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...

No abstract

SummaryMycobacteria have a unique outer membrane (OM) that is thicker than any other known biological membrane. Nutrients cross this permeability barrier by diffusion through porins. MspA is the major porin of Mycobacterium smegmatis . In this study we showed that three paralogues of MspA, namely MspB, MspC and MspD are also porins. However, only the mspA and mspC genes were expressed in the wild-type strain. None of the single deletion mutants displayed a significant OM permeability defect except for the mspA mutant. Deletion of the mspA gene caused activation of transcription of mspB and/or mspD in three independent strains by unknown chromosomal mutations. It is concluded that mspB and mspD provide backup porins for M. smegmatis . This also indicated that a minimal porin-mediated OM permeability is essential for survival of M. smegmatis . Electron microscopy in combination with quantitative image analysis of protein gels revealed that the number of pores per cell dropped from 2400 to 800 and 150 for the Δ Δ Δ Δ mspA and Δ Δ Δ Δ mspA Δ Δ Δ Δ mspC mutant (ML10) respectively. The very low number of pores correlated well with the at least 20-fold lower channel activity of detergent extracts of the ML10 strain and its 15-and 75-fold lower permeability to nutrient molecules such as serine and glucose respectively. The amount of Msp porin and the OM permeability of the triple porin mutant lacking mspA , mspC and mspD was not altered. The growth rate of M. smegmatis dropped drastically with its porin-mediated OM permeability in contrast to porin mutants of Escherichia coli . These results show that porin-mediated influx of nutrients is a major determinant of the growth rate of M. smegmatis .

SummaryThe cell wall is a major virulence factor of Mycobacterium tuberculosis and contributes to its intrinsic drug resistance. Recently, cryo-electron microscopy showed that the mycobacterial cell wall lipids form an unusual outer membrane. Identification of the components of the uptake and secretion machinery across this membrane is critical for understanding the physiology and pathogenicity of M. tuberculosis and for the development of better anti-tuberculosis drugs. Although the genome of M. tuberculosis appears to encode over 100 putative outer membrane proteins, only a few have been identified and characterized. Here, we summarize the current knowledge on the structure of the mycobacterial outer membrane and its known proteins. Through comparison to transport processes in Gram-negative bacteria, we highlight several hypothetical outer membrane proteins of M. tuberculosis awaiting discovery. Mycobacteria have a complex cell envelopeScientific interest in mycobacteria has been sparked by the medical importance of Mycobacterium tuberculosis and by properties that distinguish them from other microorganisms. In particular, mycobacteria possess a remarkably complex cell envelope consisting of a cytoplasmic membrane and a cell wall, which constitutes an efficient permeability barrier and plays a crucial role in the intrinsic drug resistance and in survival under harsh conditions [1].These microbes produce a fascinating diversity of lipids [1,2] such as the mycolic acids, exceptionally long fatty acids that account for 30% to 40% of the cell envelope mass [3,4]. Mycolic acids are covalently linked to peptidoglycan via an arabinogalactan polymer, a polysaccharide composed of arabinose and galactose subunits. In a typical arrangement, the peptidoglycan network is substituted by linear galactan molecules, which bear several branched arabinose chains [1,2]. These branches end in four arabinose dimers, each forming the head group for two mycolic acid molecules.The mycolic acid-arabinogalactan-peptidoglycan polymer is arranged to form a hydrophobic layer with other lipids and the cytoplasmic membrane [5,6]. Recently, a model describing the