Periplasmic phosphorylation of lipid A is linked to the synthesis of undecaprenyl phosphate

M Stephen Trent,Dominique Mengin‐Lecreulx,Thierry Touzé,An X Tran,Jessica V Hankins

doi:10.1111/j.1365-2958.2007.06044.x

Abstract

One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1 (lipid A 1-diphosphate). We now report an inner membrane enzyme, LpxT (YeiU), which specifically transfers a phosphate group to lipid A, forming the 1-diphosphate species. 32P-labelled lipid A obtained from lpxT mutants do not produce lipid A 1-diphosphate. In vitro assays with Kdo2-[4′-32P]lipid A as the acceptor shows that LpxT uses undecaprenyl pyrophosphate as the substrate donor. Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria. LpxT-catalysed phosphorylation is dependent upon transport of lipid A across the inner membrane by MsbA, a lipid A flippase, indicating a periplasmic active site. In conclusion, we demonstrate a novel pathway in the periplasmic modification of lipid A that is directly linked to the synthesis of undecaprenyl phosphate, an essential carrier lipid required for the synthesis of various bacterial polymers, such as peptidoglycan.

Highlights

The Gram-negative bacterial cell envelope consists of an inner membrane, an outer membrane and the periplasmic region (Nikaido, 2003)
One-third of the lipid A found in the Escherichia coli outer membrane contains an unsubstituted diphosphate unit at position 1
Inhibition of lipid A 1-diphosphate formation in wild-type bacteria was demonstrated by sequestering undecaprenyl pyrophosphate with the cyclic polypeptide antibiotic bacitracin, providing evidence that undecaprenyl pyrophosphate serves as the donor substrate within whole bacteria

Summary

Introduction

The Gram-negative bacterial cell envelope consists of an inner membrane, an outer membrane and the periplasmic region (Nikaido, 2003). Within the periplasm resides a continuous cross-linked carbohydrate polymer that forms a homogeneous layer outside the cytoplasmic membrane, known as the peptidoglycan layer (Schleifer and Kandler, 1972; van Heijenoort, 2001a). Both LPS and peptidoglycan are essential for maintaining the structural integrity of the Gram-negative cell envelope, and are generally required for viability. Biosynthesis of LPS and peptidoglycan, as well as other bacterial cell wall polymers, requires an essential carrier lipid, undecaprenyl phosphate (C55-P) (Fig. 1). An example of C55-P synthesis during the biosynthesis of peptidoglycan is shown in Fig. 1 (van Heijenoort, 2001a)

Results

Discussion

Conclusion