Identification of a protein complex that assembles lipopolysaccharide in the outer membrane of Escherichia coli

Abstract

The outer membrane of most Gram-negative bacteria is made up of LPS, and in nearly all bacteria that contain LPS it is essential for the life of the organism. The lipid portion of this molecule, lipid A, also known as endotoxin, is a potent activator of the innate immune response. More than 50 genes are required to synthesize LPS and assemble it at the cell surface. Enormous progress has been made in elucidating the structure and biosynthesis of LPS, but until recently the cellular components required for its transport from its site of synthesis in the inner membrane to its final cellular location at the cell surface remained elusive. Here we describe the identification of a protein complex that functions to assemble LPS at the surface of the cell. This complex contains two proteins: Imp, already identified as an essential outer-membrane protein implicated in LPS assembly; and another protein, RlpB, heretofore identified only as a rare lipoprotein. We show that RlpB is also essential for cell viability and that the Imp/RlpB complex is responsible for LPS reaching the outer surface of the outer membrane.

Fig. 1.

Imp copurifies with RlpB. (A) Ni-NTA column-enriched proteins from OM extracts of wild-type cells (lane 2) and wild-type cells containing the pImp-His plasmid (lane 3). The band appearing in lane 3 that is not present in lane 2 was determined to be RlpB by tandem MS. (B) Immunoprecipitated samples from whole-cell extracts prepared from wild-type cells (lane 2) and wild-type cells containing the pRlpB-His plasmid (lane 3) by using a monoclonal anti-His antibody. The protein labeled as Imp was confirmed by immunoblot. Samples were subjected to SDS/PAGE and stained with silver (A) or Coomassie blue (B). The relevant proteins that are described are labeled with arrows. Molecular mass markers (lane 1) are indicated in kDa.

Fig. 2.

Test to determine whether RlpB is essential. (A) Strain AM689 expresses rlpB from an arabinose-inducible promoter at the λatt site, allowing knockout of the endogenous rlpB gene. (B) Growth curve of AM689 grown in the presence (diamonds) or absence (squares) of arabinose as measured by optical density.

Fig. 3.

Fractionation of wild-type, Imp, RlpB, and YaeT depletion strains. Strains indicated were fractionated by sucrose density gradients as described and submitted to immunoblots using antibodies recognizing LPS, Imp, and LamB as indicated. (A) Wild-type cells grown in LB. (B) MB215 cells depleted of Imp. (C) AM689 cells depleted of RlpB. (D) JCM166 cells depleted of YaeT. Fractions were collected and loaded such that denser fractions are on the left with fractions of lighter density on the right. The 55-kDa protein is an IM protein that reacts with the Imp antibody (13). The 32-kDa protein that reacts with the Imp antibody is OmpA. The 55-kDa protein remains visible in the LamB blots because they were performed sequentially.

Fig. 4.

Immunoblots to detect LPS levels in Imp/RlpB-depleted cells and electron micrographs stained to visualize membranes in these cells. (A) MB215 was grown in the presence or absence of IPTG as indicated. Samples were taken at the time points indicated, OD-normalized, and analyzed for total cellular LPS levels by SDS/PAGE and immunoblots by using a monoclonal LPS antibody. The decrease in LPS levels at the 10-h time point occurs because of suppressor mutations that allow Imp production in the absence of inducer. (B) AM689 was grown in the presence or absence of arabinose and prepared as described for A. (C and D) Imp-depleted cells (C) and RlpB-depleted cells (D) prepared for electron microscopy according to the method of Ogura et al. (25).

Fig. 5.

LPS is modified by PagP when Imp/RlpB is depleted. Negative ion MS of lipid A species were obtained from different strains. (A) Wild-type strain MC4100 was treated with EDTA during growth following the protocol of Jia et al. (26). (B) The Imp depletion strain MB215 was grown in the absence of IPTG. (C) The RlpB depletion strain AM689 was grown in the absence of arabinose. Lipid A extraction and MS analysis followed the protocol of Zhou et al. (27).

Fig. 6.

Cell-surface accessibility of de novo synthesized LPS in Imp/RlpB-depleted cells. The Imp depletion strain MB215 (A) or the RlpB depletion strain AM689 (B) was grown with (lanes 1 and 2) or without (lanes 3 and 4) inducer. [1-¹⁴C]Acetate was added at the beginning of subculturing to label steady-state LPS (lanes 1–3) or added after Imp/RlpB was depleted (lane 4) to label de novo synthesized LPS. Of the nondepleted cultures, one was treated with EDTA following the protocol of Jia et al. (26) (lane 2) to serve as a positive control for the position of the hepta-acyl lipid A. Lipid A was isolated and analyzed by thin layer chromatography, followed by autoradiography as in Zhou et al. (27). Equal amounts of radioactive material were spotted for each sample.