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Antimicrob Agents Chemother. Dec 2007; 51(12): 4401–4409.
Published online Sep 17, 2007. doi:  10.1128/AAC.00926-07
PMCID: PMC2168023

Novel Plasmid-Mediated 16S rRNA m1A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides[down-pointing small open triangle]

Abstract

We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.

Aminoglycosides such as kanamycin, gentamicin, and neomycin bind to the A site of the 16S rRNA of the bacterial 30S ribosomal subunit and subsequently block growth through interference with protein synthesis (25). These agents have been used for the treatment of a broad range of life-threatening infections due to both gram-positive and gram-negative bacteria in human and veterinary medicine (18, 37). However, bacteria have acquired various aminoglycoside resistance mechanisms, such as through the production of aminoglycoside-modifying enzymes (acetyltransferase, nucleotidyltransferase, and phosphotransferase), the reduction of antibiotic penetration on the outer membrane protein, the acquisition of reduced affinity by changing key nucleotides within the 16S rRNA, and augmented excretion by an efflux pump system (5, 25, 36, 42).

In 2003, a plasmid-mediated 16S rRNA methyltransferase, which confers a high level of resistance to various clinically important aminoglycosides, was reported to be involved as part of a novel aminoglycoside resistance mechanism in pathogenic gram-negative rods (16, 53). At present, five types of plasmid-mediated 16S rRNA methyltransferase genes, rmtA, rmtB, rmtC, rmtD, and armA, have been found worldwide in members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. (4, 7, 10, 11, 17, 20, 34, 48, 50-52). Also, these genes are mediated by bacterium-specific recombination systems, such as transposons, and are easily translocated to other DNA target sites (17, 19, 47, 49).

The 16S rRNA methyltransferases conferring aminoglycoside resistance are supposed to have evolved as a self-defense mechanism in aminoglycoside-producing actinomycetes, including Streptomyces spp. and Micromonospora spp (9). The methylation of 16S rRNA plays a crucial role in prevention of the adverse effects of intrinsic aminoglycosides that would block their own 16S rRNA. The 16S rRNA methyltransferase conferring aminoglycoside resistance consists of two different groups, one methylates the N-7 position of G1405 and confers panresistance to aminoglycosides belonging to both the kanamycin and the gentamicin groups (3, 44), and the other methylates the N-1 position of A1408 and provides resistance to kanamycin and apramycin (3, 22, 43). Recently, it was reported that the plasmid-mediated 16S rRNA methyltranferase ArmA methylates the N-7 position of G1405 within 16S rRNA (27). On the other hand, no plasmid-mediated 16S rRNA methyltransferase which modifies the N-1 position of A1408 has so far been found in any pathogenic bacteria isolated from clinical settings and natural environments. Therefore, we screened for a new plasmid-mediated methyltransferase that methylates A1408 among bacterial species belonging to the family Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp. isolated in Japanese clinical settings. Apramycin resistance seemed to be a good indicator for the detection of an A1408 16S rRNA methyltransferase producer, since a previous study reported that the introduction of a recombinant plasmid encoding a gene for the A1408 16S rRNA methyltransferase derived from a Streptomyces sp. was also able to confer a high level of resistance to apramycin (43). The use of this screening protocol on the basis of apramycin resistance allowed us to identify a panaminoglycoside-resistant Escherichia coli strain, strain ARS3, that produces a novel plasmid-mediated methyltransferase, newly assigned NpmA, that methylates A1408 at the A site of 16S rRNA. The aim of this study was to characterize the molecular mechanism underlying the panaminoglycoside resistance conferred by NpmA.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table Table1.1. E. coli strain ARS3 was isolated in 2003 from the urine of an inpatient in a general hospital in Japan. The bacterial strains were grown in LB broth at 37°C with shaking, unless otherwise indicated. MICs were determined by the agar dilution method with Mueller-Hinton agar plates, according to the protocol recommended by the Clinical and Laboratory Standards Institute (8).

TABLE 1.
Bacterial strains and plasmids used in this study

Conjugation.

A conjugation experiment was performed as described elsewhere (48). E. coli strain CSH-2 was used as the recipient. Conjugants were selected on LB agar plates containing apramycin at 150 μg/ml and rifampin at 100 μg/ml.

Cloning of npmA.

