Permeation of tetracyclines through membranes of liposomes and Escherichiacoli

Uptake of tetracycline (tc), 2-tetracyclinonitrile (CN-tc), and 9-(N,N-dime thylglycylamido)-6-demethyl-6-deoxytetracycline (DMG-DMDOT) by liposomes co ntaining Tet repressor (TetR) and by Escherichia coil cells overexpressing TetR was examined. TetR specifically binds to tetracyclines, enhances their fluorescence and thereby allows selective detection of tetracyclines that have crossed the membranes. Analysis of the diffusion of tc and DMG-DMDOT i nto liposomes yielded permeation coefficients of (2.4 +/- 0.6) x 10(-9) cm. s(-1) and (3.3 +/- 0.8) x 10(-9) cm.s(-1), respectively. Similar coefficien ts were obtained for uptake of these tetracyclines by E. coil, indicating t hat diffusion through the cytoplasmic membrane is the rate-limiting step. T he permeation coefficients translate into half-equilibration times of appro ximately 35 +/- 15 min and explain how efflux pumps can mediate resistance against tetracyclines. Furthermore, diffusion of CN-tc into liposomes was a t least 400-fold slower than that of tc, indicating that the carboxamide gr oup at position C2 is required for efficient permeation of tc through lipid membranes and thereby explaining the lack of antibiotic activity of CN-tc.