Biomedical subjects
Publications and source records attributed to L Letellier.
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Lipid and protein segregations can be induced in E. coli cytoplasmic membranes by conformational transitions of their lipid hydrocarbon chains from a disordered to an ordered state. For E. coli strain K 1059 (an unsaturated fatty acid auxotroph) supplemented with linolenic acid, the segregation leads to large areas of membrane surfaces having distinctly different morphological characteristics (smooth compared with strongly particulated fracture faces, as visualized by freeze fracture electron microscopy). The different regions are physically separated by osmotic lysis of spheroplasts at temperatures below those of the order-disorder transition of the lipid hydrocarbon chains. The analysis of the different cytoplasmic membrane fractions provides a direct demonstration and allows a direct analysis of the segregation. As compared to the nonfractionated membranes, the membrane regions corresponding to the smooth fracture surfaces are poor in proteins, rich in lipids, and enriched in saturated fatty acids, while the membrane regions corresponding to the strongly particulated fracture surfaces are rich in proteins, poor in lipids, and enriched in unsaturated fatty acids. Quantitative information about the extent of these segregations is obtained from high-angle x-ray diffraction of the different membrane fractions and of the corresponding total lipid extracts.
The cytoplasmic and outer membranes containing either trans-delta-9-octadecenoate, trans-delta-9-hexadecenoate or cis-delta-9-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, Tt, and the range of the transition, delta-T, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60-80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25-40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.
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FhuA (M(r) = 78,900) is an Escherichia coli outer membrane protein which transports the ferric siderophore ferrichrome and is the receptor for phage T5, phi 80 and T1 and for colicin M. FhuA was purified chromatographically in non-ionic detergent (octyl glucoside). The circular dichroism spectrum indicates that FhuA is essentially organized in beta-strands like the majority of proteins of the outer membrane of Gram-negative bacteria. The structural parameters of FhuA were assessed from size exclusion chromatography, sedimention equilibrium and velocity experiments. FhuA is monomeric in solution and functional since binding of phage T5 causes the release of the phage genome, a double-stranded DNA of 121,000 base pairs, into the surrounding medium. Planar lipid bilayer experiments showed that the FhuA transporter is converted into a high conductance channel upon binding of phage T5. FhuA was reconstituted into large unilamellar vesicles (mean diameter 125 nm). Cryo-electron microscopy and fluorescence experiments, using a DNA intercalant YO-PRO 1, showed that binding of T5 to FhuA triggers the transfer of the phage genome into the proteoliposomes without altering their morphology. Two models can account for these observations, which apply both to in vitro and in vivo DNA transport. The simplest model supposes that the naked DNA is transported through the FhuA channel. Alternatively transfer of DNA might be mediated by pb2, the protein forming the phage straight fiber. pb2 would insert either directly in the membrane or inside the FhuA channel.