Lipid Flippases for Bacterial Peptidoglycan Biosynthesis

Abstract

The biosynthesis of cellular polysaccharides and glycoconjugates often involves lipid-linked intermediates that need to be translocated across membranes. Essential pathways such as N-glycosylation in eukaryotes and biogenesis of the peptidoglycan (PG) cell wall in bacteria share a common strategy where nucleotide-sugars are used to build a membrane-bound oligosaccharide precursor that is linked to a phosphorylated isoprenoid lipid. Once made, these lipid-linked intermediates must be translocated across a membrane so that they can serve as substrates in a different cellular compartment. How translocation occurs is poorly understood, although it clearly requires a transporter or flippase. Identification of these transporters is notoriously difficult, and, in particular, the identity of the flippase of lipid II, an intermediate required for PG biogenesis, has been the subject of much debate. Here, I will review the body of work that has recently fueled this controversy, centered on proposed flippase candidates FtsW, MurJ, and AmJ.

Keywords: MATE transporter; MOP exporter; MviN; YdaH; murein.

Figure 1

Structure of lipid II from E. coli. Undecaprenol is linked by a pyrophosphate to the PG building block composed of a GlcNAc-MurNAc disaccharide and an l-Ala-γ-d-Glu-A2pm-d-Ala-d-Ala stem pentapeptide. Abbreviations: GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid; A2pm, meso-diaminopimelic acid.

Figure 2

Schematic of the PG biogenesis pathway in E. coli. Synthesis of PG precursors begins in the cytoplasm where nucleotide-linked precursors UDP-N-acetylglucosamine and UDP-N-acetylmuramic acid-l-Ala-γ-d-Glu-A2pm-d-Ala-d-Ala are made. The latter precursor is linked to Und-P by MraY to generate lipid I. Then, MurG utilizes lipid I and UDP-N-acetylglucosamine to synthesize lipid II. A lipid II flippase translocates lipid II across the inner membrane (IM) so that transglycosylases (TG) can polymerize the disaccharide-pentapeptide into glycan chains. In addition, TPs catalyze peptide bonds between stem peptides that are properly oriented in adjacent glycan chains, while CPs remove the terminal d-Ala residue of stem peptides. For more detailed description, refer to relevant reviews.,,

Figure 3

Assay to measure in vitro the translocation of lipid II in liposomes. (A) As previously described,, liposomes (unilamellar vesicles) are first loaded with NBD-lipid II, which distributes symmetrically in both leaflets (stage I). At this stage, all NBD-lipid II can fluoresce, so fluorescence signal (solid blue line in graph) from NBD (green star) is maximal. Then, addition of dithionite (red circle, stage II) reduces the NBD in lipid II molecules localized in the outer leaflet of the vesicle to nonfluorescent ABD (gray star), causing a reduction in fluorescence. Residual fluorescence is completely eliminated upon treatment of vesicles with detergents in the presence of dithionite (stage III). The solid blue line in the graph represents the fluorescence signal obtained in liposomes lacking flippase activity, while the dotted green line corresponds to the fluorescence signal obtained in liposomes containing a flippase (as shown in B). (B) An illustration of how addition of a lipid II flippase (green oval) to liposomes preloaded with NBD-lipid II can drive translocation of the lipid across the bilayer (stage II, A), causing a decrease in the fluorescence signal (dotted green line, A) in the presence of dithionite.

Figure 4

A structural model of MurJ. The front view of the model structure of MurJ of E. coli showing the central cavity opened toward the periplasm. The cavity is mainly lined by TMDs 1 (blue), 2 (cyan), 7 (magenta), and 8 (red).

Figure 5

Assay to measure in vivo the translocation of lipid II in E. coli cells. (A) As previously described,, purified toxin ColM is added to actively growing E. coli cells. ColM crosses the OM to enter the periplasm, where it inhibits PG biogenesis by cleaving lipid II that has been flipped to the periplasm (lipid II_periplasmic). ColM cleaves lipid II into membrane-bound undecaprenol and soluble PP-disaccharide-pentapeptide, which is further converted by periplasmic CPs into PP-disaccharide-tetrapeptide (marked by blue box). (B) A schematic showing the experimental details of the assay., PG precursors are specifically labeled with ³H-meso-diaminopimelic acid (³H-DAP) and then treated or not with ColM. Before cell lysis occurs (drop in growth curve), cells are collected and extracted with boiling water. Species in the water-soluble fraction are separated using high-pressure liquid chromatography and radioactivity present in the ColM disaccharide-tetrapeptide product (blue box, A) is then measured. Radiolabeled lipid II that is not cleaved by ColM (lipid II_cytoplasmic) is measured after extracting the water-insoluble fraction with butanol. When flippase activity is not impaired, treatment with ColM leads to the appearance of signal in the fraction corresponding to the ColM product and the disappearance of signal from the lipid II_cytoplasmic fraction. When flippase activity is inhibited, the amount of signal in the lipid II_cytoplasmic fraction increases and the treatment of ColM does not lead to the appearance of signal corresponding to the ColM product.