Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State

Abstract

The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis*

“…Among all metabolic pathways, mycolic acid biosynthesis pathway is unique to mycobacteria and is crucial for their success as pathogens (74). Our data showed that a large proportion of enzymes in mycolic acids biosynthesis pathway were succinylated (supplemental Fig.…”

Structural elucidation and genomic scrutiny of the C60–C100 mycolic acids of Segniliparus rotundus

“…It has been previously demonstrated that the regular spacing of functional groups in mycobacterial mycolic acids facilitates folding and packing in monolayer studies (Villeneuve et al, 2005(Villeneuve et al, , 2007. This folding ability is important in allowing mycolic acids to be accommodated within the dimensions of the mycobacterial outer membrane, revealed for the first time by cryo-electron microscopy (Zuber et al, 2008). It is probable that an analogous outer membrane is present in Segniliparus.…”

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.…”