The transferable plasmid pARS3 was extracted from the E. coli conjugant and digested with restriction enzymes. The resultant fragments were ligated to cloning vectors and electroporated into E. coli strain JM109. The transformants were selected on LB agar plates supplemented with apramycin at 150 μg/ml and chloramphenicol at 30 μg/ml. The npmA gene was amplified with primer P1 (5′-CGG GAT CCA AGC ACT TTC ATA CTG ACG-3′) and primer P2 (5′-CGG AAT TCC AAT TTT GTT CTT ATT AGC-3′) (the underscored sequences indicate BamHI and EcoRI restriction sites, respectively) and cloned into the vector pMCL210.

N-terminal determination of NpmA.

The DNA fragment carrying npmA and its promoter region was amplified by PCR with primers P1 and P3 (the sequence of primer P3 is 5′-CCC AAG CTT TTA atg atg atg atg atg ATG TTT TGA AAC ATG GCC-3′ [where the underscores indicate the Hind III restriction site and the sequence with lowercase letters represents the nucleotide sequence of C-terminal histidine tag]). Primer P3 was designed so that five histidine codons could be added to the 3′ end of npmA. The resultant fragments were ligated to pMCL210 and introduced into E. coli JM109. The cells were cultured in 1 liter of LB broth containing chloramphenicol at 30 μg/ml, disrupted with a French press, and centrifuged at 100,000 × g for 1 h. The supernatant containing the recombinant protein was loaded onto a HisTrap HP column (Amersham Biosciences) and purified according to the manufacturer's instructions. The N-terminal sequence of the purified protein was obtained by Edman degradation in a model Shimadzu PPSQ-23 automated protein sequencer.

Overexpression and purification of histidine-tagged NpmA.

The npmA gene was amplified with primer P4 (5′-GGA ATT CCA TAT GTT AAT ACT CAA AGG AA-3′), which introduced an NdeI restriction site at the 5′ end, and primer P3, which introduced a HindIII restriction site and five histidine codons at the 3′ end. The amplified fragments were cloned into the pCold-IV vector (Takara) and introduced into E. coli BL21(DE3)pLysS. The purification of recombinant protein was performed as described above, with some modifications. After the step of nickel-nitrilotriacetic acid chromatography, the eluted protein was dialyzed against 50 mM sodium phosphate buffer (pH 6.4). Furthermore, the protein was applied to a cation-exchange HiTrap S HP column (Amersham Biosciences). Finally, the eluted protein was concentrated and the buffer was exchanged with 50 mM sodium phosphate buffer (pH 7.4).

Methylation assay.

Both the 30S and the 50S subunits of E. coli JM109 were prepared as described previously (27). After ultracentrifugation with 10 to 30% sucrose density gradients, the 30S and 50S subunit fractions were collected. The purity of each subunit was checked by denatured agarose gel electrophoresis of the rRNA derived from the material. The methylation assay was carried out at 35°C, as follows. Thirty picomoles of substrate, 30 pmol of His5-NpmA, and 7.5 μCi of S-adenosyl-l-[methyl-3H]methionine (76 Ci/mmol, 1 mCi/ml) were adjusted to 300 μl with methylation buffer (50 mM HEPES-KOH, pH 7.5; 10 mM MgCl2; 100 mM NH4Cl; 5 mM 2-mercaptoethanol). Aliquots were taken at 0, 5, 15, 30, and 45 min and purified with an RNeasy mini kit (Qiagen), according to the instructions provided by the manufacturer. The samples were counted with a scintillation counter.

RNase protection assay.

One picomole of [3H]methyl-labeled 16S rRNA was hybridized with 100 pmol of a deoxyoligonucleotide (positions 1421 to 1392 [5′-CAC TCC CAT GGT GTG ACG GGC GGT GTG TAC-3′] and positions 1507 to 1478 [5′-TAC CTT GTT ACG ACT TCA CCC CAG TCA TGA-3′]) in 50 μl of hybridization buffer (40 mM morpholineethanesulfonic acid, pH 6.4; 400 mM NaCl; 9 mM EDTA; 80% [vol/vol] formamide) The sample was incubated at 90°C for 10 min, cooled at room temperature for 15 min, and diluted with 450 μl of RNase buffer (10 mM Tris-HCl, pH 7.5; 300 mM NaCl; 5 mM EDTA) containing RNase T1 (Roche). The digestion was performed at 37°C for 1 h. The reaction was stopped by adding 4.5 ml of 10% ice-cold trichloroacetic acid, and the reaction mixture was placed on ice for 10 min. The samples were passed through cellulose nitrate filters. The filters were dissolved in scintillation fluid, and the radioactivity was measured.

