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.

SummaryMycolic acids, long-chain (C 70 ±C 90 ) a-alkyl, bhydroxy fatty acids, are characteristic cell envelope components of mycobacteria; similar but shorterchain substances occur in corynebacteria and related taxa. These compounds apparently play an important role in the physiology of these bacteria. The deduced N-terminal region of PS1, one of the two major secreted proteins of Corynebacterium glutamicum encoded by the csp1 gene, is similar to the antigens 85 complex of Mycobacterium tuberculosis which has been shown to be associated in vitro with a mycoloyltransferase activity onto trehalose. Overexpression of PS1 in the wild-type strain of C. glutamicum suggested the implication of the protein in the transfer of corynomycolates, evidenced by an increase esterification of the cell wall arabinogalactan with corynomycolic acid residues and an accumulation of trehalose dicorynomycolates. Overexpression of truncated forms of PS1 demonstrated that the crucial region for transfer activity of the protein involves all the region of homology with antigens 85. To establish the putative mycoloyltransferase activity of PS1, a csp1-inactivated mutant of C. glutamicum was biochemically characterized. Inactivation of the gene resulted in: (i) a 50% decrease in the cell wall corynomycolate content; (ii) the alteration of the permeability of the C. glutamicum cell envelope; (iii) the decrease of the trehalose dicorynomycolate content; (iv) the accumulation of trehalose monocorynomycolate; and (v) the appearance of a glycolipid identified as 6-corynomycoloylglucose. Complementation of the mutant by the csp1 gene fully restored the wild-type phenotype. Finally, a mycoloyltransferase assay established that PS1 possesses a trehalose mycoloyltransferase activity. To define the in vivo function of antigens 85, the csp1-inactivated mutant was complemented with the fbpA, fbpB or fbpC genes. Complementation with the different fbp genes restored the normal cell wall corynomycolate content and permeability, but did not affect either the fate of trehalose corynomycolates or the occurrence of glucose corynomycolate. Thus, PS1 is one of the enzymes that transfer corynomycoloyl residues onto both the cell wall arabinogalactan and trehalose monocorynomycolate, whereas in the whole bacterium the mycobacterial antigens 85A, 85B and 85C can transfer mycolates only onto the cell wall acceptor in C. glutamicum.