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Figure 9 | BMC Microbiology

Figure 9

From: Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli

Figure 9

The electrogenicity of MdtM-catalysed Na + /H + and K + /H + antiport. The electrogenicity of MdtM-catalysed Na+/H+ and K+/H+ antiport at alkaline pH was probed by Oxonol V fluorometry of inverted vesicles generated from E. coli TO114 cells transformed with pMdtM (A, C & E) or, as a negative control, pD22A (B & D). Inverted vesicles isolated from BW25113 cells were used as a positive control (F). Respiration-dependent formation of Δψ was initiated by addition of lactate at the time indicated. Once steady-state Δψ was achieved, antiport was initiated by addition of 100 mM Na+ gluconate (A & B) or 100 mM K+ gluconate (C & D) as indicated. Vesicles were depolarised by addition of CCCP or valinomycin in the presence of K+ as indicated. Fluorescence measurements on TO114 inverted vesicles were conducted at either pH 9.0 (for detection of K+/H+ antiport; panels C & D) or pH 9.25 (for detection of Na+/H+ antiport; panels A & B), whereas positive control measurements using vesicles derived from BW25113 cells were done at pH 8.5 to ensure detection of the activity of the electrogenic antiporter, NhaA (panel F). The Oxonol V fluorescence is presented as a percentage of the initial fluorescence prior to establishment of the steady-state Δψ. The traces shown are representative of experiments performed in triplicate on two separate preparations of inverted vesicles.

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