Primer extension.

One microgram of 16S rRNA extracted from the 30S subunits methylated in vitro was hybridized with 50 pmol of a primer (5′-biotin-CCA ACC GCA GGT TCC CCT ACG G-3′) complementary to nucleotides 1530 to 1509 at 65°C for 10 min. The elongation was performed with Transcriptor reverse transcriptase (Roche) at 43°C for 1 h. The cDNA transcripts were analyzed on an 8% polyacrylamide gel containing 8 M urea.

HPLC assay of methylated adenine residue.

16S rRNA was extracted from the 30S subunits of E. coli. Sixty micrograms of extracted 16S rRNA was digested with nuclease P1 (3 U; Wako) and alkaline phosphatase (0.08 U; Takara) in 120 μl of a reaction mixture containing 25 mM HEPES-KOH (pH 7.5) at 37°C for 6 h. The resulting mixture was analyzed by high-performance liquid chromatography (HPLC) with an HRC-ODS column (4.6 mm [inner diameter] by 250 mm; Shimadzu). The solvent system consisted of 5 mM ammonium acetate (pH 5.3) (solvent A) and 30% acetonitrile (solvent B) and was used as follows: 0% to 50% solvent B from 0 to 100 min, 50% to 99% solvent B from 100 to 110 min, and 99% solvent B from 110 to 130 min, with an effluent rate of 600 μl/min at 30°C.

Aminoglycoside binding to 30S subunit.

Sixty picomoles of the wild-type or the modified 30S subunits was incubated in 100 μl of dimethylsulfate (DMS) buffer (80 mM sodium cacodylate, pH 7.2; 100 mM NH4Cl; 20 mM MgCl2; 1 mM dithiothreitol; 0.5 mM EDTA) at 42°C for 10 min. Addition of aminoglycosides (final concentration range, 1 μM to 1,000 μM) was followed by incubation at 37°C for 30 min and then on ice for 10 min. DMS (2 μl, 1:6 in ethanol) was added, and the mixture was incubated at 37°C for 10 min. The reaction was quenched by adding 25 μl of stop buffer (1.5 M sodium acetate, 1 M 2-mercaptoethanol). After ethanol precipitation, modified rRNA was obtained by extraction with phenol three times and chloroform twice. Reduction with sodium borohydride and aniline-induced strand scission were performed as described previously (27). A primer extension analysis was carried out as described above.

Nucleotide sequence accession number.

The open reading frame of npmA was deposited in the EMBL and GenBank databases through the DDBJ database and has been assigned accession number AB261016.

RESULTS

Characteristics of E. coli strain ARS3.

The MICs of various aminoglycosides for parent E. coli strain ARS3 are shown in Table Table2.2. This strain demonstrated resistance to structurally diverse aminoglycosides. The panaminoglycoside-resistant phenotype of strain ARS3 was successfully transferred to the E. coli CSH-2 recipient strain at a frequency of 2 × 10−8 per donor by conjugation. The transconjugant acquired a transferable plasmid (pARS3), which was estimated to be about 115 kb in size by summation of the sizes of the EcoRI digestion products, and demonstrated resistance to various aminoglycosides (Table (Table22).

TABLE 2.
Antimicrobial susceptibilities of parental strain, transconjugant, and transformant

Genetic determinant of aminoglycoside resistance on transferable plasmid pARS3.

A cloning experiment was performed to confirm the genetic aminoglycoside resistance determinant, which is mediated by pARS3. As a result, one recombinant plasmid (plasmid pMCL-H) was obtained by selection with apramycin and chloramphenicol, and both strands of the 3,946-bp HindIII insert were entirely sequenced. The schematic organization of probable genes found in the cloned fragment is shown in Fig. Fig.1.1. To identify the gene responsible for apramycin resistance, Tn5 (Tetr) insertion mutants of clone pMCL-H were generated. A total of 12 insertion mutants were obtained (Fig. (Fig.1),1), and 3 of them that carried a Tn5 insertion in orf6 lost apramycin resistance. Recombinant plasmid pMCL-BE, which contained only orf6 and its putative promoter region, showed apramycin resistance, as was seen in clone pMCL-H.

