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.Streptomycetes are bacteria characterized by a high degree of morphological and biochemical differentiation. Streptomyces hygroscopicus grows as branched hyphae, and it is able to form an aerial mycelium and spores. It produces several secondary metabolites, especially the antibiotic turimycin. The vegetative cells are 3-to 20-m-long hyphal units surrounded by a cytoplasmic membrane and a typical 30-nm-thick cell wall. From this species, we could isolate an L-form strain, which is able to propagate as cell wall-less, spherical, or pleomorphic cells on agar media as well as in liquid media under shaking conditions (2, 12).The stable protoplast type L-form strain is the result of an adaptive process. It has lost, irreversibly, the capability to resynthesize cell wall structures, and it represents a genetically stable mutant showing extreme pleomorphic alterations. These involve cell and colony morphology, growth behavior, biochemical activities, and an incapability to form spores and secondary metabolites (12, 13). In particu...

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), collisioninduced 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.The phospholipids play an important role in stability, fluidity, and structural alterations of membranes. A class of phospholipids consists of various molecular species. Because of their importance in the function of biological membranes, there have been many attempts to elucidate the composition of the molecular phospholipid species. However, knowledge about the molecular phospholipid composition of bacteria is, with a few exceptions, still restricted because of difficulties with traditional techniques (1,5,14,15). Electrospray ionization (ESI) coupled with collision-induced dissociation (CID) and tandem mass spectrometry (MS-MS; i.e., ESI-MS-CID-MS) (4,11,12) is a relatively new method that has been used for the elucidation of bacterial phospholipid species in only a few cases (17)(18)(19).We used this technique to characterize, for the first time, the molecular phospholipid species of streptomycetes. The phospholipids were isolated from purified cytoplasmic membranes of the cell wall-less L form of Streptomyces hygroscopicus. These cells have been grown without a cell wall and a periplasmic compartment for 20 year...

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 g 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 immunogoldlabeled 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.The overexpression of recombinant proteins, which remain bound to the outer surface of the bacterial cells as accessible and functional active molecules, offers new applications in biotechnology and medicine. Among these are the development of diagnostics and vaccines, adhesin-receptor interaction studies, the generation of peptide libraries, the immobilization of enzymes, and the expression of heavy metal-binding peptides and antibody fragments (6,10,25,33). The surface display systems follow various strategies for anchoring. In gramnegative bacteria, outer membrane proteins (21), pili and flagella (26), modified lipoproteins (6, 7, 11), ice nucleation proteins (17, 23), and autotransporters (19,27,35) have been used as anchors. In gram-positive bacteria, surface anchors have been derived from lipoproteins, cell wall proteins, or S-layer proteins (24,32,33). However, depending on the displayed protein and the desired application, each system has its own advantages and disadvantages, such as the size limitation of the displayed protein, mislocalization or formation of inclusion bodies, association with lipopolysaccharides (LPS), or destabilization of the outer membrane (10,17,22). There is still a need for further developments to increase the repertoire of applications of surface display systems (21).Stable protoplast type L-form bacteria have up to now not been considered as a system for surface display, although they exhibit several interesting features for such applications. They have lost irreversibly the ability to form cell wall structures and periplasmic compartments, and their cells are surrounded only by a cytoplasmic membrane (CM). Cell biological properties of the strains and the lipid composition of their CM are well characterized (13,16). Furthermore, the L-form strains of Proteus mirabilis LVIWEI and Escherichia coli LWFϩWEI have been used for the efficient overexpr...