM K_D, 4.8 µM), which methylates G1207 in 16S rRNA (31). It is also similar to the affinity for the cofactor measured by other methods for RsmD and RlmE (SAM K_m, 26 µM), both endogenous MTs from E. coli that also recognize highly structured substrates in 30S and 50S ribosomal subunits, respectively (1, 3). In contrast, no binding of SAM could be detected for the G135, D156, and D182 mutants. Based on a survey of various MTs of known structure and the roles of equivalent residues in these proteins (11, 12, 17, 28, 31), we speculate that D156 and D182 may position the adenine component of SAM via contacts with the ribose hydroxyl groups and adenine base, respectively, and G135 may directly influence the positioning of the methyl group. As each position is extremely highly conserved, whatever their precise roles, we infer that these positions and those around them in the Sgm model will fulfill the same functions across the entire FmrO family of enzymes.

We further hypothesized that residues K199, E205, R236, and E267 should have a role in methyl group transfer. Each residue was again mutated to alanine, and the function of the mutated protein was tested in vivo and in vitro. As before, MICs for gentamicin were dramatically reduced (20- to 50-fold) and MT activity was abolished for each mutant enzyme. These proteins were, however, still competent in SAM cofactor binding. Together, these results support the hypothesis that this group of amino acids contributes directly to Sgm catalytic function, though it is not possible to eliminate a potential additional role in substrate recognition.

The precise site of methylation has not been determined but has been inferred indirectly to be G1405 in 16S rRNA by virtue of the protection against further methylation of 30S subunits by Sgm following in vivo protection by the m⁷G1405 MT KgmB (18; Ilic-Tomic et al., unpublished). The methylation reaction is highly substrate specific and requires intact 30S ribosomal subunit (20), as has been observed for two endogenous E. coli MTs, RsmE (3) and YebU (2). This implies that recognition of the target site is at least partially related to the subunit structure around the target nucleotide. It is also possible that docking to ribosomal proteins, such as the nearby S12, in addition to rRNA is also necessary. Our model and analysis of conserved amino acids indicate that the N-terminal part of Sgm is likely to be involved in target site recognition. However, in addition to these uncertainties with regard to recognition mechanism, there is also a precedence for modifications of MT residues involved in target recognition having only mild or moderate phenotypes (22, 31), making detailed analysis of their roles difficult.

The aminoglycoside resistance spectrum for Sgm was previously assessed only in homologous host strains, i.e., in high-G+C bacteria such as "Streptomyces lividans" and "Micromonospora melanospora." The present study therefore provides a first demonstration that a high level of resistance can be achieved by expression of a gene from an aminoglycoside producer in a heterologous host, the gram-negative bacterium E. coli. Aminoglycoside resistance determinants such as Sgm can be easily transferred to pathogenic strains by means of mobile genetic elements (14), making a complete understanding of their structures, activities, and target recognition essential. We have demonstrated that homology-based analysis coupled with mutational analysis of Sgm can provide a platform for a detailed structure-function analysis of these enzymes prior to the determination of the high-resolution X-ray crystallographic structure(s) of FmrO family members. These approaches could ultimately provide a deeper understanding of how FmrO MT enzymes recognize their rRNA/ribosomal protein binding site(s) on the 30S subunit with such exquisite specificity.

 
This work was supported by grant no. 078374 from the Wellcome Trust. B.V. also acknowledges support from the Ministry of Science of the Republic of Serbia (grant no. 143056).

 
* Corresponding author. Mailing address: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom. Phone: (0161) 3064218. Fax: (0161) 2365201. E-mail: Graeme.L.Conn{at}manchester.ac.uk

