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Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane

Significance Protein secretion is an essential determinant of bacterial physiology and virulence. Members of the Corynebacteriales order have evolved a complex cell envelope containing two membranes, a plasma membrane and an outer membrane, called the mycomembrane, which harbors mycolic acids and outer membrane proteins (OMPs) of unusual structure. Here, we have investigated the biogenesis of OMPs in Corynebacterium glutamicum and deciphered the role of O-mycoloylation in targeting OMPs to the mycomembrane. Partially enabled by our methodology, we found that the posttranslational state of major OMPs determined their presence in the outer membrane vs. the extracellular medium. We have also uncovered a short linear amino acid motif for O-acylation of proteins that seems to be preserved throughout the kingdoms. The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.