Formation of supported lipid bilayers of charged E. coli lipids on modified gold by vesicle fusion.

We describe a simple way of fusing E. coli lipid vesicles onto a gold surface. Supported lipid bilayers on metal surfaces are interesting for several reasons: transducing a biological signal to an electric readout, using surface analytical tools such as Surface Plasmon Resonance (SPR), Infrared Reflection Absorption Spectroscopy, Neutron Reflectivity or Electrochemistry. The most widely used method to prepare supported lipid membranes is fusion of preexisting liposomes. It is quite efficient on hydrophilic surfaces such as glass, mica or SiO2, but vesicle fusion on metals and metal oxide surfaces (as gold, titanium oxide or indium tin oxide), remains a challenge, particularly for vesicles containing charged lipids, as is the case of bacterial lipids. We describe a simple method based on modifying the gold surface with a charged mercaptopropionic acid self-assembled monolayer and liposomes partially solubilized with detergent. The formed bilayers were characterized using a Quartz Crystal Microbalance with dissipation (QCM-D) and Atomic Force Microscopy (AFM). Some advantages of this protocol are that the stability of the self-assembled monolayer allows for repeated use of the substrate after detergent removal of the bilayer and that the amount of detergent required for optimal fusion can be determined previously using the lipid-detergent solubility curve.

Mutations on FtsZ lateral helix H3 that disrupt cell viability hamper reorganization of polymers on lipid surfaces.

FtsZ filaments localize at the middle of the bacterial cell and participate in the formation of a contractile ring responsible for cell division. Previous studies demonstrated that the highly conserved negative charge of glutamate 83 and the positive charge of arginine 85 located in the lateral helix H3 bend of Escherichia coli FtsZ are required for in vivo cell division. In order to understand how these lateral mutations impair the formation of a contractile ring,we extend previous in vitro characterization of these mutants in solution to study their behavior on lipid modified surfaces. We study their interaction with ZipAand look at their reorganization on the surface. We found that the dynamic bundling capacity of the mutant proteins is deficient, and this impairment increases the more the composition and spatial arrangement of the reconstituted system resembles the situation inside the cell: mutant proteins completely fail to reorganize to form higher order aggregates when bound to an E.coli lipid surface through oriented ZipA.We conclude that these surface lateral point mutations affect the dynamic reorganization of FtsZ filaments into bundles on the cell membrane, suggesting that this event is relevant for generating force and completing bacterial division.