Efflux of antibiotics by Escherichia coli AG100 is performed by a variety of efflux pumps, ensuring survival of the bacterium in widely diverse media. At pH 5, efflux is independent of metabolic energy during the period of time the assay is conducted; at pH 8 it is totally dependent upon metabolic energy. Because calcium ions (Ca(2+)) are important for membrane transport channels and the activity of ATPases that provide energy functions, the role of Ca(2+) in the extrusion of an efflux pump substrate under conditions that challenge the bacterium was investigated. Real-time accumulation and efflux of ethidium bromide (EtBr) by E. coli K-12 AG100 strain [argE3 thi-1 rpsL xyl mtl Δ(gal-uvrB) supE44] was determined by a semi-automated fluorometric method in the presence and absence of Ca(2+) and agents that are known to inhibit access of calcium to enzymes that provide energy. Chlorpromazine (CPZ), an inhibitor of calcium binding to proteins (calcium-dependent enzymes), and ethylene diamine tetra-acetic acid (EDTA), a chelator of Ca(2+), increased accumulation and efflux of EtBr at pH 8 but not at pH 5. Ca(2+) reverses these effects when the assay is conducted at pH 8. In conclusion, the activity of the efflux pump system of E. coli is dependent upon metabolic energy at pH 8. Because at pH 8 hydrolysis of ATP is favoured and contributes protons for activation of the AcrAB-TolC efflux pump, CPZ is suspected of having its effects on accumulation/efflux of EtBr by indirectly affecting ATPase activity that is dependent upon Ca(2+).
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