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.

Three strains of Corynebacterium producing various amounts of PS2 S-layer protein were studied. For all strains, more PS2 was produced if the bacteria were grown in minimal medium supplemented with lactate than if they were grown in minimal medium supplemented with glucose. The consumption of substrate and PS2 production was studied in cultures with mixed carbon sources. It was found that the inhibitory effect of glucose consumption was stronger than the stimulatory effect of lactate in one strain, but not in the other two strains. The regulation of gene expression involved in S-layer formation may involve metabolic pathways, which probably differ between strains. S-layer organization was also studied by freeze-fracture electron microscopy. It was found that low levels of PS2 production correlated with the partial covering of the cell surface by a crystalline array. Finally, it was found that PS2 production was mainly regulated by changes in gene expression and that secretion was probably not a limiting step in PS2 accumulation.

By using appropriate Corynebacterium glutamicum-Escherichia coli shuttle plasmids, the gene encoding the fibronectin-binding protein 85A (85A) from Mycobacterium tuberculosis was expressed in C. glutamicum, also an actinomycete and nonsporulating gram-positive rod bacterium, which is widely used in industrial amino acid production. The 85A gene was weakly expressed in C. glutamicum under the control of the ptac promoter from E. coli, but it was produced efficiently under the control of the promoter of the cspB gene encoding PS2, one of the two major secreted proteins from C. glutamicum. The 85A protein was produced in various forms, with or without its own signal sequence and with or without the signal sequence and the NH 2-terminal (18-amino-acid) mature sequence of PS2. Western blot analysis with monoclonal antibodies raised against the M. tuberculosis antigen 85 complex showed that recombinant 85A protein was present in the corynebacterial cell wall extract and also released in extracellular culture medium. NH 2-terminal microsequencing of recombinant 85A secreted by C. glutamicum showed that signal peptide was effectively cleaved off at the predicted site. The recombinant 85A protein was biologically active in vitro, inducing significant secretion of Th1 T-cell cytokines, particularly interleukin-2 and gamma interferon, in spleen cell cultures from mice vaccinated with live Mycobacterium bovis BCG. Heterologous expression of mycobacterial antigens in C. glutamicum now offers a potent tool for further immunological characterization and large scale preparation of these recombinant proteins.