Lipoproteins are well known Virulence factors of bacterial pathogens in general and of Mycobocterium tuberculosis (Mtb), the causative agent of tuberculosis, in particular Lipoprotein lipidation between Gram-positive and Gram-negative bacteria differs significantly as these are di- and triacylated, respectively Little is known about the lipid anchor Of mycobacterial lipoproteins. We reported recently that mycobacterial LppX. a lipoprotein involved in synthesis of cell wall components is triacylated, although mycobacteria are classified as GC-rich Gram-positive bacteria We here exploited the model organism Mycobacterium smegmatis for the expression of Mtb LprF and characterized N-terminal modifications at the molecular level. LprF is a putative lipoprotein of Mtb involved in signaling of potassium-dependent osmotic stress LprF is extensively modified in a mycobacterium-specific manner by a thioether-linked diacylglyceryl residue with one ester-bound tuberculostearic- and one C16 0 fatty acid and additionally by a third N-linked C16:0 fatty acid, and a hexose (C) 2009 Elsevier Inc. All rights reserved