Fusion of small unilamellar vesicles with viable EDTA-treated Escherichia coli cells.
Fusion characteristics of EDTA-treated Escherichia coli cells with small unilamellar vesicles were investigated, using a membrane fusion assay based on resonance energy transfer. Ca2+-EDTA treatments of Escherichia coli O111:B4 (wild type), E. coli C600 (rough), and E. coli D21f2 (deep rough) which permeabilize the outer membrane by inducing the release of lipopolysaccharide and outer membrane proteins resulted in fusion activity of the intact and viable bacteria with small unilamellar vesicles. No fusion activity was observed when the EDTA treatment was omitted. Fusion could be elicited at low pH and by a combination of a higher pH and Ca2+. The low-pH-induced fusion was composed of a fast and a slow reaction. The latter and the Ca2+-induced fusion could be completely inhibited by trypsin treatments of the EDTA-treated cells, which also resulted in the simultaneous disappearance of two outer membrane protein bands (50 and 58 kilodaltons) and the appearance of proteins banding at 22, 52, and 54 kilodaltons. The most efficient fusion was obtained with negatively charged liposomes composed of cardiolipin. In contrast to the Ca2+-induced fusion, fusion was observed at low pH with small unilamellar vesicles containing lipids with decreased negative charge (phosphatidylserine). Fluorescent and phase-contrast microscopy revealed that essentially all bacteria were engaged in fusion. We propose that a Ca2+-EDTA treatment of E. coli cells results in the appearance of phospholipids and the exposure of a protein(s) in the outer leaflet of the outer membrane, both of which could mediate fusion with liposomes.
Marvin
H J
HJ
Department of Biochemistry, University of Groningen, The Netherlands.
ter Beest
M B
MB
Hoekstra
D
D
Witholt
B
B
D001425:Bacterial Outer Membrane Proteins / Q000502:physiology; D002118:Calcium / Q000494:pharmacology; D002308:Cardiolipins; D004492:Edetic Acid / Q000494:pharmacology*; D004926:Escherichia coli / Q000166:cytology* / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008081:Liposomes; D008561:Membrane Fusion / Q000187:drug effects; D008856:Microscopy, Fluorescence
D001425:Bacterial Outer Membrane Proteins; D002308:Cardiolipins; D008051:Lipid Bilayers; D008081:Liposomes; D004492:Edetic Acid; D002118:Calcium