The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis

Abstract

Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C₅₅-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that <i>Aquifex aeolicus</i> LpxE structurally resembles YodM in <i>Bacillus subtilis</i>, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak <i>in vitro</i> activity on C₅₅-PP, and rescues <i>Escherichia coli</i> deficient in PGP and C₅₅-PP phosphatase activities; deletion of <i>lpxE</i> in <i>Francisella novicida</i> reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C₅₅-PP phosphatase activity. Suppression of plasmid-borne <i>lpxE</i> in <i>F. novicida</i> deficient in chromosomally encoded C₅₅-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope.<b>IMPORTANCE</b> Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.