The bacterial membrane has been suggested as a good target for future antibiotics, so it is important to understand how naturally occurring antibiotics like antimicrobial peptides (AMPs) disrupt those membranes. The interaction of the AMP magainin 2 (MAG2) with the bacterial cell membrane has been well characterized using supported lipid substrates, unilamellar vesicles, and spheroplasts created from bacterial cells. However, to fully understand how MAG2 kills bacteria, we must consider its effect on the outer membrane found in Gram-negative bacteria. Here, we use atomic force microscopy (AFM) to directly investigate MAG2 interaction with the outer membrane of Escherichia coli and characterize the biophysical consequences of MAG2 treatment under native conditions. While propidium iodide penetration indicates that MAG2 permeabilizes cells within seconds, a corresponding decrease in cellular turgor pressure is not observed until minutes after MAG2 application, suggesting that cellular homeostasis machinery may be responsible for helping the cell maintain turgor pressure despite a loss of membrane integrity. AFM imaging and force measurement modes applied in tandem reveal that the outer membrane becomes pitted, more flexible, and more adhesive after MAG2 treatment. MAG2 appears to have a highly disruptive effect on the outer membrane, extending the known mechanism of MAG2 to the Gram-negative outer membrane.

Programmed À1 frameshifting (À1 PRF) is an important biological process for the modification of gene expression enabled by the presence of RNA secondary and tertiary structures. However, the mechanism for this process is still under active study. Here, we investigate the role of mechanical force in À1 PRF by characterizing mechanically induced folding and unfolding of RNA pseudoknots using an enhanced atomic force microscopy (AFM) based singlemolecule force spectroscopy (SMFS) assay featuring $10 ms resolution. The pioneering SMFS study of RNA psuedoknots associated with À1 PRF used a custom-built optical trap. Unexpectedly, this study indicated PRF efficiency was correlated with the presence of alternative folding pathways (rather than with average unfolding force), indicating a complex role of RNA psuedoknots in À1 PRF involving folding dynamics. Here, we found a new folding intermediate in an RNA pseudoknot associated with the sugarcane yellow leaf virus (ScYLV) that was not observed in the original optical-trapping based assay. We speculate that the shorter linkers and stiffer force probe in our AFM assay (relative to an optical trap) are the primary reasons for this enhanced state resolution. Our initial measurements of contour length and folding kinetics indicate this folding intermediate is an RNA hairpin that is part of the overall pseudoknot structure. We observed this intermediate every time the pseudoknot folds, indicating that this intermediate is obligate for folding. Overall, our results indicate that the folding dynamics of RNA pseudoknots are significantly more complex than previously observed. Because of the role of the folding dynamics of RNA pseudoknots in À1 PRF, we expect these new insights into RNA pseudoknot folding dynamics will provide a deeper understanding into the mechanisms of À1 PRF.

Bacterial biofilms have long been recognized as a source of persistent infections and industrial contamination with their intransigence generally attributed to their protective layer of extracellular polymeric substances (EPS). EPS, consisting of secreted nucleic acids, proteins, and polysaccharides, make it difficult to fully eliminate biofilms by conventional chemical or physical means. Since most bacteria are capable of forming biofilms, understanding how biofilms respond to new antibiotic compounds and components of the immune system has important ramifications. Antimicrobial peptides (AMPs) are both potential novel antibiotic compounds and part of the immune response in many different organisms. Here, we use atomic force microscopy to investigate the biomechanical changes that occur in individual cells when a biofilm is exposed to the AMP magainin 2 (MAG2), which acts by permeabilizing bacterial membranes. While MAG2 is able to prevent biofilm initiation, cells in an established biofilm can withstand exposure to high concentrations of MAG2. Treated cells in the biofilm are classified into two distinct populations after treatment: one population of cells is indistinguishable from untreated cells, maintaining cellular turgor pressure and a smooth outer surface, and the second population of cells are softer than untreated cells and have a rough outer surface after treatment. Notably, the latter population is similar to planktonic cells treated with MAG2. The EPS likely reduces the local MAG2 concentration around the stiffer cells since once the EPS was enzymatically removed, all cells became softer and had rough outer surfaces. Thus, while MAG2 appears to have the same mechanism of action in biofilm cells as in planktonic ones, MAG2 cannot eradicate a biofilm unless coupled with the removal of the EPS.

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over a certain threshold. The ion leakage caused by daptomycin is transient. It occurs only when daptomycin binds the membrane for the first time; afterwards they cease to induce ion leakage. The ion leakage effect of daptomycin is not transferrable from one membrane to another. The membrane binding of daptomycin is reduced in the gel phase versus the fluid phase. Cholesterol also weakens the membrane binding of daptomycin. The combination of membrane concentration threshold and differential binding is significant. This could be a reason why daptomycin discriminates between eukaryotic and prokaryotic cell membranes.