Surface charge density is a critical characteristic that affects the physiology of microorganisms. A considerable portion of the cell’s metabolic energy is devoted to maintaining that surface charge, which, due to the presence of phosphates, carboxyls, and various other functional groups, tends to be primarily anionic in most environments. In general, surface charge plays a vital role in how a microorganism interacts with its local environment. Understanding a microorganism’s response to acute changes in environmental factors necessitates monitoring variations to their surface charge. Experimentally, this is a non-trivial task, in part because microorganisms tend to exist in colloidal suspension. Quantification of surface charge therefore requires the ability to selectively detect representative signals related to electric charge from a specific surface of interest.
In our study, we used the nonlinear optical method, second harmonic light scattering (SHS) to quantitatively determine the number of cations adsorbed to the anionic sites of the exterior surface of representative Gram-negative (Escherichia coli) and Gram-positive (Lactobacillus rhamnosus) bacteria. Time-resolved SHS is a surface-sensitive technique that is capable of sequentially monitoring the interactions (e.g., adsorption and transport) of non-centrosymmetric molecules with biological interfaces, including phospholipid membranes. By monitoring saturated adsorption of cations on the bacterial outer surfaces, we deduced the number of anionic surface binding sites and hence the surface charge density.
The cover art featured on the June 15 issue of Biophysical Journal depicts the surface interactions of the non-centrosymmetric cationic dye, malachite green (MG), with poly-anionic lipopolysaccharide (LPS) strands on the outer leaflet of E. coli’s outer membrane. The electrostatic interaction between the cation and the anionic bacterial surface resulted in oriented adsorption of the dye molecules on the membrane surface and produced a detectable second harmonic signal. Two photons of 800 nm (red) light are shown on the bacterial surface, which subsequently results in the scattering of a single 400 nm (violet) photon from the surface-oriented dyes. The density of adsorbed cations is encoded in the magnitude of the measured SHS signal intensity, which is known to scale as the square of the adsorbate density. For the mc4100 strain of E. coli, it was revealed that each LPS held 5.5 MG cations, suggesting that each strand contained five to six phosphate groups. Additional information about our research can be found on our group website at: https://thedaigroup.weebly.com.
- Michael J. Wilhelm, Mohammad Sharifian Gh., Tong Wu, Yujie Li, Chia-Mei Chang, Jianqiang Ma, and Hai-Lung Dai