MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis

Abstract

Peptidoglycan (PG) is a polysaccharide matrix that protects bacteria from osmotic lysis. Inhibition of its biogenesis is a proven strategy for killing bacteria with antibiotics. The assembly of PG requires disaccharide-pentapeptide building blocks attached to a polyisoprene lipid carrier called lipid II. Although the stages of lipid II synthesis are known, the identity of the essential flippase that translocates it across the cytoplasmic membrane for PG polymerization is unclear. We developed an assay for lipid II flippase activity and used a chemical genetic strategy to rapidly and specifically block flippase function. We combined these approaches to demonstrate that MurJ is the lipid II flippase in Escherichia coli.

MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

“…Collectively, our data lead us to propose that Amj is the founding member of a new family of lipid II flippases. An additional conclusion that emerges from our study and previous work (6,8) is that the MOP family member SpoVB required for synthesis of spore cortex PG (27) is a lipid II flippase. It is unclear why B. subtilis possesses a sporulation-specific flippase, but one possibility is that the spatial and temporal requirements for PG synthesis during development cannot be met by the vegetative enzymes.…”

“…Tn-seq was performed as described previously (38,39). Lipid II flippase activity in E. coli was measured as described previously (6). Detailed protocols; tables of strains, plasmids, and oligonucleotide primers; and a description of strain and plasmid construction are provided in SI Experimental Procedures and Table S1.…”

“…The enzymes that transport lipid II across the membrane have been the subject of extensive research and speculation (3)(4)(5)(6). Recent work in Escherichia coli has provided strong evidence that the polytopic membrane protein MurJ is required for lipid II transport across the membrane, and is likely to be a lipid II flippase (6). MurJ is a member of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily (7).…”

“…The molecule is then translocated to the outer face of the membrane, where the disaccharide-peptide monomer is incorporated into the existing PG by cell wall synthetic machineries composed of penicillin-binding proteins and additional factors (2). The enzymes that transport lipid II across the membrane have been the subject of extensive research and speculation (3)(4)(5)(6). Recent work in Escherichia coli has provided strong evidence that the polytopic membrane protein MurJ is required for lipid II transport across the membrane, and is likely to be a lipid II flippase (6).…”

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development

“…Next, the FemXAB transpeptidases catalyse the coupling of a pentaglycin-moiety to the D-lysine of the pentapeptide u it a d fu the o e, the a idatio of the -D-glutamate of the pentapeptide unit by the MurT/GatD two-enzyme complex leading to the corresponding D-isoglutamin (Münch et al 2012;Zapun et al 2013). This modified lipid II now contains the complete peptidoglycan monomer linked via a pyrophosphate to the bactoprenol membrane anchor, which is translocated through the phospholipid membrane by the MurJ flippase and presented on the extracellular membrane surface (Mohammadi et al 2011;Butler et al 2013a;Sham et al 2014;Meeske et al 2015). On the extracellular membrane surface the peptidoglycan precursor is released, incorporated into the growing peptidoglycan layer and crosslinked (Zapun et al 2013) with each other catalyzed by the PBPs (Sauvage et al 2008).…”