Metabolic Tagging Reveals Surface-Associated Lipoproteins in Mycobacteria

Mycobacteria such as the causative agent of tuberculosis, <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>), encode over 100 bioinformatically predicted lipoproteins. Despite the importance of these post-translationally modified proteins for mycobacterial survival, many remain experimentally unconfirmed. Here we characterized in <i>Mtb</i> and <i>M. smegmatis</i> (<i>Msm</i>) the metabolic incorporation of several modified fatty acids as a facile method of adding chemical groups that enable downstream applications such as detection and enrichment of lipid-modified proteins. We further showed for azido palmitic acid in <i>Msm</i> that incorporation is an active process dependent on the lipoprotein biosynthesis pathway and that a subset of these lipid-modified proteins are associated with the mycobacterial cell surface. Because mycobacteria do not encode known lipoprotein transporters, these data have implications for uncovering the roles of lipoproteins and the possible transport processes involved. Our findings and the tools we validated will enable the further study of pathways related to lipoprotein function in mycobacteria and other bacteria in which lipoproteins remain poorly understood.