The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

No abstract

MagA to lipid bilayers with different charge and fluidity properties was studied with dual polarization interferometry, an optical technique capable of measuring real-time simultaneous changes in mass and birefringence (an optical parameter representing bilayer order). The dependence of birefringence vs. mass were categorised into several discrete mechanistic profiles and new multiple-state kinetic models were developed and fitted to the experimental data, with a three-state model with lateral bilayer expansion providing distinctly superior fits to simpler model types. Overall this is the first study to quantitatively analyse complex peptide-membrane binding data in terms of specific states of membrane disruption by an antimicrobial peptide.

No abstract