Functional Analysis of the Protein Machinery Required for Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli

Abstract

The cell envelope of gram-negative bacteria consists of an inner (IM) and an outer membrane (OM) separated by an aqueous compartment, the periplasm, which contains the peptidoglycan layer. The OM is an asymmetric bilayer, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) facing outward (29, 32). The OM is an effective permeability barrier that protects the cells from toxic compounds, such as antibiotics and detergents, thus allowing bacteria to inhabit several different and often hostile environments. LPS is responsible of most of the permeability properties of the OM and consists of the lipid A moiety (a glucosamine-based phospholipid) linked to the short core oligosaccharide and the distal O-antigen polysaccharide chain. The core oligosaccharide can be further divided into an inner core, composed of 3-deoxy-Dmanno-octulosanate (KDO) and heptose, and an outer core, which has a somewhat variable structure. LPS is essential in most gram-negative bacteria, with the notable exception of Neisseria meningitidis (39).The biogenesis of the OM implies that the individual components are transported from the site of synthesis to their final destination outside the IM by crossing both hydrophilic and hydrophobic compartments. The machinery and the energy source that drive this process are not yet understood.The lipid A-core moiety and the O-antigen repeat units are synthesized at the cytoplasmic face of the IM and are separately exported via two independent transport systems, namely, the O-antigen transporter Wzx (13, 17) and the ATP binding cassette (ABC) transporter MsbA that flips the lipid A-core moiety from the inner leaflet to the outer leaflet of the IM (12,28,45). O-antigen repeat units are then polymerized in the periplasm by the Wzy polymerase and ligated to the lipid A-core moiety by the WaaL ligase (reference 29 and references therein). Escherichia coli K-12 LPS is missing the O antigen, as an IS5 insertion disrupts its synthesis (18). Very recently, a modified LPS in which repeating units of colanic acid, a cell surface polysaccharide synthesized by enteric bacteria in the presence of envelope-damaging stresses (42), are ligated to the core oligosaccharide in a WaaL-dependent manner has been described (21).How LPS reaches the OM is less well understood. A protein complex in the OM of E. coli composed of LptD (formerly Imp), an essential ␤-barrel OM protein (6), and LptE (formerly RlpB), an essential OM lipoprotein, has recently been implicated in LPS assembly (43). Depletion of either protein results in similar OM biogenesis defects, including increased LPS levels, abnormal membrane structures, and activation of the OM enzyme PagP (43). These findings indicate that the LptD/LptE complex is responsible for LPS assembly at the outer surface of the OM (43). LptD has also been shown to be

Nonconsecutive disulfide bond formation in an essential integral outer membrane protein

“…To determine whether the defect in LptD biogenesis is specific to the loss of LptE, we analyzed cells depleted for different Lpt factors, because depletion of any Lpt factor results in the same lethal envelope defects that could indirectly compromise disulfide bond formation (26,38). An LptF/G double depletion strain (39) revealed that depletion of LptFG does not result in defective oxidation of LptD (Fig.…”

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

“…LptE is anchored to the inner leaflet of the OM via an N-terminal lipid moiety. To understand the role of this lipid anchor in bringing the LptD/E complex together in vivo, we engineered a plasmid in which the coding sequencing of LptE, excluding its signal sequence and the N-terminal lipidated cysteine (a.a. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], is placed after the sequence encoding the pelB leader peptide. This plasmid constitutively expresses a soluble version of LptE in the periplasm that is no longer lipidated at the N terminus.…”

“…1). Current models propose that another ABC transporter consisting of LptB, LptC, LptF, and LptG (formerly YhbG, YrbK, YjgP, and YjgQ, respectively) extracts LPS from the IM, initiating the transport and assembly of LPS to its final destination (9)(10)(11)(12). Because LPS is amphipathic, it is unlikely to diffuse across the periplasm unassisted.…”

“…Because LPS is amphipathic, it is unlikely to diffuse across the periplasm unassisted. LptA (formerly YhbN), a periplasmic protein, is thought to mediate LPS transit across this aqueous compartment by an unknown mechanism (9,10,(13)(14)(15). Once LPS arrives at the OM, a protein complex consisting of LptD and LptE (formerly Imp and RlpB, resp.)…”

Mechanisms of O-Antigen Structural Variation of Bacterial Lipopolysaccharide (LPS)

“…For example, in the case of the transporter ABC, LptBFG and LptA and LptC translocate the LPS to the internal side of the external membrane such that the proteins LptD and LptE place it on the surface of the membrane. It has been observed an absence of LptA or LptB or both causes the accumulation of LPS in the periplasm [11][12][13][14][15][16][17].…”