![]() ![]() To test this model, we first expressed a functional MurG-Dendra2 fusion in M. These observations suggested a model where lipid II synthesis is segregated from subsequent steps of cell wall assembly ( Figure 1A). We also noted that the polar enrichment of MurG-RFP resembles that of the validated IMD marker mCherry-GlfT2 or GlfT2-GFP ( Hayashi et al., 2016 Meniche et al., 2014), but that nascent peptidoglycan at the mycobacterial poles primarily abuts rather than colocalizes with mCherry-GlfT2 ( Hayashi et al., 2018). While PM-CW-resident proteins distribute along the perimeter of live mycobacteria, IMD-resident proteins are enriched toward sites of polar cell elongation with additional presence along the sidewalls ( Hayashi et al., 2016 Hayashi et al., 2018). Reanalysis of our proteomics data ( Hayashi et al., 2016) suggested that Mycobacterium smegmatis MurG is enriched in the IMD while sequentially acting transglycosylases and transpeptidases associate with the PM-CW. The proteome and lipidome of IMD are distinct from those of the PM-CW ( Hayashi et al., 2016 Morita et al., 2005). In mycobacteria, intracellular membrane domains (IMD, formerly called PMf, Morita et al., 2005) can be separated from the conventional plasma membrane (PM-CW, for plasma membrane associated with cell wall) by sucrose density gradient fractionation. ![]() More recent in vivo data has localized Bacillus subtilis MurG to regions of increased fluidity (RIFs, Müller et al., 2016 Strahl et al., 2014), one of three classes of membrane domains that have been described in bacteria to date. Early in vitro work in Staphylococcus aureus and Escherichia coli indicated that a fluid microenvironment might stimulate the activities of MurG and the upstream, lipid I synthase MraY ( Norris and Manners, 1993). Lipid II is then flipped to the outer leaflet by MurJ where its disaccharide-pentapeptide cargo is inserted into the existing cell wall by membrane-bound transglycosylases and transpeptidases ( Zhao et al., 2017). The final lipid-linked precursor for peptidoglycan synthesis, lipid II, is made by the glycosyltransferase MurG in the inner leaflet of the plasma membrane. smegmatis, indicating that horizontal compartmentalization of precursors may be a general feature of bacillary cell wall biogenesis. Our findings are applicable to rod-shaped bacteria that are phylogenetically distant from M. These data support a model in which the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. The cell wall-organizing protein DivIVA and the cell wall itself promote domain homeostasis. Membrane partitioning likely contributes to robust, orderly peptidoglycan synthesis, suggesting that these domains help template peptidoglycan synthesis. By tracking enzymes, substrates, and products of peptidoglycan biosynthesis in Mycobacterium smegmatis, we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on limited pools of lipid-linked precursors that generate and are attracted to membrane disorder. Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. ![]()
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