Determination of Intrinsic Bacterial Surface Acidity Constants using a Donnan Shell Model and a Continuous p Ka Distribution Method

Abstract

Intrinsic acidity constants (p K a int) for Bacillus subtilis (Gram+) and Escherichia coli (Gram−) cells were calculated from potentiometric titration data at different salt concentrations. Master curves were generated by replotting charge excess data as a function of pH S (pH at the location of surface reactive sites) where pH S was determined as a function of Donnan potential, Ψ DON. This potential decreased in magnitude with increasing ionic strength, from −48.5±0.2 to −3.5±0.0 mV for B. subtilis and −47.9±0.3 to −3.5±0.0 mV for E. coli at 0.01 and 0.5 M K +, respectively, indicating an efficient surface charge neutralization by counterions. A fully optimized continuo us (FOCUS) p K a distribution method revealed four binding sites on B. subtilis and E. coli surfaces from the master curves with p K a int values of 3.59±0.38, 4.33±0.57, 5.94±0.66, and 8.64±0.57 for B. subtilis and 3.73±0.44, 4.85±0.71, 6.56±0.64, and 8.79±0.62 for E. coli. These were assigned to functional groups according to reported p K a ranges of 2.0–6.0 (carboxylic acid), 3.2–3.5 (phosphodiesters), 5.6–7.2 (phosphoric acid), and 9.0–11.0 (amine groups). Average points of zero salt effect (pH pzse) for B. subtilis experiments were 6.63±0.21 and 6.42±0.08 as a function of pH bulk and pH S, respectively. Under the same criteria, E. coli calculations yielded 5.73±0.23 and 5.45±0.05. An understanding of metal and proton reactivity on bacterial cell surfaces can be addressed quantitatively through the use of electrostatic and chemical equilibrium modeling techniques proposed in this study. The results are consistent with those of electrical force microscopy studies used to document the intrinsic electrochemical heterogeneity of bacterial cell surfaces.