O-glycosylation occurs at Ser and Thr residues respectively. Although glycosylations of the tryptic or AspN-digested N-terminal peptides of LprF and LppX were identified, the exact glycosylation

site within the peptide could not be determined. No glycosylations were found for N-terminal fragments of LpqH and LpqL. This possibly is due to the use of proteases which have cleavage sites close to the N-terminus and therefore the peptide fragment may be too short to include O-glycosylation sites. The information about the exact molecular nature and function of the glycosylation is scarce, but its influence on subcellular lipoprotein localization and its protection from proteolytic degradation are proposed [45, 62]. In B. subtilis lipoprotein CAL101 glycosylation is discussed to control a lipoprotein “shaving” mechanism and thus their release into the culture medium [63]. In our study, glycosylations were found also in lipoproteins from the Δlnt mutant, demonstrating that N-acylation is not a prerequisite for glycosylation. Lnt independent glycosylation was also I BET 762 demonstrated in C. glutamicum[16]. In C. glutamicum Cg-Ppm1 is responsible for glycosylation. Cg-ppm1 (Ppm buy AMN-107 synthase) and Cg-ppm2 (Lnt) are similar organized as MSMEG_3859 (Ppm synthase) and MSMEG_3860 (Lnt) in

M. smegmatis (Figure 2). Deletion of the Lnt domain of BCG_2070c obviously did not abolish Ppm activity encoded in the same ORF. Of note, Lnt is dispensable while Ppm is 4-Aminobutyrate aminotransferase essential in M. tuberculosis[64]. In Gram-negative bacteria, the efficient lipoprotein transport to the outer membrane depends on the localization of lipoproteins (Lol) transport system and there is good evidence that N-acylation by Lnt facilitates lipoprotein translocation in E. coli[6, 65]. Lnt is essential in E. coli, however deletion of lnt was possible upon overexpression of proteins from the Lol system, indicating an important role

of N-acylation in targeting lipoproteins to the outer membrane [9]. Mycobacteria have an outer membrane mycolic acid bilayer [66–68] and are known to localize lipoproteins to the cell surface [66]. Nevertheless, no mechanisms for translocation or transport systems are identified and whether N-acylation and glycosylation, alone or in combination are involved in the translocation of specific lipoproteins to the mycolate layer is not known so far. In the present study we show that lipoproteins from M. bovis BCG, the live vaccine for tuberculosis are triacylated and we identified the lipid modifications at the molecular level. BCG_2070c is a functional homologue of E. coli Lnt, but differs in substrate specificity. The identification of N-linked tuberculostearic acid shows for the first time, to our knowledge, that mycobacteria-specific fatty acids are used by mycobacterial Lnts.