Functional analysis of the protein machinery required for transport of lipopolysaccharide to the outer membrane of Escherichia coli

Abstract

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

FIG. 1.

Subcellular localization of LptC. BW25113/pGS108 cells were induced with IPTG, disrupted, and fractionated as described in Materials and Methods. Samples of periplasmic (P), cytoplasmic (S), IM, and OM fractions were analyzed by SDS-PAGE and Western blotting with anti-His₆ antibodies (upper panel). The same fractions were analyzed with anti-YidC antibodies as an IM marker (lower panel). T, total protein fraction.

FIG. 2.

Cell morphology upon depletion of LptA-LptB, LptE, LptD, and LptC. Cells grown in the presence (+) or in the absence (−) of arabinose were prepared for electron microscopy as described in Materials and Methods. Scale bars, 0.5 μm.

FIG. 3.

Membrane fractionation of cells depleted of LptA-LptB, LptD, LptE, and LptC. FL907, AM661, AM689, and FL905 cultures were grown with arabinose to an OD₆₀₀ of 0.2, harvested, and resuspended in an arabinose-supplemented or arabinose-free medium. About 1 h after the cultures had reached the maximal OD₆₀₀ (OD₆₀₀ between 0.2 and 0.6), cells were pulse-labeled for 2 min with [³H]GlnNAc and chased for 5 min with 0.4% nonradioactive GlnNAc; the nondepleted cultures were pulse-labeled when the same OD₆₀₀ was reached, as described in Materials and Methods. Total membranes prepared from cells were fractionated by sucrose density gradient. Fractions were collected from the top of the gradient and immunoblotted using antibodies recognizing LPS, LamB, and a 55-kDa IM protein as indicated. Fractions were also analyzed for total incorporated radioactivity. The panels on the left show the percentages of the total incorporated radioactivity for nondepleted (▪) and depleted (○) mutant cells. The panels on the right show the LamB and OmpA profiles of nondepleted (+ ara) and depleted (− ara) mutant cells. The OmpA protein cross-reacts with the LamB antibody. (A) FL907 cells depleted and not depleted of LptA-LptB. (B) AM661 cells depleted and not depleted of LptD. (C) AM689 cells depleted and not depleted of LptE. (D) FL905 cells depleted and not depleted of LptC.

FIG. 4.

(a) MALDI-MS spectrum of the product OS1. The main molecular ion at m/z 1796.7 consists either of hexa-acylated lipid A or the complete core region of wild-type E. coli K-12 LPS, whereas the higher-molecular-mass ion peaks can be assigned to the same oligosaccharide that in addition bears one or more bis-acetylated colanic acid repeating units (Δm/z 1038) lacking a pyruvate group. (b) ¹H NMR spectrum of the O-deacetylated OS1 product, in which the great heterogeneity of the sample due to nonstoichiometric substitutions and reducing KDO arrangements is evident. (Inset) Anomeric assignments of the colanic acid single repeating unit as shown in Table S1 in the supplemental material. (c) Repeating unit of colanic acid. Residues are α-configured unless stated otherwise.

FIG. 5.

WaaL dependence of anomalous LPS production. The strains carrying a functional O-antigen ligase (+ waaL) or in which the O-antigen ligase is disrupted (− waaL) were grown with arabinose (+ ara) or without arabinose (− ara) as described in Material and Methods. The LPS profile was determined by Western blotting using anti-LPS WN1 222-5 antibodies. (A) LptE depletion, 300 min. (B) LptD depletion, 360 min. (C) LptAB depletion, 240 min. (D) LptC depletion, 360 min.

FIG. 6.

Model for the transport of LPS. The lipid A-core moiety is synthesized in the cytoplasm and flipped over the IM by MsbA. LptA, LptB, and LptC are part of a protein machine that transports LPS across the periplasm to the OM. The two additional transmembrane components recently identified (31), LptF and LptG, are postulated to complete the IM-bound ABC transporter. The LptD/LptE complex is thought to mediate the insertion of the newcomer LPS into the OM. The former names of the proteins are indicated in parentheses.