FIG. 1.
Schematic presentation of open reading frames (ORFs) in the cloned fragment conferring aminoglycoside resistance. Open reading frames are shown as arrows indicating the transcription orientation. The positions where Tn5 was inserted are indicated by open ...

The deduced amino acid sequences of ORF6 exhibited low-level identities (<31%) to the chromosomally encoded 16S rRNA methyltransferases KamA, KamB, KamB2, KamB3, KamC, and Amr of actinomycetes that produce aminoglycosides. Several studies already revealed that some of these 16S rRNA methyltransferases of actinomycetes methylate the N-1 position of nucleotide A1408 in 16S rRNA and confer intrinsic aminoglycoside resistance to bacteria (3, 22, 43). Therefore, it is probable that the product of orf6, NpmA, has 16S rRNA methyltransferase activity and confers panresistance to aminoglycosides in a manner similar to that seen in aminoglycoside-producing actinomycetes. NpmA has a conserved residue (D) and the consensus GXGXG motif, which is considered the hallmark S-adenosylmethionine (SAM)-binding site of Rossman fold SAM-dependent methyltransferases (Fig. (Fig.2)2) (26). SAM is often used as the source of the methyl group in methyltransferase reactions in various organisms (26).

FIG. 2.
Alignment of deduced amino acid sequences of NpmA with those of KamA and KamB. Chromosomal 16S rRNA methyltransferases (KamA and KamB) were found in aminoglycoside-producing actinomycetes (24, 32). Identical amino acids in all proteins are highlighted ...

Genetic environments of npmA.

The structures of the flanking regions of npmA were determined (Fig. (Fig.1).1). The genes for orf7 (which encodes a probable ABC transporter substrate binding protein) and orf8 (which encodes a truncated mobilization protein) were located at the 3′-end region of npmA. Three open reading frames, orf3 (which encodes a hypothetical protein), orf4 (which encodes a possible replication protein), and orf5 (which encodes a hypothetical protein), existed at the 5′-end region of npmA. The 9.1-kb region containing orf3 to orf8 was flanked by two IS26 elements in direct orientation and composed a transposable element (12). The sequences around the 9.1-kb transposable element have significant sequence similarities to the sequences of a part of various multidrug resistance plasmids deposited in the EMBL/GenBank/DDBJ databases.

Antibiotic susceptibilities.

The MICs of the aminoglycosides for the NpmA-producing E. coli transformant are shown in Table Table2.2. The introduction of npmA-carrying plasmid pMCL-BE conferred resistance to both 4,6-disubstituted 2-deoxystreptamines, consisting of the kanamycin and gentamicin groups, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. In addition, NpmA augmented the MIC of apramycin, whose structure is far different from those of the 4,6- and 4,5-disubstituted 2-deoxystreptamines. On the other hand, NpmA did not confer resistance to the non-A-site binders streptomycin and spectinomycin. On the whole, NpmA could confer resistance to various aminoglycosides which bind to the A site of the decoding region in 16S rRNA.

N-terminal sequence of NpmA.

As shown in Fig. Fig.2,2, the exact locations of the N termini of A1408 methyltransferases are still controversial. For example, the N-terminal position of the Kam family of proteins, including KamB and KamC, was previously reported to be position M61, shown in Fig. Fig.22 (22). This fact, however, indicates the lack of a SAM-binding motif, which plays a crucial role in methyltransferase activity among the mature Kam family of enzymes. Most recently, Koscinski et al. reanalyzed the amended amino acid sequences of a Kam family protein and revealed that the SAM-binding motif is perfectly conserved in the missing N-terminal sequences of the Kam family of proteins (24). In this study, in order to determine the exact position of the N terminus in NpmA experimentally, the recombinant NpmA protein was purified from E. coli cells harboring pMCL-BH and was subjected to Edman protein sequencing. The N terminus of NpmA was exactly determined to be MLILK (Fig. (Fig.2),2), although TTG is uncommon as a bacterial initiation codon.

Methylation of 30S subunits by NpmA.

