On the other hand, bacteria have acquired various resistance mech

On the other hand, bacteria have acquired various resistance mechanisms to cope with aminoglycosides. Plasmid-mediated 16S rRNA methyltransferases (MTases), which confer a high level of resistance VX-809 to various aminoglycosides, especially to those containing 4,6-disubstituted 2-deoxystreptamine (2-DOS), have been widely distributed among pathogenic microorganisms belonging to the family Enterobacteriaceae and glucose nonfermentative Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii isolated from clinical and livestock-farming environments (Chen et al., 2007; Yamane et al., 2007). RmtA (Yokoyama et al., 2003), RmtB (Doi et al., 2004), RmtC (Wachino et al., 2006), RmtD (Doi et al., 2007),

RmtE (Davis et al., 2010), ArmA (Galimand et al., 2003), and NpmA (Wachino et al., 2007) have so far been reported as plasmid-mediated 16S rRNA MTases conferring aminoglycoside resistance, but methylation sites have only been determined as G1405 for RmtB and ArmA, and A1408 for NpmA (Liou et al., 2006; Perichon et al., 2007; Wachino et al., 2007). As for RmtA, RmtC, RmtD, and RmtE, the site of methylation in the 16S rRNA has not been

described. Plasmid-mediated 16S rRNA MTases have only been found in Gram-negative pathogenic bacteria, and not in Gram-positives. Z-VAD-FMK mw It remains controversial whether or not 16S rRNA MTase as described above is functional and confers aminoglycoside resistance in Gram-positives as well as in Gram-negatives, although it was revealed previously that armA controlled under the original promoter could confer aminoglycoside resistance PD184352 (CI-1040) in Bacillus subtilis (Liou et al., 2006). Therefore, in this study, we aimed to determine exactly the residue modified by RmtC, and investigated whether RmtC can provide aminoglycoside resistance in Gram-positive pathogens. The rmtC gene

was amplified with the P1 primer (5′-GGA ATT CCATATGAA AAC CAA CGA TAA TT-3′: NdeI restriction site added), the P2 primer (5′-GCTCTAGAT TAC AAT CTC GAT ACG ATA-3′: XbaI restriction site added), and the pET-His-rmtC vector (Wachino et al., 2006) as a DNA template. The amplified fragments were digested with endonucleases, cloned into pCold-II vector (Takara), and introduced into Escherichia coli BL21(DE3)pLysS. Cells were grown until A600 nm 0.5 at 37 °C in Luria–Bertani medium. After the addition of isopropyl-β-d-1-thiogalactopyranoside (0.5 mM), cells were grown at 15 °C for 24 h, and disrupted with a French press. Protein purification using nickel-nitrilotriacetic acid was performed according to the manufacturer’s instructions (GE Healthcare). The eluted recombinant protein was loaded on size-exclusion chromatography column Superdex™ 200 10/300GL (GE Healthcare), and eluted with 20 mM phosphate buffer (pH 7.4) containing 0.5 M NaCl and 1 mM dithiothreitol. Finally, the purified protein (His6-RmtC) was concentrated using an Amicon Ultra-15 Centricon (Millipore).

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