Cell wall peptidoglycan, a mesh of polysaccharides crosslinked by short 16 peptides, encases the bacterial cell and protects it from turgor pressure lysis. 17 Peptidoglycan synthesis is an effective antibiotic target. Assembly of the biopolymer 18 occurs in close association with the plasma membrane, but higher order organization of 19 the process has not been described. In mycobacteria, intracellular membrane domains 20 comprise biochemically and spatially distinct regions within the conventional plasma 21 membrane. We find that lipid-linked peptidoglycan precursors are made in these 22 domains and then trafficked to the conventional plasma membrane for insertion into the 23 2 cell wall. Disorganization of the membrane rapidly delocalizes and then halts 24 peptidoglycan assembly. Our data show that membrane compartmentalization is an 25 essential feature of mycobacterial cell wall biogenesis. 26 27 Main Text: Many antibiotics target peptidoglycan synthesis, a well-conserved pathway 28 that spans the cytoplasm, plasma membrane and periplasm. The polyprenol-linked, 29 disaccharide-pentapeptide monomer lipid II is completed by the glycosyltransferase 30 MurG in the inner leaflet of the plasma membrane (Fig. 1A). Lipid II is then flipped to the 31 outer leaflet by MurJ and integrated into the cell wall by membrane-bound 32 transglycosylases and transpeptidases from the penicillin-binding protein (PBP) and 33 shape, elongation, division, and sporulation (SEDS) families (1-4). 34The plasma membrane is a heterogeneous mixture of lipids and proteins. 35 Mycobacteria, for example, have intracellular membrane domains (IMD, formerly called 36 the PMf (5) for plasma membrane free of cell wall) that are separable from the 37 conventional plasma membrane (designated the PM-CW, for plasma membrane tightly 38 associated with cell wall) by sucrose density gradient fractionation. The proteome and 39 lipidome of IMD are distinct from PM-CW (5, 6). While PM-CW-resident proteins localize 40 along the perimeter of live mycobacteria, IMD-resident proteins localize along sidewall 41 but are enriched adjacent to sites of polar cell elongation ( 6, 7). 42 Our proteomics analysis indicated that MurG is present in the IMD while 43 sequentially-acting PBPs preferentially associate with the PM-CW (6). We also 44 observed in situ that the subpolar enrichment of MurG-RFP resembles that of the 45 validated IMD marker mCherry-GlfT2 or 8) but that nascent cell wall at 46 3 the mycobacterial poles primarily abuts rather than colocalizes with mCherry-GlfT2 (7). 128 phosphatidylethanolamine, a major phospholipid of mycobacterial plasma membrane, 129 phosphatidylinositol mannoside, a cell envelope glycolipid, and menaquinone, the 130 primary lipid electron carrier of the mycobacterial respiratory chain, are partitioned 131 across the IMD and PM-CW (5, 6, 21). In the absence of a standard set of membrane-132 bound organelles, plasma membrane compartmentalization may be a general bacterial 133 strategy for organizing pathways with lipid-l...