Disclosure of the mycobacterial outer membrane: Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure

Abstract

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...

Lipoprotein biogenesis in Gram-positive bacteria: knowing when to hold ‘em, knowing when to fold ‘em

“…Moreover, cell fractionation experiments and recent advances in electron microscopy have lent some credibility to the controversial concept of a Gram-positive 'periplasm' [4][5][6]. Cryoelectron microscopy has also provided evidence supporting the presence of an outer membrane permeability barrier in the mycolic acid-containing actinomycete bacteria Mycobacterium smegmatis, Mycobacterium bovis and Corynebacterium glutamicum [7,8]. In addition, since the last review of Gram-positive lipoproteins 13 years ago [3] our understanding of the diverse functions of these proteins has been greatly advanced by the availability of whole genome sequence data.…”

Viability states of bacteria—Specific mechanisms of selected probes

“…Although Mycobacteria belong to the gram1 bacteria, which are generally assumed to have no OM recent studies showed the existence of such a membrane for some members of the genera (15). The mycobacterial OM is described to comprise a bilayer which is even thicker than the IM but of different structure than that of gram2 bacteria.…”

“…Nevertheless, mycolic acids are located in the inner leaflet of the OM causing a highly ordered structure (16). The addition of detergents like Tween 60 may initiate alteration of fluidity of the mycolic acid layer (16), which in consequence may turn to be leaky to amphiphilic molecules (15).…”

Global transcriptional response to vancomycin in Mycobacterium tuberculosis

“…In spite of a good knowledge of the chemical composition of the mycobacterial surface, not much is known about its organization and physiology (Barry, 2001). The recent demonstration of the presence of an outer membrane will surely boost research in this field (Hoffmann et al, 2008;Zuber et al, 2008).…”

“…In spite of a good knowledge of the chemical composition of the mycobacterial surface, not much is known about its organization and physiology (Barry, 2001). The recent demonstration of the presence of an outer membrane will surely boost research in this field (Hoffmann et al, 2008;Zuber et al, 2008).One strategy to study bacterial surface physiology is to characterize the variation of the bacterial transcriptional profile in response to exposure to compounds able to perturb surface homeostasis. This approach has been successfully used to study the global transcriptional response of Bacillus subtilis and Staphylococcus aureus in response to several inhibitors of peptidoglycan biosynthesis (Cao et al, 2002;Mascher et al, 2003;Utaida et al, 2003), and in M. tuberculosis to study the transcriptional response to the Abbreviations: HCL, hierarchical clustering; SAM, significance analysis of microarrays.…”