uridine-diphosphate-n-acetylmuramic-acid and (2-sulfonatoethyl)methanethiosulfonate

Peptidoglycan (PG) is the unique cell shape-determining component of the bacterial envelope, and is a key target for antibiotics. PG synthesis requires the transmembrane movement of the precursor lipid II, and MurJ has been shown to provide this activity in Escherichia coli. However, how MurJ functions in vivo has not been reported. Here we show that MurJ localizes both in the lateral membrane and at midcell, and is recruited to midcell simultaneously with late-localizing divisome proteins and proteins MraY and MurG. MurJ septal localization is dependent on the presence of a complete and active divisome, lipid II synthesis and PBP3/FtsW activities. Inactivation of MurJ, either directly by mutation or through binding with MTSES, did not affect the midcell localization of MurJ. Our study visualizes MurJ localization in vivo and reveals a possible mechanism of MurJ recruitment during cell division.

Topics: Bacterial Proteins; Cell Division; Cell Wall; Escherichia coli; Escherichia coli Proteins; Membrane Proteins; Mesylates; Peptidoglycan; Phospholipid Transfer Proteins; Transferases; Transferases (Other Substituted Phosphate Groups); Uridine Diphosphate N-Acetylmuramic Acid

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.

Topics: Cell Wall; Escherichia coli; Escherichia coli Proteins; Mesylates; Models, Molecular; Peptidoglycan; Phospholipid Transfer Proteins; Protein Conformation; Uridine Diphosphate N-Acetylmuramic Acid