With the recent success of the heterologous expression of mycobacterial antigens in corynebacteria, in addition to the importance of these bacteria in biotechnology and medicine, a better understanding of the structure of their cell envelopes was needed. A combination of molecular compositional analysis, ultrastructural appearance and freeze-etch electron microscopy study was used to arrive at a chemical model, unique to corynebacteria but consistent with their phylogenetic relatedness to mycobacteria and other members of the distinctive suprageneric actinomycete taxon. Transmission electron microscopy and chemical analyses showed that the cell envelopes of the representative strains of corynebacteria examined consisted of (i) an outer layer composed of polysaccharides (primarily a high-molecular-mass glucan and arabinomannans), proteins, which include the mycoloyltransferase PS1, and lipids ; (ii) a cell wall glycan core of peptidoglycan-arabinogalactan which may contain other sugar residues and was usually esterified by corynomycolic acids ; and (iii) a typical plasma membrane bilayer. Freeze-etch electron microscopy showed that most corynomycolate-containing strains exhibited a main fracture plane in their cell wall and contained low-molecular-mass porins, while the fracture occurred within the plasma membrane of strains devoid of both corynomycolate and pore-forming proteins. Importantly, in most strains, the amount of cell wall-linked corynomycolates was not sufficient to cover the bacterial surface ; interestingly, the occurrence of a cell wall fracture plane correlated with the amount of non-covalently bound lipids of the strains. Furthermore, these lipids were shown to spontaneously form liposomes, indicating that they may participate in a bilayer structure. Altogether, the data suggested that the cell wall permeability barrier in corynebacteria involved both covalently linked corynomycolates and non-covalently bound lipids of their cell envelopes.

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane ␤-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.The widespread type II secretion systems (T2SS) 3 of Gram-negative bacteria allow the secretion of hydrolytic enzymes (lipases, amylases) or virulence factors, collectively referred to as exoproteins, into the external medium (1, 2). These exoproteins are first translocated by the Sec (3) or Tat (4) translocons into the periplasm. They are then specifically transported through the outer membrane by an ATP and proton-motive force-dependent machinery (the secreton) (5, 6) composed of 12-15 proteins (1, 2). The secreton components include several integral inner membrane proteins, pseudopilins (proteins with structural features similar to those of type IV pilins (7)) and an integral outer membrane protein called secretin. Besides their role in protein secretion by the T2SS (e.g. Klebsiella oxytoca protein PulD (8) and Pseudomonas aeruginosa protein XcpQ (9, 10)) and the type III secretion system (e.g. Yersinia enterocolitica protein YscC (10)), secretins are also required for filamentous bacteriophage secretion (e.g. bacteriophage f1 protein pIV (11)) and type IV pilus assembly (e.g. Neisseria meningitidis and P. aeruginosa PilQ (9, 12)).According to electron microscopy, 12-14 identical secretins form ring-like complexes with an internal channel (estimated diameters range from 5 nm (PilQ, YscC) to 10 nm (XcpQ) (9, 13, 14)), large enough to accommodate their substrates (9,10,15,16). Negative stain analysis of PulD (17) and cryoelectron microscopy of pIV (18) revealed a central channel plug. Incorporation of secretins into the Escheri...