Corynebacterium glutamicumis a diderm bacterium extensively used in the industrial-scale production of amino acids. Corynebacteria belong to the bacterial familyMycobacteriaceae, which is characterized by a highly unusual cell envelope with an outer membrane consisting of mycolic acids. Despite the occurrence of this distinctive cell envelope in several bacterial pathogens, includingCorynebacterium diphtheriae, Mycobacterium tuberculosis, andMycobacterium leprae, its ultrastructural and molecular details remain elusive.To address this, we investigated the cell envelope ofC. glutamicumusing electron cryotomography and cryomicroscopy of focused ion beam-milled cells. Our high-resolution images allowed us to accurately map the different components of the cell envelope into the tomographic density. Our data reveal thatC. glutamicumhas a variable cell envelope, with the outermost layer comprising the surface (S-)layer, which decorates the mycomembrane in a patchy manner. We further isolated and resolved the structure of the S-layer at 3.1 Å resolution using single particle electron cryomicroscopy. Our structure shows that the S-layer ofC. glutamicumis composed of a hexagonal array of the PS2 protein, which interacts directly with the mycomembrane via a coiled coil-containing anchoring segment. Bioinformatic analyses revealed that the PS2 S-layer is sparsely yet exclusively present within theCorynebacteriumgenus and absent in other genera of theMycobacteriaceaefamily, suggesting distinct evolutionary pathways in the development of their cell envelopes.Our structural and cellular data collectively provide a high-resolution topography of the unusualC. glutamicumcell surface, features of which are shared by many pathogenic and microbiome-associated bacteria, as well as by several industrially significant bacterial species. This study, therefore, provides a strong experimental framework for understanding cell envelopes that contain mycolic acids.
