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We cloned the MLSB resistance determinant by PCR from a clinical isolate of Enterococcus faecalis 373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of E. faecalis 373, we analyzed the cloned gene, designated ermAMR, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.

To study the role of mRNA termination in the regulation of ermK, we introduced mismatches into terminators by in vitro mutagenesis. In wild-type ermK, only truncated transcription products were detected in the absence of induction. In contrast, only the full-length transcript was synthesized in the terminator 1 and terminator 2 double mutants, even in the absence of erythromycin. These results indicate that the expression of ermK is primarily regulated by transcriptional attenuation rather than translational attenuation. We also tested the possible contribution of translational attenuation control to the regulation of ermK by constructing a triple mutant (terminator 1 plus terminator 2 plus the methylase Shine-Dalgarno region). A higher level of beta-galactosidase synthesis was seen in the triple mutant. Therefore, unlike with previously described attenuators, it can be concluded that both transcriptional and translational attenuation contribute to the regulation of ermK, although transcriptional attenuation plays a larger role.