Enhanced Membrane Permeability in E. coli Induced by Extracellular Adenosine Triphosphate

Abstract

It is well known that the addition of adenosine triphosphate (ATP) to the extracellular suspension of a variety of cell types increases the relative permeability of the cell membrane to a number of molecular species. We have previously demonstrated the use of time-resolved second-harmonic laser scattering (SHLS) and bright-field transmission microscopy (TM) as complementary real-time probes of molecular uptake in living cells (1,2) which are capable of revealing membrane-specific transport kinetics. Herein, we employ SHLS and TM to quantify the ATP-induced enhancement of membrane permeability in living Gram-negative bacteria. Specifically, we characterize the variation of the uptake rate of the cationic triphenyl methane dye malachite green (MG) into E. coli as a function of the concentration of ATP added to the extracellular suspension. As expected, due to the presence of the outer membrane (OM) porin channels, no change in the OM transport kinetics is observed. Conversely, transport across the cytoplasmic membrane (CM) is shown to increase by nearly an order of magnitude. Additionally, the presence of ATP results in an increase of MG adsorption onto the CM, suggesting that the permeability enhancement likely arises from a potential differential across the membrane. To gain mechanistic insight into the observed enhancement, we conduct control experiments using liposomes generated from lipids isolated from E. coli, including liposomes 1) composed solely of lipids, 2) with internalized ATP, 3) reconstituted with nucleoside transporters, and/or 4) reconstituted with ATP binding cassette cation transporters (and associated internalized cations). Subsequently, using model systems of gradually increasing complexity, the effectiveness of each membrane component on the observed permeability enhancement is compared.1. Zeng, J., et al., (2013) Biophys. J., 104:139-145.2. Wilhelm, M.J., et al., (2014) Chem. Phys. Lett., 605-606:158-163.