Probing Bacterial Electrosteric Interactions Using Atomic Force Microscopy

Abstract

Atomic force microscopy (AFM) was used to probe the effects of pH, ionic strength, and the presence of bacterial surface polymers on interaction forces between individual, negatively charged bacteria and silicon nitride. Bacterial surface polymers dominated interactions between bacteria and AFM silicon nitride tips. The measured forces were represented well by an electrosteric repulsion model accounting for repulsion between the tip and bacterial polymers but were much larger in magnitude and extended over longer distances (100's of nanometers) than predicted by DLVO theory. The equilibrium length ( L o ) of the polymers was allowed to vary with solution chemistry to account for intramolecular electrostatic interactions between individual polymer units. The effects of the variables pH and ionic strength on bacterial interaction forces were investigated independently. Pseudomonas putida KT2442 was studied in 1 mM MOPS buffer at pH values of 2.2, 4.75, 7.00, and 8.67. Burkholderia cepacia G4 was studied in 1 mM MOPS buffer at pH values of 2.2, 4.75, and 6.87. Then, the pH was held constant at 4.5 or 4.75, and the ionic strength was studied in 0.01, 1, or 100 mM MOPS buffer (for each microbe). For KT2442 (in 1 mM MOPS buffer), L o increased from 230 to 750 nm as pH increased from 4.75 to 8.67. For G4 (in 1 mM MOPS buffer), L o increased from 350 nm at pH 2.2 to 1040 nm at pH 7.0. Varying the ionic strength between 0.01 and 100 mM did not affect the equilibrium length of the polymers nearly as much as pH. Partially removing polysaccharides from the bacterial surfaces resulted in lower repulsive forces that decayed much more rapidly. The magnitude of the measured forces in these experiments and the equilibrium lengths predicted by the electrosteric model are comparable to other force measurements and size estimates on polymers and polysaccharides.