E. coli BL21(DE3)pLysS and the pCold-IV expression vector were used for the overexpression and purification of NpmA. E. coli BL21(DE3)pLysS carrying pCold-IV is susceptible to apramycin (MIC, 3.9 μg/ml), while E. coli BL21(DE3)pLysS carrying pCold-NpmA exhibited a very high level of resistance to apramycin (MIC, >1,000 μg/ml) in the microdilution susceptibility test. This result indicated that the histidine-tagged NpmA (His5-NpmA) still has methylation activity and is responsible for apramycin resistance in E. coli BL21(DE3)pLysS. An optimized culture condition yielded 8 mg of purified protein per 1 liter of bacterial culture, and the purified enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie brilliant blue staining (Fig. (Fig.3A3A).

FIG. 3.
Purification of NpmA and methylation assays. (A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified His5-NpmA protein. Lanes: M, protein size marker; 1, purified His5-NpmA. (B) Methyl acceptor activities of 30S subunits, 50S subunits, ...

The methylation assay was performed with three different substrates, 50S subunits, 30S subunits, and 16S rRNA dissociated from the 30S subunits, to explore the activity of NpmA and its substrate specificity. NpmA was able to incorporate about 0.8 pmol of methyl groups into 1 pmol of 30S subunits after 45 min of incubation, whereas no significant incorporation of methyl groups into each 50S subunit or the dissociated 16S rRNA molecule was detected under the same experimental conditions (Fig. (Fig.3B).3B). These results demonstrated that NpmA has optimal methyltransferase activity toward the properly assembled 30S subunit.

RNase protection assay.

As described above, the in silico analysis indicated that NpmA exhibits amino acid sequence similarity to various chromosomally encoded A1408 16S rRNA methyltransferases of aminoglycoside-producing actinomycetes. This suggested that NpmA would also modify the same position within 16S rRNA, as reported previously (3, 22, 43). To determine the exact position of methylated nucleotide, a hybridization protection study was first carried out with deoxynucleotides that were complementary to a part of the 16S rRNA sequence. Two oligomers from positions 1392 to 1421 and positions 1478 to 1507 were prepared to span the aminoglycoside-binding A-site region within the 16S rRNA. The hybridization with the oligomer from positions 1392 to 1421 served to keep the radioactivity of [3H]methyl-labeled 16S rRNA after RNase T1 digestion, while the oligomer from positions 1478 to 1507 was ineffective in protecting against RNase T1 digestion (Fig. (Fig.4A).4A). This finding indicated that the position of the methylated nucleotide is located within the region from residue 1392 to residue 1421 in the 16S rRNA.

FIG. 4.
Nuclease protection assay and primer extension analysis. (A) Nuclease protection assay with [3H]-methyl-labeled 16S rRNA and DNA oligonucleotides (oligo) complementary to the regions from positions 1392 to 1421 or positions 1478 to 1507. The values are ...

Primer extension.

Methylated 16S rRNA, prepared from 30S subunits which were incubated with His5-NpmA in the presence of the methyl donor SAM, was used as the template RNA in reverse transcriptase extension. The extension terminated at position C1409, indicating that methylation surely occurs at position A1408 (Fig. (Fig.4B).4B). In contrast, no termination signal was observed at the same position when unmethylated 16S rRNA was used for the reverse transcription experiment (Fig. (Fig.4B4B).

HPLC assay of methylated adenine residue.

We determined the type of detailed modification by HPLC. When wild-type 16S rRNA was treated with nuclease P1 plus alkaline phosphatase, there was no peak corresponding to 1-methyladenosine (m1A), due to the lack of an innate m1A nucleoside in the 16S rRNA of a K-12-derived E. coli strain (Fig. (Fig.5A).5A). On the other hand, the m1A peak was clearly observed when the 16S rRNA methylated by NpmA was analyzed (Fig. (Fig.5B).5B). These results clearly demonstrate that NpmA actually methylates the N-1 position of adenosine. Thus, NpmA is an adenine N-1 methyltransferase. Each peak corresponding to m5C, m7G, m3U, and m2G was detected with almost equal intensity between the wild-type and the methylated 16S rRNAs.

FIG. 5.
HPLC analysis of methylated adenine residue. Purified 16S rRNA was completely digested with nuclease P1 and alkaline phosphatase and subjected to HPLC. (A) Wild-type 16S rRNA; (B) 16S rRNA methylated by NpmA. The m1A peak indicates the formation of a ...

Binding of aminoglycosides to 30S subunits assayed by footprinting.

