The mycomembrane proteins PorH and ProtX are inserted at polar growth zones and linked to the cell wall

The Mycobacteriales order of bacteria includes important pathogens such as Mycobacterium tuberculosis. These organisms are surrounded by a unique cell envelope architecture that includes a two-layered cell wall composed of peptidoglycan (PG) and arabinogalactan. They also build an outer membrane called the mycomembrane that is made of mycolic acids. Mycolate outer membrane proteins (MOMPs) reside within the mycomembrane and a subset are thought to form pores that allow essential nutrients to permeate the envelope. However, little is known about the structure of these proteins or the mechanism by which they are assembled. Here, we investigate MOMP assembly in the model organism Corynebacterium glutamicum (Cglu) using PorH as a model MOMP. PorH is encoded in an operon with the MOMP PorA, and the two small, alpha-helical proteins have been proposed to form hetero-oligomeric pores in the mycomembrane. Consistent with this proposal, AlphaFold2 predicts a high confidence structure of a hetero-oligomeric pore formed by five copies each of PorH and its partner PorA, and we show that PorA is required for the surface assembly of PorH. Using a fluorescence assay for detection of surface-exposed PorH or another MOMP called ProtX, we found that MOMP assembly occurs within zones of active PG synthesis at the cell poles. We also discovered that PorH and ProtX are linked to the cell wall. Thus, like Gram-negative bacteria, Cglu and potentially other members of Mycobacteriales order, coordinate outer membrane protein assembly with PG biogenesis and use proteins to connect the mycomembrane and the cell wall. SIGNIFICANCEDiderm bacteria in the Mycobacteriales order have a distinctive outer layer called the mycomembrane. Proteins that reside within the mycomembrane play critical roles in virulence and cell viability. However, how proteins are assembled into the mycomembrane has remained an outstanding question in the field. Here, we investigate the biogenesis of mycomembrane proteins in the model organism Corynebacterium glutamicum. We show that these proteins are inserted into the mycomembrane in a manner that correlates with polar growth and are attached to the cell wall. Many features of these mycomembrane proteins are shared between species in the Mycobacteriales, suggesting that our findings may be conserved in other species within this order.