The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg2+ and Ca2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.

The Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo-like conditions.

Alkoxyamines are heat-labile molecules, widely used as in-situ source of nitroxides in polymer and materials sciences. Here we show that the one-electron oxidation of an alkoxyamine leads to a cation radical intermediate that even at room temperature rapidly fragments releasing a nitroxide and carbocation. Digital simulations of experimental voltammetry and current-time transients suggest the unimolecular decomposition which yields the "unmasked" nitroxide (TEMPO) is exceedingly rapid and irreversible. High-level quantum computations indicate the collapse of the alkoxyamine cation radical is likely to yield a neutral nitroxide radical and a secondary phenylethyl cation. However, this fragmentation is predicted to be slow and energetically very unfavorable. To attain qualitative agreement between the experimental kinetics and computational modelling for this fragmentation step the explicit electrostatic environment within the double layer must be accounted for. Single-molecule break-junction experiments in a scanning tunneling microscope using solvent of low dielectric (STM-BJ technique) corroborate the role played by electrostatics forces on the lysis of the alkoxyamine CON bond. This work highlights the electrostatic aspects played by charged species in a chemical step that follows an electrochemical reaction, defines the magnitude of this catalytic effect by looking at an independent electrical technique in non-electrolyte systems (STM-BJ), and suggests a redox on/off switch to guide the cleavage of alkoxyamines at an electrified interface.

The membrane interactions of the antimicrobial peptide aurein 1.2 were studied using a range of biophysical techniques to determine the location and the mechanism of action in DMPC (dimyristoylphosphatidylcholine) and DMPC/DMPG (dimyristoylphosphatidylglycerol) model membranes that mimic characteristics of eukaryotic and prokaryotic membranes, respectively. Neutron reflectometry and solid-state NMR revealed subtle changes in membrane structure caused by the peptide. Quartz crystal microbalance with dissipation, vesicle dye leakage and atomic force microscopy measurements were used to investigate the global mode of peptide interaction. Aurein 1.2 displayed an enhanced interaction with the anionic DMPC/DMPG membrane while exhibiting primarily a surface interaction with both types of model membranes, which led to bilayer disruption and membrane lysis. The antimicrobial peptide interaction is consistent with the carpet mechanism for aurein 1.2 with discrete structural changes depending on the type of phospholipid membrane.

Predicting or manipulating charge-transfer at semiconductor interfaces, from molecular electronics to energy conversion, relies on knowledge generated from a kinetic analysis of the electrode process, as provided by cyclic voltammetry. Scientists and engineers encountering non-ideal shapes and positions in voltammograms are inclined to reject these as flaws. Here we show that non-idealities of redox probes confined at silicon electrodes, namely full width at half maximum <90.6 mV and anti-thermodynamic inverted peak positions, can be reproduced and are not flawed data. These are the manifestation of electrostatic interactions between dynamic molecular charges and the semiconductor’s space-charge barrier. We highlight the interplay between dynamic charges and semiconductor by developing a model to decouple effects on barrier from changes to activities of surface-bound molecules. These findings have immediate general implications for a correct kinetic analysis of charge-transfer at semiconductors as well as aiding the study of electrostatics on chemical reactivity.

The outer membrane (OM) of gram-negative bacteria is an unusual asymmetric bilayer with an external monolayer of lipopolysaccharide (LPS) and an inner layer of phospholipids. The LPS layer is rigid and stabilized by divalent cation cross-links between phosphate groups on the core oligosaccharide regions. This means that the OM is robust and highly impermeable to toxins and antibiotics. During their biogenesis, OM proteins (OMPs), which function as transporters and receptors, must integrate into this ordered monolayer while preserving its impermeability. Here we reveal the specific interactions between the trimeric porins of Enterobacteriaceae and LPS. Isolated porins form complexes with variable numbers of LPS molecules, which are stabilized by calcium ions. In earlier studies, two high-affinity sites were predicted to contain groups of positively charged side chains. Mutation of these residues led to the loss of LPS binding and, in one site, also prevented trimerization of the porin, explaining the previously observed effect of LPS mutants on porin folding. The high-resolution X-ray crystal structure of a trimeric porin-LPS complex not only helps to explain the mutagenesis results but also reveals more complex, subtle porin-LPS interactions and a bridging calcium ion.gram-negative bacteria | lipopolysaccharide | X-ray crystal structure | porin | outer membrane

Lipopolysaccharides (LPS) make up approximately 75% of the Gram-negative bacterial outer membrane (OM) surface, but because of the complexity of the molecule, there are very few model OMs that include LPS. The LPS molecule consists of lipid A, which anchors the LPS within the OM, a core polysaccharide region, and a variable O-antigen polysaccharide chain. In this work we used RcLPS (consisting of lipid A plus the first seven sugars of the core polysaccharide) from a rough strain of Escherichia coli to form stable monolayers of LPS at the air–liquid interface. The vertical structure RcLPS monolayers were characterized using neutron and X-ray reflectometry, while the lateral structure was investigated using grazing incidence X-ray diffraction and Brewster angle microscopy. It was found that RcLPS monolayers at surface pressures of 20 mN m–1 and above are resolved as hydrocarbon tails, an inner headgroup, and an outer headgroup of polysaccharide with increasing solvation from tails to outer headgroups. The lateral organization of the hydrocarbon lipid chains displays an oblique hexagonal unit cell at all surface pressures, with only the chain tilt angle changing with surface pressure. This is in contrast to lipid A, which displays hexagonal or, above 20 mN m–1, distorted hexagonal packing. This work provides the first complete structural analysis of a realistic E. coli OM surface model.

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.