The interaction between the 30S subunit and aminoglycosides was monitored by determining the changes in the chemical modification pattern of 16S rRNA by using DMS. The RNA footprints at G1494 were analyzed by primer extension with reverse transcriptase (Fig. (Fig.6).6). The apparent protection of G1494 was observed when wild-type 30S subunits were treated with gentamicin, neomycin, and ribostamycin at concentrations of 100 μM and 1,000 μM, indicating the certain binding of aminoglycosides to the 30S subunits. On the other hand, no decrease in the signal on G1494 was observed at 100 μM and 1,000 μM when m1A1408-methylated 30S subunits were used under the same reaction conditions, indicating the interruption of aminoglycoside binding by methylation at A1408. Although a slight increase in the signal at position G1494 in the m1A1408-methylated 30S subunits was observed in a gentamicin concentration-dependent manner, the precise reason for the phenomenon remains uncertain from the findings of the present study.

FIG. 6.
Footprinting for confirmation of aminoglycoside binding to 30S subunits. The gentamicin, neomycin, and ribostamycin footprints are indicated at the position of G1494 (N-7) in the 16S rRNA. Dideoxy sequencing lanes (lanes C, A, U, and G) were generated ...

DISCUSSION

A careful review of the recent literature enabled us to understand the binding mode between aminoglycosides and the target site through the resolution structure investigated by X-ray crystallography and nuclear magnetic resonance imaging (6, 13-15, 29, 38, 39, 40, 54). Basically, aminoglycosides such as 4,5- and 4,6-disubstituted 2-deoxystreptamines form specific hydrogen bonds with the nucleotides in decoding region A-site within 16S rRNA (Fig. (Fig.4C4C and Fig. 7C and D). Thus, mutations and modifications in the key nucleotides that perturb the hydrogen bond result in the loss of binding between aminoglycosides and 16S rRNA and lead to resistance to aminoglycosides in bacteria. The production of methyltransferase, which converts guanosine to 7-methylguanosine (m7G) at position 1405 in 16S rRNA, is one representative mechanism of aminoglycoside resistance in the manner described above. The genes encoding guanine N-7 methyltransferases, which methylate position G1405, have been found on the chromosomes of aminoglycoside-producing actinomycetes and on the plasmids of various pathogenic gram-negative pathogens isolated in both clinical and veterinary settings (7, 10, 11, 17, 20, 34, 48, 53). Furthermore, it is well known that adenine N-1 methyltransferases which modify position A1408 belong to another group of 16S rRNA methyltransferases that confer resistance to multiple aminoglycosides. However, this has so far been found exclusively on the chromosomes of aminoglycoside-producing actinomycetes. The present study is the first to describe the emergence of plasmid-mediated adenine N-1 methyltransferase, which confers panaminoglycoside resistance among pathogenic bacteria.

FIG. 7.
Predicted interaction between aminoglycosides and 16S rRNA in the 30S ribosomal subunit. (A) Complex structure of 16S rRNA (black) and 23S rRNA (orange) in the 70S ribosome from E. coli (PDB codes 2AVY and 2AW4) (41). The positions of G1405 (magenta) ...

Position A1408 plays a crucial role in the binding of 4,6- and 4,5-disubstituted 2-deoxystreptamines, because the puckered sugar ring I of these agents is inserted into the A-site helix to form hydrogen bonds to Watson-Crick sites N-1 and N-6 of the universally conserved A1408 among bacteria (Fig. 7C and D) (6, 15, 46). Thus, alternation of A1408 to G leads to a repulsive interaction with the 6′-NH3 group at ring I of 2-deoxystreptamines, while 2-deoxystreptamines carrying a 6′-OH group can still interact with 16S rRNA by accepting a hydrogen bond from the N-1 and N-2 positions of G1408 (35, 46). Likewise, methylation at the N-1 position of A1408 by NpmA will disturb the formation of the hydrogen bond toward the N-6′ or O-6′ of ring I of aminoglycosides, and this would, in turn, reduce the binding affinities of aminoglycosides to the target. In fact, the NpmA-producing strains demonstrate resistance to various aminoglycosides that combine the N-1 of A1408 through ring I. On the other hand, NpmA production did not confer resistance to non-A-site binders, such as streptomycin and spectinomycin. The results of susceptibility testing are well consistent with their footprinting patterns, with protection against DMS modification at G1494.

