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OmpA, a predominant outer membrane (OM) protein in Escherichia coli, affects virulence, adhesion, and bacterial OM integrity. However, despite more than 50 y of research, the molecular basis for the role of OmpA has remained elusive. In this study, we demonstrate that OmpA organizes the OM protein lattice and mechanically connects it to the cell wall (CW). Using gene fusions, atomic force microscopy, simulations, and microfluidics, we show that the β-barrel domain of OmpA is critical for maintaining the permeability barrier, but both the β-barrel and CW-binding domains are necessary to enhance the cell envelope's strength. OmpA integrates the compressive properties of the OM protein lattice with the tensile strength of the CW, forming a mechanically robust composite that increases overall integrity. This coupling likely underpins the ability of the entire envelope to function as a cohesive, resilient structure, critical for the survival of bacteria. [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly- L -lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane. [ABSTRACT FROM AUTHOR]

Recent advances in biomolecular design require accurate measurements performed in native or near-native environments in real time. Atomic force microscopy (AFM) is a powerful tool to observe the dynamics of biologically relevant processes at aqueous interfaces with high spatial resolution. Here, we describe imaging protocols to characterize the effects of peptide materials on phospholipid membranes in solution by AFM. These protocols can be used to determine the mechanism and kinetics of membrane-associated activities at the nanoscale.