Protein–Protein Interactions Between Peptidoglycan, Lipopolysaccharide, and Phospholipid Biosynthesis Enzymes in Escherichia coli

Gram-negative bacteria are protected by a three-layered cell envelope and must tightly coordinate the biosynthesis of phospholipids (PL), peptidoglycan (PG) and lipopolysaccharides (LPS) to maintain envelope integrity. In Escherichia coli, the inner membrane protein LapB (YciM) plays a critical dual role in LPS homeostasis by acting as a scaffold for cytoplasmic LPS and PL biosynthesis enzymes and by promoting FtsH-dependent proteolysis of the key enzyme LpxC. Because LPS and PG biosynthesis compete for the shared precursor UDP-GlcNAc, we investigated whether MurA, catalyzing the first committed step of PG biosynthesis, is an integral component of the LapB complex. Using bacterial two-hybrid analysis, pull-down assays and microscale thermophoresis, we demonstrate that MurA directly interacts with LapB, LpxA, LpxC, LpxD, and FabZ. Together, these findings support a model in which PL, PG, and LPS biosynthesis enzymes are engaged in a protein-protein interaction hub that synchronizes the production of all three layers of the Gram-negative cell envelope in E. coli.