Lipoprotein LptE is required for the assembly of LptD by the beta-barrel assembly machine in the outer membrane of Escherichia coli

Abstract

Most Gram-negative bacteria contain lipopolysaccharide (LPS), a glucosamine-based phospholipid, in the outer leaflet of the outer membrane (OM). LPS is unique to the bacterial OM and, in most cases, essential for cell viability. Transport of LPS from its site of synthesis to the cell surface requires eight essential proteins, MsbA and LptABCDEFG. Although the key players have been identified, the mechanism of LPS transport and assembly is not clear. The stable LptD/E complex is present at the OM and functions in the final stages of LPS assembly. Here, we have identified the mutant allele lptE6, which causes a two-amino-acid deletion in the lipoprotein LptE that affects its interaction with LptD. Highly specific suppressor mutations were isolated not only in lptD but also in bamA, which encodes the central component of the β-barrel assembly machine. We show that lptE6 and both suppressor mutations affect the assembly of the LptD/E complex and suggest that the lipoprotein LptE interacts with LptD while this protein is being assembled by the β-barrel assembly machine.

Fig. 1.

Levels of LptE and LptD. Whole-cell samples were obtained from AM604, AM604 ΔlptE (plptE), AM604 ΔlptE (plptE^low), AM604 ΔlptE (plptE6), AM604 ΔlptE lptD7 (plptE6), and AM604 ΔlptE bamA8 (plptE6) cells that were grown to stationary phase and subjected to SDS/PAGE and immunoblotting for LptE (Top), LptD (Middle), and DegP (Bottom). The asterisk indicates degradation products of DegP.

Fig. 2.

The lptE6 mutation directly affects the interaction between LptE and LptD. Ni-NTA affinity purification is shown, using AM604 containing pET23/42 (lane1), pET23/42scT-lptE-his (lane 2), or pET23/42lptE6-his (lane 3). Samples before (whole-cell lysates) and after (Ni-NTA eluates) purification were subjected to immunoblotting using anti-LptD and anti-His antibodies. Positions of relevant molecular markers are indicated in kilodaltons.

Fig. 3.

The suppressors of lptE6 improve LptD assembly. Whole-cell samples from AM604 ΔlptE (plptE), AM604 ΔlptE (plptE6), AM604 ΔlptE lptD7 (plptE6), and AM604 ΔlptE bamA8 (plptE6) grown to an OD₆₀₀ of ∼1.0 were boiled in the presence (Upper) or the absence of β-ME (Lower) and Western blot analysis of LptD was carried out to detect reduced LptD (LptD_RED) or oxidized LptD (LptD_OX), respectively.

Fig. 4.

Effects of lptD7 and bamA8 on LptD and LptE levels. Western blot analysis is shown of LptD and LptE and DegP on whole-cell samples prepared from AM604, AM604 lptD7, and AM604 bamA8 after overnight growth. The asterisk indicates degradation products of DegP.

Fig. 5.

Genetic interactions between lpt and bam. (A) Genetic interactions (dashed lines) between genes encoding members of the Lpt and Bam complexes exist. lptD4213 is suppressed by mutations in bamA and bamB (27, 28). lptE6 is suppressed by mutations in lptD and bamA. Physical interactions in the LptDE (12) and BamABCDE (30, 40) complexes are shown with solid lines. (B) LptE and LptD are targeted to the OM by different pathways. Lipoprotein LptE is targeted to LolB by the Lol pathway (26). OMP LptD is targeted to the BamABCDE complex (24). Defects in LptD assembly caused by the presence of LptE6 are suppressed by mutations in the gene encoding BamA, suggesting that LptD and LptE interact at the Bam complex during biogenesis of the LptDE complex.