Characterization of the in vivo acceptors of the mycoloyl residues transferred by the corynebacterial PS1 and the related mycobacterial antigens 85

Abstract

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.

Genetic dissection of trehalose biosynthesis in Corynebacterium glutamicum: inactivation of trehalose production leads to impaired growth and an altered cell wall lipid composition

“…Unusually for Gram-positive bacteria, their cell walls contain a characteristic hydrophobic layer (Minnikin & O'Donnell, 1984;Nikaido et al, 1993). It was shown that this layer plays an important role in drug and substrate permeability (Jarlier & Nikaido, 1990;Puech et al, 2000). In contrast to the Gram-negative bacteria, where the outer membrane is composed of phospholipids and lipopolysaccharides, the predominant constituents of the outer lipid layer of corynebacteria and related taxa are the mycolic acid esters.…”

Fluorescent Phospholipid Analogs as Microscopic Probes for Detection of the Mycolic Acid-Containing Layer inCorynebacterium glutamicum: Detecting Alterations in the Mycolic Acid-Containing Layer Following Ethambutol Treatment

“…15) These homologous proteins mediate the transfer of the mycolate residue to trehalose monomycolate. [16][17][18] A C. glutamicum homolog of mycobacterial polyprenyl monophosphomannose synthetase, which is involved in mycolate-layer formation, was also identified. 19) Recently, an emb gene encoding a possible target enzyme of EMB was cloned from C. glutamicum.…”

A Role of thecspAGene Encoding a Mycolyltransferase in the Growth under Alkaline Conditions ofCorynebacterium glutamicum

“…It has been reported that a cspA mutant derived from the C. glutamicum CGL2005 strain contains 50% less cell wall-bound corynomycolates. 12) A cspA mutation also caused a similar reduction of cell wall-bound corynomycolates in the C. glutamicum ATCC13032 strain. 16) Corynebacteria and mycobacteria are phylogenetically related and share a similar cell-wall structure that contains (coryno)mycolic acids.…”

“…It has been reported that mutations of cspA resulted in decreases in the amount of corynomycolate covalently bound to arabinogalactan, and non-covalently bound trehalose dicorynomycolate. 12,16) It is known that the mycolate layer functions as a permeability barrier against antibacterial agents and against host defense systems. [7][8][9] To identify defects in the mycolate layer of the ÁcspA mutant strains, the antibacterial susceptibility of wildtype strain ATCC13869 and the ÁcspA and ÁcspB mutant strains were investigated.…”

“…Tel: +81-45-924-5770; Fax: +81-45-924-5820; E-mail: mwachi@bio.titech.ac.jp ynomycolate. 12) Mutations of the cspA gene (also called csp1, cop1, or cmytA) result in a decrease in the amounts of corynomycolate covalently bound to arabinogalactan, a decrease in the amount of trehalose dicorynomycolate, and a simultaneous accumulation of trehalose monocorynomycolate in the cell-wall fractions. 12) A homolog of cspA was also found in Corynebacterium ammoniagenes ATCC6872.…”