Abstract
Tetracycline uptake into inverted membrane vesicles from Tn10-bearing Escherichia coli cells required divalent cations. The degree of the stimulation of tetracycline uptake by various divalent cations showed the following decreasing order: Co2+ greater than Mn2+ greater than Mg2+ greater than Cd2+ greater than Ca2+. This order is consistent with the increasing order of the dissociation constants for metal chelate complexes of tetracycline. The Hill constants for the tetracycline uptake rate with various divalent cation concentrations were one. These observations strongly suggested that a 1:1 complex of tetracycline and a divalent cation was transported by a tetracycline resistance protein. This notion was confirmed by our observations that 60Co2+ was actively taken up with tetracycline by the membrane vesicles prepared from resistant cells. In the absence of tetracycline, no uptake of 60Co2+ was observed. It is clear that the 60Co2+ uptake was mediated by the tetracycline resistance protein, because the membrane vesicles from tetracycline-sensitive cells did not show the uptake of 60Co2+ and tetracycline. The 60Co2+ uptake was inhibited in the presence of other divalent cations, without any significant effect on tetracycline uptake, indicating that these cations are also transported with tetracycline by the tetracycline resistance protein.
Highlights
The e°Co2+ uptake was inhibited in the presence of other divalent cations, without any significant effect on tetracycline uptake, indicating that these cations are transported with tetracycline by the tetracycline resistance protein
A wide variety of divalent cations including Mg2+was examined as to their effects on tetracycline uptake into inverted membrane vesicles prepared from tetracycline-resistant cells (Fig. 1)
The experiments described in the present paper showed that a divalent cation was transported with tetracycline by the TnlO tetracycline resistance protein, probably as a metalchelate complex
Summary
Cells were harvested by centrifugation at 4 “C, washed once with 50 mM MOPS-KOH buffer (DH 6.6) containing 10. The supernatant was centrifuged at 43,000 rpm for 1 h and the vesicles precipitated were washed once with 50 mM MOPS-KOH buffer ~1 of the membrane vesicle suspension (3.5 mg protein/ml) and 0.5 rl of 250 mM NADH were mixed, followed by incubation at 30 “C for 1 min. The tetracycline uptake was initiated by mixing the suspension with 40 ~1 of 50 mM MOPS-KOH buffer (pH 7.5) containing the divalent cations (CoC12, MnC12, MgSO,, CdC12, or Ca&) and 10 PM [3H]tetracycline (266,400 dpm/nmol). Divalent Cation Transport by the Tetracycline Resistance Protein twice with 2 ml of the same buffer, and the radioactivity was measured with a liquid scintillation counter
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