Quantitative determination of ion distributions in bacterial lipopolysaccharide membranes by grazing-incidence X-ray fluorescence

Abstract

A model of the outer membrane of Gram-negative bacteria was created by the deposition of a monolayer of purified rough mutant lipopolysaccharides at an air/water interface. The density profiles of monovalent (K þ ) and divalent (Ca 2þ ) cations normal to the lipopolysaccharides (LPS) monolayers were investigated using grazingincidence X-ray fluorescence. In the absence of Ca 2þ , a K þ concentration peak was found in the negatively charged LPS headgroup region. With the addition of CaCl 2 , Ca 2þ ions almost completely displaced K þ ions from the headgroup region. By integrating the experimentally reconstructed excess ion density profiles, we obtained an accurate measurement of the effective charge density of LPS monolayers. The experimental findings were compared to the results of Monte Carlo simulations based on a coarse-grained minimal model of LPS molecules and showed excellent agreement.monolayer | Monte Carlo simulation | electrostatics | biological interface B iological surfaces expose a variety of charged macromolecules that interact with various sorts of ions under physiological conditions. However, despite the crucial role of charged macromolecules in modulating the interaction between cells and their surrounding environments, the quantitative understanding of electrostatics at such soft, complex interfaces still remains a general scientific challenge. For example, the outer membrane surface of Gram-negative bacteria is mainly composed of lipopolysaccharides (LPSs) (1), whose negatively charged saccharide head groups stabilize the structural integrity of bacteria and protect bacteria against their environment. Several in vivo studies (2, 3) demonstrated that bacteria increase their resistance against cationic antimicrobial peptides (e.g., protamine) in the presence of divalent cations (Ca 2þ , Mg 2þ ). Therefore, for the development of peptide-based antibiotics (4), it is important to understand the molecular mode of action of antimicrobial peptides.A number of theoretical models for the interactions of LPS molecules with divalent cations (5-7), suggested that the ions would bind to the charged 2-keto-3-deoxyoctonoic acid (KDO) groups (the "inner core") thereby stabilizing the membrane. Recently, we measured grazing-incidence X-ray scattering from a monolayer of rough mutant LPS from Salmonella enterica sv. Minnesota at an air/water interface and demonstrated the Ca 2þ -induced increase in the electron density near the inner core (8). These observations were supported by the results of Monte Carlo (MC) simulations of a coarse-grained model (8). A further challenge would be to extend such a strategy to wild-type LPSs that possess polydisperse, specific O-polysaccharide chains (O-side chains). Pink et al. (9) carried out MC simulations of a minimal model of the more complex, wild-type LPSs from Pseudomonas aeruginosa (PAO1) and concluded that divalent cations would induce a collapse of the negatively charged O-sidechains toward the membrane surface. Because it is difficult in practice to deposit LPSs wit...

Interaction of protamine with gram‐negative bacteria membranes: possible alternative mechanisms of internalization in Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa

“…When Ca 2+ ions are present, the Ptm is essentially unbound and exhibits a distribution between~9 and~16 nm. This result is in accord with predictions [36] relating to the MIC of P. aeruginosa and recently confirmed [53][54][55][56] employing grazing-incidence X-ray scattering to investigate monolayers of LPS taken from the outer membranes of Salmonella enterica and P. aeruginosa. Figure 4A for a (65:35) membrane and the Na + (black, f) and Ptm (black, g) distributions for a (75:25) membrane.…”

“…The intent of representing some of the ions by a Debye screening length, -κ 1 , was so that we could carry out our simulations using a screened potential to avoid having to model infinite-range interactions using standard techniques, such as the Ewald approach [39]. We have successfully used and discussed this approach elsewhere [36,[53][54][55][56], where the value of κ = 0.5 nm 1 was also used. Briefly, the criterion for choosing κ is related to maximizing the region of space in which the ions are represented as discrete charges and not described by the continuum (meanfield) theory, while keeping the simulation time within an acceptable value.…”

Atom-scale depth localization of biologically important chemical elements in molecular layers

“…1, we safely neglected the depth dependence of the fluorescence attenuation, because in the present study the target elements are confined in nanometric layers. For a given incident angle θ, Φ(θ, z) follows from the interfacial scattering length density (SLD) profile and can be computed from a suitable slab model representation of the SLD via the phase-correct summation of all reflected and transmitted partial waves (23), as has been described previously (14). As shown in the inset of Fig.…”

“…The angle-dependent fluorescence intensity thus contains information on the interfacial element distribution (7). This principle has been exploited for the study of interfacial phenomena involving solid, liquid, and gas phases (8)(9)(10)(11)(12)(13)(14)(15)(16). High resolution perpendicular to the interface is implied by SWXF experiments in Bragg reflection configuration, in which case planar, nanometric multilayers are used to create strongly modulated standing waves above the terminal surface close to the Bragg condition (9,10).…”

Use of Proteomic Analysis To Elucidate the Role of Calcium in Acetone-Butanol-Ethanol Fermentation by Clostridium beijerinckii NCIMB 8052

“…The binding of Ca 2ϩ and Mg 2ϩ to the negatively charged surface lipopolysaccharides (LPS) in Gram-negative bacteria generates ionic cross bridges, which exert a stabilizing effect on the outer cell membrane (41)(42)(43)(44). Notwithstanding variations in the cell wall/membrane makeup and structure between Gram-positive and Gram-negative bacteria (45), we considered whether the formation of Ca 2ϩ -mediated cross bridges in the Gram-positive bacterium C. beijerinckii might partly contribute to the effects of Ca 2ϩ on ABE fermentation.…”