Published ahead of print on 27 June 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Agarwalla, S., J. T. Kealey, D. V. Santi, and R. M. Stroud. 2002. Characterization of the 23 S ribosomal RNA m⁵U1939 methyltransferase from Escherichia coli. J. Biol. Chem. 277:8835-8840.[Abstract/Free Full Text]
2
2. Andersen, N. M., and S. Douthwaite. 2006. YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407. J. Mol. Biol. 359:777-786.[CrossRef][Medline]
3
3. Basturea, G. N., and M. P. Deutscher. 2007. Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE. RNA 13:1969-1976.[Abstract/Free Full Text]
4
4. Beauclerk, A. A. D., and E. Cundliffe. 1987. Sites of action of two ribosomal RNA methylases responsible for resistance to aminoglycosides. J. Mol. Biol. 193:661-671.[CrossRef][Medline]
5
5. Bujnicki, J. M., and L. Rychlewski. 2001. Sequence analysis and structure prediction of aminoglycoside-resistance 16S rRNA:m⁷G methyltransferases. Acta Microbiol. Pol. 50:7-17.[Medline]
6
6. Crooks, G. E., G. Hon, J. M. Chandonia, and S. E. Brenner. 2004. WebLogo: a sequence logo generator. Genome Res. 14:1188-1190.[Abstract/Free Full Text]
7
7. Cuff, J. A., M. E. Clamp, A. S. Siddiqui, M. Finlay, and G. J. Barton. 1998. JPred: a consensus secondary structure prediction server. Bioinformatics 14:892-893.[Abstract/Free Full Text]
8
8. Cundliffe, E. 1992. Resistance to macrolides and lincosamides in Streptomyces lividans and to aminoglycosides in Micromonospora purpurea. Gene 115:75-84.[CrossRef][Medline]
9
9. Doi, Y., and Y. Arakawa. 2007. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides. Clin. Infect. Dis. 45:88-94.[CrossRef][Medline]
10
10. Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32:1792-1797.[Abstract/Free Full Text]
11
11. Fauman, E. B., R. M. Blumenthal, and X. Cheng. 1999. Structure and evolution of AdoMet-dependent methyltransferases, p. 1-38. In X. Cheng and R. M. Blumenthal (ed.), S-Adenosylmethionine-dependent methyltransferases: structures and functions. World Scientific Publishing, River Edge, NJ.
12
12. Foster, P. G., C. R. Nunes, P. Greene, D. Moustakas, and R. M. Stroud. 2003. The first structure of an RNA m⁵C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate. Structure 11:1609-1620.[Medline]
13
13. Frishman, D., and P. Argos. 1995. Knowledge-based protein secondary structure assignment. Proteins 23:566-579.[CrossRef][Medline]
14
14. Galimand, M., S. Sabtcheva, P. Courvalin, and T. Lambert. 2005. Worldwide disseminated armA aminoglycoside resistance methylase gene is borne by composite transposon Tn1548. Antimicrob. Agents Chemother. 49:2949-2953.[Abstract/Free Full Text]
15
15. Garnier, J., D. J. Osguthorpe, and B. Robson. 1978. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120:97-120.[CrossRef][Medline]
16
16. Greenfield, N. J. 1996. Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal. Biochem. 235:1-10.[CrossRef][Medline]
17
17. Hallberg, B. M., U. B. Ericsson, K. A. Johnson, N. M. Andersen, S. Douthwaite, P. Nordlund, A. E. Beuscher IV, and H. Erlandsen. 2006. The structure of the RNA m5C methyltransferase YebU from Escherichia coli reveals a C-terminal RNA-recruiting PUA domain. J. Mol. Biol. 360:774-787.[CrossRef][Medline]
18
18. Holmes, D. J., and E. Cundliffe. 1991. Analysis of a ribosomal-RNA methylase gene from Streptomyces tenebrarius which confers resistance to gentamicin. Mol. Gen. Genet. 229:229-237.[CrossRef][Medline]
19
19. Ili Tomi, T., S. Markovi, and B. Vasiljevi. 2005. Expression and purification of the Sgm protein from E. coli. J. Serb. Chem. Soc. 70:817-822.[CrossRef]
20
20. Kojic, M., L. Topisirovic, and B. Vasiljevic. 1992. Cloning and characterization of an aminoglycoside resistance determinant from Micromonospora zionensis. J. Bacteriol. 174:7868-7872.[Abstract/Free Full Text]
21
21. Landau, M., I. Mayrose, Y. Rosenberg, F. Glaser, E. Martz, T. Pupko, and N. Ben-Tal. 2005. ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33:W299-302.[Abstract/Free Full Text]
22
22. Maravi, G., J. M. Bujnicki, M. Feder, S. Pongor, and M. Flogel. 2003. 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. 31:4941-4949.[Abstract/Free Full Text]
23
23. Mashhoon, N., M. Carroll, C. Pruss, J. Eberhard, S. Ishikawa, R. A. Estabrook, and N. Reich. 2004. Functional characterization of Escherichia coli DNA adenine methyltransferase, a novel target for antibiotics. J. Biol. Chem. 279:52075-52081.[Abstract/Free Full Text]
24
24. Ogle, J. M., D. E. Brodersen, W. M. Clemons, Jr., M. J. Tarry, A. P. Carter, and V. Ramakrishnan. 2001. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292:897-902.[Abstract/Free Full Text]
25
25. Ohta, T., and M. Hasegawa. 1993. Analysis of the self-defense gene (fmrO) of a fortimicin A (astromicin) producer, Micromonospora olivasterospora: comparison with other aminoglycoside-resistance-encoding genes. Gene 127:63-69.[CrossRef][Medline]
26
26. Powell, L. M., D. T. Dryden, D. F. Willcock, R. H. Pain, and N. E. Murray. 1993. DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine. J. Mol. Biol. 234:60-71.[CrossRef][Medline]
27
27. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.[Abstract]
28
28. Schluckebier, G., M. Ogara, W. Saenger, and X. D. Cheng. 1995. Universal catalytic domain structure of AdoMet-dependent methyltransferases. J. Mol. Biol. 247:16-20.[CrossRef][Medline]
29
29. Schneider, T. D., and R. M. Stephens. 1990. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 18:6097-6100.[Abstract/Free Full Text]
30
30. Sreerama, N., S. Y. Venyaminov, and R. W. Woody. 1999. Estimation of the number of -helical and β-strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 8:370-380.[Medline]
31
31. Sunita, S., E. Purta, M. Durawa, K. L. Tkaczuk, J. Swaathi, J. M. Bujnicki, and J. Sivaraman. 2007. Functional specialization of domains tandemly duplicated within 16S rRNA methyltransferase RsmC. Nucleic Acids Res. 35:4264-4274.[Abstract/Free Full Text]
32
32. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
33
33. Wachino, J. I., K. Shibayama, H. Kurokawa, K. Kimura, K. Yamane, S. Suzuki, N. Shibata, Y. Ike, and Y. Arakawa. 2007. Plasmid-mediated novel m¹A1408 methyltransferase, NpmA, for 16S rRNA found in clinically isolated Escherichia coli resistant to structurally diverse aminoglycosides. Antimicrob. Agents Chemother. 51:4401-4409.[Abstract/Free Full Text]

---

Journal of Bacteriology, September 2008, p. 5855-5861, Vol. 190, No. 17
0021-9193/08/$08.00+0     doi:10.1128/JB.00076-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Savic, M., Lovric, J., Tomic, T. I., Vasiljevic, B., Conn, G. L. (2009). Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics. Nucleic Acids Res 0: gkp575v1-gkp575 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Supplemental material Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Savic, M. Articles by Conn, G. L. Search for Related Content PubMed PubMed Citation Articles by Savic, M. Articles by Conn, G. L.