The cell walls of mycobacteria form an exceptional permeability barrier, and they are essential for virulence. They contain extractable lipids and long-chain mycolic acids that are covalently linked to peptidoglycan via an arabinogalactan network. The lipids were thought to form an asymmetrical bilayer of considerable thickness, but this could never be proven directly by microscopy or other means. Cryo-electron tomography of unperturbed or detergenttreated cells of Mycobacterium smegmatis embedded in vitreous ice now reveals the native organization of the cell envelope and its delineation into several distinct layers. The 3D data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, Myobacterium bovis bacillus Calmette-Gué rin, and Corynebacterium glutamicum identified the outermost layer as a morphologically symmetrical lipid bilayer. The structure of the mycobacterial outer membrane necessitates considerable revision of the current view of its architecture. Conceivable models are proposed and discussed. These results are crucial for the investigation and understanding of transport processes across the mycobacterial cell wall, and they are of particular medical relevance in the case of pathogenic mycobacteria.bacterial cell wall ͉ Corynebacterium glutamicum ͉ Mycobacterium bovis ͉ Mycobacterium smegmatis ͉ mycolic acid layer M ycobacteria have evolved a complex cell wall, comprising a peptidoglycan-arabinogalactan polymer with covalently bound mycolic acids of considerable size (up to 90 carbon atoms), a variety of extractable lipids, and pore-forming proteins (1-3). The cell wall provides an extraordinarily efficient permeability barrier to noxious compounds and contributes to the high intrinsic resistance of mycobacteria to many drugs (4). Because of the paramount medical importance of Mycobacterium tuberculosis, the ultrastructure of mycobacterial cell envelopes has been intensively studied during recent decades. The current view of the cell wall architecture is essentially based on a model suggested by Minnikin (5). He proposed that the covalently bound mycolic acids form the inner leaflet of an asymmetrical bilayer. Other lipids extractable by organic solvents were thought to form the outer leaflet, either intercalating with the mycolates (5, 6) or forming a more clearly defined interlayer plane (7). Elegant x-ray diffraction studies proved that the mycolic acids are oriented parallel to each other and perpendicular to the plane of the cell envelope (8). Furthermore, freeze-fracture studies showed a second fracture plane in electron micrographs (9), indicating the existence of a hydrophobic bilayer structure external to that of the cytoplasmic membrane. Mutants or treatments affecting mycolic acid biosynthesis and the production of extractable lipids resulted in an increase of cell wall permeability in various mycobacteria and related microorganisms (10-12) and a drastic decrease of virulence, underlining the importance of the integrity of the cell wall for intracellular survival...

Membrane-enclosed organelles, a defining characteristic of eukaryotic cells, are lost during differentiation of specific cell types such as reticulocytes (an intermediate in differentiation of erythrocytes), central fibre cells of the eye lens, and keratinocytes. The degradation of these organelles must be tightly regulated with respect to both the time of activation and the specificity of membrane degradation. The expression of 15-lipoxygenase (15-LOX) peaks in reticulocytes immediately before organelle degradation. Here we show that 15-LOX integrates into the membranes of various organelles, allowing release of proteins from the organelle lumen and access of proteases to both lumenal and integral membrane proteins. In addition, by sparing the plasma membrane, 15-LOX shows the required specificity for organellar membranes. Thus, the action of 15-LOX provides a mechanism by which the natural degradation process can be explained. This conclusion is supported by our finding that lipoxygenase expression in the eye lens is restricted to the region at which organelle degradation occurs.

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MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water‐filled channels in vitro. Immunogold electron microscopy and an enzyme‐linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA‐specific primers and reverse‐transcribed RNA. Detergent extracts of the ΔmspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild‐type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the ΔmspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine‐ and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the ΔmspA mutant. The minimal permeability coefficient of the ΔmspA mutant for glucose was 7.2 × 10−8 cm s−1, which is the lowest value reported so far for bacteria. This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.

The three-dimensional structure of the Acetogenium kivui surface layer (S-layer) has been determined to a resolution of 1.7 nm by electron crystallographic techniques. Two independent reconstructions were made from layers negatively stained with uranyl acetate and Na-phosphotungstate. The S-layer has p6 symmetry with a center-to-center spacing of approximately 19 nm. Within the layer, six monomers combine to form a ring-shaped core surrounded by a fenestrated rim and six spokes that point towards the axis of threefold symmetry and provide lateral connectivity to other hexamers in the layer. The structure of the A. kivui S-layer protein is very similar to that of the Bacillus brevis middle wall protein, with which it shares an N-terminal domain of homology. This domain is found in several other extracellular proteins, including the S-layer proteins from Bacilus sphaericus and Thermus thermophilus, Ompa from Thermotoga maritima, an alkaline cellulase from Bacillus strain KSM-635, and xylanases from Clostridium thermocellum and Thermoanaerobacter saccharolyticum, and may serve to anchor these proteins to the peptidoglycan. To our knowledge, this is the first example of a domain conserved in several S-layer proteins.Acetogenium kivui (19) is a hydrogen-oxidizing, acetogenic bacterium (18) that is moderately thermophilic and grows optimally at 66°C. In spite of its gram-negative staining behavior, its cell wall has gram-positive characteristics. Like many other bacteria, gram positive as well as gram negative, it is covered by a regularly arrayed surface layer (S-layer). This layer has a hexagonal structure and consists of a single 80-kDa protein whose gene has been cloned and sequenced (21). The S-layer protein is modified at four tyrosine residues by long glycan chains that are composed of glucose, galactosamine, and an as-yet-unidentified sugar-related component (22) 8578-2652. Fax: (089) 8578-2641. for S-layer homology) is conserved in several other proteins and discuss possible implications for its function. MATERUILS AND METHODSBacterial strain and growth conditions. A. kivui was obtained from the German collection of Microorganisms (DSM 2030), Braunschweig, Germany. Cells were grown anaerobically in the medium described by Leigh et al. (18), buffered with 50 mM phosphate (pH 6.5) and supplemented with yeast extract (2.0 g per liter), tryptone (2.0 g per liter), and glucose (5.0 g per liter). The growth temperature was between 60 and 640C.S-layer preparation. Cells were harvested in the logarithmic growth phase by centrifugation at 4,500 x g and washed once in distilled water. The peptidoglycan was digested by adding 10 to 20 mg of lysozyme to 100-ml aliquots of cell suspension and incubating the mixture for 6 to 8 h at room temperature. The tilt series chosen for processing comprised 14 projections each. The actual tilt angles ranged from -0.3 to 78.30 (UA) and from 2.3 to 80.9°(PTA). No significant radiation damage was accumulated while recording the tilt series, as the power spectra of nominal 00 tilts...