A novel glycylcycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, is neither transported nor recognized by the transposon Tn10-encoded metal-tetracycline/H+ antiporter

Abstract

A novel tetracycline derivative, DMG-DMDOT [9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline] , is one of the glycylcyclines which have a broad antibacterial spectrum, including many tetracyclineresistant bacteria (R.T. Testa, P.J. Petersen, N.V. Jacobus, P.-E. Sum, V.J. Lee, and F.P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). The mechanism by which DMG-DMDOT overcomes efflux-based tetracycline resistance was investigated. Tetracycline-resistant Escherichia coli cells carrying an R plasmid encoding the tet(B) gene, which encodes the typical tetracycline efflux pump [TetA(B)] of gram-negative bacteria, were as susceptible to DMG-DMDOT as was the tetracycline-susceptible host. When mid-log-phase cells carrying the tet(B) gene were incubated with a subbactericidal concentration of DMG-DMDOT (0.5 micrograms/ml) for 2 h, a significant amount of the TetA(B) protein was detected in the cell membrane by Western blotting (immunoblotting) with an anti-carboxyl-terminal antibody, similar to the case in which tetracycline was used as the inducer, indicating that the tet repressor, TetR, can recognize DMG-DMDOT as an efficient inducer. Everted membrane vesicles prepared from cells producing the TetA(B) protein showed absolutely no transport activity for DMG-DMDOT. Furthermore, the presence of excess DMG-DMDOT had no effect on the tetracycline transport activity of the everted vesicles, indicating that DMG-DMDOT is not recognized as a substrate by the TetA(B) protein.