Quantifying adsorption and transport in Gram-positive cell walls by second-harmonic light scattering

Elucidating molecule-membrane interactions within bacterial cell walls is essential for understanding proliferation of bacteria in the environment and developing effective therapeutics. Quantitative measurements of molecular adsorption and transport have been reported for individual membrane layers in Gram-negative bacteria, yet no analogous investigation exists for Gram-positive bacteria, which feature a substantially different cell-envelope architecture. In this work, we employed time-resolved second-harmonic light scattering to quantify the adsorption and transport kinetics of a small antimicrobial cation, malachite green, at each of the surface layer (SL), peptidoglycan mesh (PM), and cytoplasmic membrane (CM) within the cell walls of three Gram-positive organisms: Lysinibacillus sphaericus, which possesses a canonical crystalline S-layer; and Bacillus subtilis and Lacticaseibacillus rhamnosus, whose outermost cell-wall barriers are composed of surface-associated proteins and teichoic acids. The transport rates measured for all three bacteria reveal a consistent permeability hierarchy in which transport through the SL and PM is rapid, whereas translocation across the CM is two orders of magnitude slower, constituting the rate-limiting bottleneck for cytosolic entry. In comparing the adsorption and transport behaviors across the three Gram-positive strains studied here and previously reported Gram-negative bacteria, we find that despite profound differences in cell-envelope architecture, molecular uptake is regulated in a similar, hierarchical manner, with outer layers governing accessibility at similar rates and the lipid bilayer controlling cytosolic entry with a much slower rate. The SLs of Gram-positive bacteria behave similarly to the porin ion channels of Gram-negative bacteria in transporting hydrophobic molecular cations. Moreover, the adsorption free energies (approximately -9.5 to -12 kcal · mol^-1) determined for the various interfaces within the Gram-positive and Gram-negative cell envelopes are remarkably similar, with the sole notable distinction being the substantially higher surface charge density of the Gram-negative outer membrane relative to the Gram-positive SL.