Abstract
The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of > or = 60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter approximately 100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.
Highlights
Coli, have no internal membranes but only the inner and outer membranes on each side of the enclosing peptidoglycan layer
We report here that overexpression of a foreign lipid glycosyltransferase (GT)4 can yield massive formation of membrane vesicles in the cytoplasm of E. coli, which seems coupled to the sequence and structural features of this GT enzyme
E. coli like many other Gram-negative bacteria can form and shed to the surroundings vesicles from their outer membranes [19, 20]
Summary
Cloning and Expression Hosts—The cloning of the monoglycosyldiacylglycerol synthase (ALmgs) and the diglycosyldiacylglycerol synthase (ALdgs) genes from A. laidlawii has been described previously [34, 35]. Fractions were collected using a syringe and diluted to double volume with TEA buffer before transfer to 1.5-ml ultracentrifuge tubes These tubes were centrifuged at 150,000 ϫ g for 40 min at 4 °C to collect the membranes, which were dispersed in 50 mM HEPES (pH 8) and stored at Ϫ80 °C until further analyzed. The suspension was incubated on ice for 15 min before the addition of 2 ml of buffer containing 50 mM TEA (pH 7.5), 1 mM EDTA, 0.1 mg/ml DNase, 1 mM DTT, and 1.5 mg/ml Pefabloc This suspension was passed through a French press at 1000 p.s.i. for three cycles before the cell debris was pelleted at 11,000 rpm and at 4 °C for 20 min. Data for the molecular composition of E. coli interface is Ϸ2000 Å2, according to the structure model and were taken from the CyberCell data base
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