Additionally, it is speculated that m1A1408 methylation will fundamentally affect the formation of the A1408·A1493 base pair pocket, which is essential for aminoglycoside binding. However, a dynamic conformational change in RNA structure might impair a number of ribosomal innate functions, including decoding, aminoacyl transfer, and translocation. Actually, A1493 participates in codon-anticodon recognition during the tRNA selection step and involves a conformational change from a “tucked-in” form to a “flipped-out” form (28, 31). Although the effect of m1A1408 methylation on the innate rRNA function remains uncertain, it seems unlikely that m1A1408 methylation would be a serious disadvantage for bacterial proliferation, because there is no significant difference in the doubling times between NpmA-producing E. coli and wild-type E. coli strains under culture conditions with both rich and minimal medium compositions (data not shown). A growth competition assay may be required to elucidate the accurate biological cost induced by m1A1408 methylation in bacteria.

The methylation reaction by an innate C1407 16S rRNA methyltransferase, YebU, of E. coli is specific for the 30S subunits and not for the naked 16S rRNA molecule (1). Docking of YebU onto the 30S subunit revealed several contacts between the methyltransferase domain of YebU and ribosomal protein S12 as well as 16S rRNA (21). Hallberg et al. concluded that interactions of YebU with ribosomal protein would explain the substrate specificity seen in YebU (21). Obviously, the accessibility of YebU to the 30S subunit would be supported by the fact that the C1407 position is exposed in the 30S subunit as well as in the 16S rRNA. As expected, the substrate specificity of NpmA is similar to that of YebU (Fig. (Fig.3B),3B), and the explanation for this specificity might partially be the same reason suggested for YebU (Fig. 7A and B). A similar substrate specificity was also observed in a part of the aminoglycoside-resistant G1405 16S rRNA methyltransferase group (27). Methylation at an exposed position such as A1408 would occur in the late stage, during the assembly of the 30S subunit.

The G+C content of A1408 16S rRNA methyltransferase genes from aminoglycoside-producing actinomycetes is greater than 70%, whereas that of npmA is 34%. This discrepancy would make it unlikely that the origin of npmA is aminoglycoside producers with high G+C contents. A similar discrepancy was also observed in the case of the G1405 16S rRNA methyltransferases of actinomycetes and pathogenic bacteria. Liou et al. indicated that aminoglycoside producers with low G+C contents, such as Bacillus circulans, which naturally produces butirosin, might be the candidate sources of plasmid-mediated 16S rRNA methyltransferases (27). Although questions remain as to the presence of a 16S rRNA methyltransferase that confers aminoglycoside resistance in the genus Bacillus, the gene products of a putative ABC transporter substrate binding protein (orf7) and a mobilization protein (orf8) located at the 3′ end of npmA certainly have relatively low levels of identity to those of Bacillus spp. The detailed characterization of 16S rRNA methyltransferases in aminoglycoside-producing Bacillus spp. demonstrating low G+C contents might provide clues to the identification of the origin of plasmid-mediated 16S rRNA methyltransferases, including npmA.

In conclusion, to our knowledge this is the first time that a novel plasmid-mediated m1A1408 16S rRNA methyltransferase, NpmA, was identified in a panaminoglycoside-resistant E. coli clinical isolate. Indeed, methylation at A964 (pactamycin resistance) (2), G1405 (kanamycin-gentamicin resistance), and A1408 (kanamycin-apramycin resistance) and the loss of methylation at G527 (streptomycin resistance) (33), C1409 (capreomycin resistance) (23), and A1518-A1519 (kasugamycin resistance) (45) have been reported so far to be mechanisms of resistance to 30S subunit-targeting drugs in bacteria. However, these mechanisms have not been fully understood, especially in pathogenic bacteria that tend to be continuously or intermittently exposed to various aminoglycosides in both clinical and livestock farming environments. Further study is warranted to clarify the molecular mechanisms underlying the panaminoglycoside resistance that has been acquired by pathogenic bacteria.

Acknowledgments

We are grateful to Kumiko Kai and Yoshie Taki for their technical assistance.

This study was supported by the Ministry of Health, Labor, and Welfare of Japan (grant H18-Shinkou-11). The research activity of J. Wachino was supported by a scholarship for young scientists provided by the Japan Society for the Promotion of Science.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 September 2007.

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