Characterization of the active transport of chlorotetracycline in Staphylococcus aureus by a florescence technique

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Abstract
The antibiotic chlorotetracycline (CTC) is used as a fluorescent chelate probe to investigate its active transport in respiring Staphylococcus aureus cells. CTC chelation to magnesium or calcium leads to fluorescence enhancement. This enhancement is further increased when the polarity of its environment is decreased, as occurs when the complex moves from an aqueous environment into a membrane. Upon addition of CTC to a dispersion of S. aureus cells, a time dependent fluorescence enhancement is detected which is a monitor of the transport of the CTC‐divalent cation complex into the membrane. This uptake has been shown to be energy dependent and exhibits saturation kinetics with an apparent Km of 107 ± 20 μM by the same technique. The initial rates of antibiotic uptake are shown to have a pH optimum between 5.5 and 6.5. The effects of exogenously added EDTA and paramagnetic Mn2+ indicate that the CTC‐divalent cation complex is transported to the inside of the membrane. Exogenously added magnesium inhibits the accumulation process. This implies that the membrane CTC binding site involves a divalent cation sequestered away from the surface of the membrane, and only free CTC is bound to that site. The uptake of CTC is also temperature dependent with a maximal rate at 40°. Arrhenius plots of the initial fluorescence enhancement rates are found to be biphasic with a 27° transition temperature. The break in the plots presumably reflects an order‐disorder transition involving the fatty acids of the cell membrane. Thus, transport of the CTC involves movement through the fatty acid region of the membrane. This movement is facilitated by the more fluid state of the membrane above the transition temperature.