Single-cell analysis of lipopolysaccharide-mediated heterogeneity in Escherichia coli adhesion and mechanics.

Bacterial populations that exhibit phenotypic subgroups of cells with different biophysical properties are better adapted to colonize diverse surfaces, thereby facilitating survival and dissemination in varied environments. In this study, we applied atomic force microscopy (AFM) to investigate lipopolysaccharide (LPS)-mediated heterogeneity in the adhesion and mechanics of individual Escherichia coli ATCC 25922 cells within a clonal population. Our results, based on both single-cell and population level analyses, revealed that partial LPS removal by ethylenediaminetetraacetic acid (EDTA) substantially altered the bacterial cell surface, rendering it smoother and featureless. EDTA-induced disorganization of the outer membrane not only diminished both adhesion forces and cell elasticity but also markedly reduced the structural diversity of the cell envelope, thereby decreasing cell-to-cell heterogeneity. Notably, the population no longer exhibited cells with a strongly adherent and stiff phenotype. Our findings indicate that the structural and chemical diversity of the outer membrane, primarily conferred by LPS, is a key determinant of phenotypic heterogeneity, with important implications for bacterial adhesion, environmental adaptation, and responses to antimicrobial stressors. Overall, this study emphasizes the essential role of LPS in modulating bacterial surface interactions and mechanical behavior, and demonstrates the utility of AFM-based force spectroscopy with colloidal probes as a quantitative platform for analyzing single-cell variability.