Evolution of membrane permeability in the life cycle of a bacterial culture

Introduction Bacterial membranes are dynamic interfaces that adapt to changing physiological and environmental conditions. Throughout the growth cycle of a bacterial culture, nutrient depletion and accumulation of metabolic byproducts impose progressively tighter environmental constraints. Bacteria respond through coordinated remodeling of the cell envelope, including changes in membrane lipid composition and protein abundance or activity. However, how these changes affect molecular transport across individual barriers of the Gram-negative cell envelope throughout the bacterial life cycle, and how they contribute to bacterial adaptation to antimicrobial stress, have yet to be characterized. Methods We employed time-resolved second-harmonic light scattering (SHS) to quantify the transport kinetics of malachite green (MG), a cationic antimicrobial compound, across the outer membrane (OM), peptidoglycan mesh (PM), and cytoplasmic membrane (CM) of living Escherichia coli throughout the lag, exponential, stationary, and death phases. Extracellular pH was monitored as a temporal indicator of environmental change, and complementary antimicrobial susceptibility assays were performed to examine phase-dependent responses to MG exposure. Results MG transport was highest in the lag and early exponential phases and declined progressively thereafter: transport across the CM decreased by more than three orders of magnitude from the lag phase to the death phase, whereas OM transport decreased by approximately 60-fold. In contrast, MG transport kinetics across the PM remained relatively stable across growth phases. Adsorption free energies at the membrane interfaces varied by less than 1 kcal/mol, suggesting that the transport changes were not primarily associated with altered interfacial adsorption energetics. Antimicrobial susceptibility, as measured by cell viability, decreased progressively from the lag phase to the death phase. Discussion The pronounced and barrier-specific changes in MG transport kinetics demonstrate that molecular transport across the bacterial envelope is a dynamic property that changes substantially throughout the bacterial life cycle. The strong suppression of OM and CM transport from the lag to death phases is consistent with growth-phase-dependent remodeling of OM porins and CM lipid composition reported in previous studies. The correlation observed between reduced MG transport and susceptibility to MG further suggests that, for antimicrobial compounds whose activity requires cellular entry, efficacy may decrease as transport rate decreases.