[Show abstract][Hide abstract] ABSTRACT: We describe a novel membrane surface display system that allows the anchoring of foreign proteins in the cytoplasmic membrane (CM) of stable, cell wall-less L-form cells of Escherichia coli and Proteus mirabilis. The reporter protein, staphylokinase (Sak), was fused to transmembrane domains of integral membrane proteins from E. coli (lactose permease LacY, preprotein translocase SecY) and P. mirabilis (curved cell morphology protein CcmA). Both L-form strains overexpressed fusion proteins in amounts of 1 to 100 microg ml(-1), with higher expression for those with homologous anchor motifs. Various experimental approaches, e.g., cell fractionation, Percoll gradient purification, and solubilization of the CM, demonstrated that the fusion proteins are tightly bound to the CM and do not form aggregates. Trypsin digestion, as well as electron microscopy of immunogold-labeled replicas, confirmed that the protein was localized on the outside surface. The displayed Sak showed functional activity, indicating correct folding. This membrane surface display system features endotoxin-poor organisms and can provide a novel platform for numerous applications.
Applied and Environmental Microbiology 03/2002; 68(2):525-31. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A comparative analysis of the lipid compositions and fatty acids in the cytoplasmic membranes of Streptomyces hygroscopicus and its stable cell wall-less L form has been carried out to detect the differences which may be involved in the altered properties of the L-form membranes. Because only quantitative differences could be found (8), we analyzed the lipid components at the molecular level. Electrospray ionization (ESI), collision-induced dissociation (CID), and tandem mass spectrometry (MS-MS) were used for qualitative detection and quantitative determination of the molecular lipid species in phosphatidylethanolamine (PE1), lyso-cardiolipin (LCL), and cardiolipin (CL). Each phospholipid, isolated by preparative high-performance liquid chromatography showed several homologous molecular ion groups (PE1, four groups; LCL, six groups; CL, six groups) in the negative ESI-MS spectra. The sizes of the peaks represent their relative amounts in the corresponding phospholipid classes. Structural details about individual components of the molecular ion groups were obtained by mass selection and CID with MS-MS. Product ions derived from CID (daughter ions) give information about the molecular weights of the acyl constituents. The qualitative and quantitative compositions of the molecular species were determined by combining the data from the fatty acid pattern obtained by gas chromatography (GC), the relative quantities of the molecular ion groups, and the acyl constituents detected in these molecular ions. Because the ESI-MS-CID-MS data do not allow us to distinguish between n, iso, and anteiso fatty acids of the same molecular weight, it has been assumed that the ratio of these equal-numbered fatty acids determined by GC analysis of the isolated fatty acids is also present in the CID-MS peaks. In this way, 18 species were found in PE1, 43 species were estimated in LCL, and 59 species were ascertained for CL.
Journal of Bacteriology 07/1997; 179(11):3437-42. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cells of an L-form strain of Streptomyces hygroscopicus have been grown for 20 years without a cell wall. Their cytoplasmic membranes have high stability and an unusual structural polymorphism. To clarify the importance of the lipid components for these membrane properties, a comparative analysis has been carried out with purified membranes of L-form cells, of parent vegetative hyphal cells (N-form cells), and of protoplasts derived from the latter. The phospholipid classes and fatty acids were determined by thin-layer chromatography (TLC), two-dimensional TLC, high-performance liquid chromatography, gas chromatography, and mass spectrometry. The qualitative compositions of cardiolipin (CL), lyso-cardiolipin (LCL), phosphatidylethanolamine (PE1 and PE2), lyso-phosphatidylethanolamine (LPE), phosphatidylinositolmannoside (PIM), phosphatidic acid (PA), dilyso-cardiolipin-phosphatidylinositol (DLCL-PI), and the 13 main fatty acids were the same in the three membrane types. However, significant quantitative differences were observed in the L-form membrane. They consist of a three- to fourfold-higher content of total, extractable lipids, 20% more phospholipids, an increased content of CL and PIM, and a reduced amount of the component DLCL-PI. Furthermore, the L-form membrane is characterized by a higher content of branched anteiso 15:0 and anteiso 17:0 fatty acids compared to that of the membranes of the walled vegetative cells. These fatty acids have lower melting points than their straight and iso-branched counterparts and make the membrane more fluid. The phospholipid composition of the protoplast membrane differs quantitatively from that of the N form and the L form. Whereas the phospholipid classes are mostly similar to that of the N form, the fatty acid pattern tends to be closer to that of the L-form membrane. The membranes of both the L-form cells and the protoplasts need to be more fluid because of their spherical cell shape and higher degree of curvature compared with N-form membranes.
Journal of Bacteriology 07/1997; 179(11):3430-6. · 3.19 Impact Factor