Molecular efflux is a process by which a bacterium actively removes unwanted compounds from its interior in order to preserve viability. This mechanism is used by bacteria to stave off active antimicrobial attacks or prevent overindulging on otherwise helpful nutrients. Herein, we quantify the influence of efflux on the molecular uptake kinetics of an exogenous molecule - the bacterial uptake of the hydrophobic ion, malachite green (MG), by a colloidal suspension of P. Aeruginosa. MG is a quaternary ammonium cation and is known to be weakly antibiotic towards Gram-negative strains. Above a critical concentration, MG ions should be recognized by the RND efflux pumps of P. Aeruginosa and removed from the cell. As a test of this hypothesis, we used time-resolved second harmonic light scattering (SHS) to monitor the concentration dependent interactions of MG ions with the membranes of P. Aeruginosa. Indeed, above ca. 10 mM MG, the MG ions were recognized by the efflux pumps and were being removed from the cells. The effect of a second effluxable compound, hexane at the saturation concentration in water which is expected to reduce the efficacy of the efflux pumps, was also examined. Indeed, when hexane was present, the deduced MG transport rate was observed to increased closer to the non-efflux value. Importantly, we note that when the effect of efflux on transport is included in the kinetic model for quantitative description of the bacterial membrane transport, we were able to 1) accurately account for the MG concentration dependent changes in the membrane transport rate and 2) deduce the rate of efflux of MG. Finally, application of competitive efflux for deducing the propensity for efflux and determining the efflux rate of the non-SHS active molecule will be presented.

Influence of efflux on molecular transport across bacterial membranes

ACS Spring 2022

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