Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids

Abstract

Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics.

Supported Lipid Bilayers of Escherichia coli Extracted Lipids and Their Calcium Dependence

“…PLE [PE: PG: CA = 67: 23.2: 9.8 (wt/wt)] and lissamine rhodamine B-dipalmitoylphosphatidylethanolamine (Rb-DPPE, Ex/Em: 560/583 nm) were purchased from Avanti Polar Lipids, Inc., (Alabaster, AL, USA), and used without purification. The lipid ratio among PE, PG and CA corresponds to that in E. coli (Merz et al, 2008;Lopes et al, 2010;Lind et al, 2015). Chloroform solutions of PLE and Rb-DPPE were mixed in a glass vial.…”

“…As a bacterial model cell system, reconstituted Escherichia coli (E. coli) cell membrane has been studied (Dodd et al, 2008;Merz et al, 2008;Lopes et al, 2010;Unsay et al, 2013;Lind et al, 2015;Phan and Shin, 2015;Hsia et al, 2016;Konarzewska et al, 2017;Márquez and Vélez, 2017). Understanding the bacterial membrane system helps in revealing the mechanism of molecular recognition and material transportation during bacterial infection for medical technology.…”

“…There are several previous studies on the formation of SLBs containing E. coli lipids such as polar lipid extract (PLE) or total lipid extract (TLE) (Dodd et al, 2008;Merz et al, 2008;Lopes et al, 2010;Unsay et al, 2013;Lind et al, 2015;Hsia et al, 2016;Konarzewska et al, 2017;Márquez and Vélez, 2017). Solid substrates, which are generally used for SLB formation, e.g., glass, oxidized layer on a Si wafer, and mica, are negatively charged.…”

“…Solid substrates, which are generally used for SLB formation, e.g., glass, oxidized layer on a Si wafer, and mica, are negatively charged. Because of the electrostatic repulsion between negatively charged E. coli lipids and the substrates, divalent cation such as Ca 2+ is necessary to form SLB (Merz et al, 2008;Lind et al, 2015;Márquez and Vélez, 2017). Previous QCM and fluorescence microscope studies showed that ∼2 mM Ca 2+ is sufficient for the formation of E. coli lipid SLB (Merz et al, 2008;Lind et al, 2015;Márquez and Vélez, 2017).…”

“…Because of the electrostatic repulsion between negatively charged E. coli lipids and the substrates, divalent cation such as Ca 2+ is necessary to form SLB (Merz et al, 2008;Lind et al, 2015;Márquez and Vélez, 2017). Previous QCM and fluorescence microscope studies showed that ∼2 mM Ca 2+ is sufficient for the formation of E. coli lipid SLB (Merz et al, 2008;Lind et al, 2015;Márquez and Vélez, 2017). Divalent cations bridge negatively charged substances by effectively shielding electrostatic interaction (Rajendran et al, 2011;Ido et al, 2013), and also positively affect the SLB formation from lipid vesicles (Hansma and Laney, 1996;Rivetti et al, 1996;Reviakine and Brisson, 2000;Pastré et al, 2003;Seantier and Kasemo, 2009).…”