Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

Abstract

The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level of lptB is a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries Pa-LptC, the highly divergent LptC orthologue from Pseudomonas aeruginosa We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carrying Pa-LptC which is otherwise defective in interactions with the E. coli LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery.

Importance: The asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. In Escherichia coli the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine.

FIG 1

Comparison of E. coli and P. aeruginosa LptC amino acid sequences and structures. Amino acid sequence alignment of LptC from E. coli (Ec-LptC) and P. aeruginosa (Pa-LptC). Amino acid identity (asterisk) and similarity (colon and period) are labeled. Leader peptide (LP) and transmembrane regions are indicated. Regions 1, 2, and 3 swapped in chimera constructions are delimited by arrowheads at the end of double underlining. Sequences corresponding to the MEME motifs are overlined and color coded as follows: blue, motif 1 (residues 94 to 143); red, motif 2 (residues 37 to 81); green, motif 3 (residues 152 to 180). Relevant LptC mutations are indicated above and below the E. coli and P. aeruginosa sequences, respectively. Arrows indicate the mutated amino acid (for point mutations), the first deleted amino acid (for deletions), and the first amino acid at the right of the inserted transposon. ST-190, transposon insertion in ST-190 (24); 23553 and 39714 indicate two transposon insertions in Pa-lptC (http://ausubellab.mgh.harvard.edu). o and o indicate amino acids photo-cross-linked to LPS and LptA, respectively (9, 16). Tilde marks indicate amino acids in β strands, progressively numbered (17).

FIG 2

Complementation test of LptC depletion mutants with different E. coli and P. aeruginosa wild-type or chimeric LptC constructs. Cultures of FL905 (araBp-lptC) strains freshly transformed with pGS100 derivatives expressing Ec-LptC (pGS103, CCC), Pa-LptC (pGS111, PPP), LptC chimeras (pGS201, CPP; pGS202, PCC; pGS203, CPC; pGS204, PCP; pGS206, CCP; pGS207, PPC), or a truncated Ec-LptC protein missing region 3 (pGS208, CCΔ) and grown in LD-chloramphenicol-arabinose were serially diluted 1:10 in microtiter wells and replica plated in agar plates with (+ ara) or without (− ara) arabinose or with glucose (+ glu) to fully repress the araBp promoter. The log of the serial dilutions is indicated on the right of the panel.

FIG 3

Assembly of the Lpt complex by Pa-LptC and LptC chimeras. Dodecyl β-d-maltoside-solubilized total membranes from AMM04 strains harboring pGS100 derivatives expressing the His-tagged proteins of interest were affinity purified using a Talon metal affinity resin, as described in Materials and Methods. Proteins were then fractionated by SDS-PAGE and immunoblotted with suitable antibodies to detect the corresponding proteins in the fraction. (A) Assembly of the Lpt IM complex. Samples were prepared from AMM04 harboring the pGS100 derivatives pGS108 (expressing Ec-LptC-H, CCC), pGS200 (expressing Pa-LptC-H, PPP), or those expressing His-tagged LptC chimeras, namely, pGS201H (CPP-H), pGS202H (PCC-H), pGS203H (CPC-H), pGS204H (PCP-H), pGS206H (CCP-H), and pGS207H (PPC-H). Immunoblotting was performed with anti-LptF and anti-His antibodies to detect LptF and the different His-tagged LptC forms, respectively, which display different electrophoretic mobility on SDS-PAGE. (B) Assembly of the transenvelope Lpt complex. Samples were prepared from AM604 harboring pGS108 (Ec-LptC-H, CCC), pGS200 (Pa-LptC-H, PPP), the His-tagged LptC CCP chimera (pGS206H), or pGS100 expressing the His tag (−) as a negative control. Immunoblotting was performed with the antibodies anti-His (to detect the different LptC forms) and anti-LptA, anti-LptB, anti-LptF, and anti-LptD.

FIG 4

Growth of the ST-190 conditional expression mutant with different levels of LptA and/or LptB expression. Cultures of ST-190 transformed with pGS100 derivatives expressing the genes indicated on the top of each lane (lptC carried on pGS103; lptAB carried on pGS416; lptA carried on pGS321; lptB carried on pGS428) and grown in LD-chloramphenicol-arabinose were serially diluted 1:10 in microtiter wells and replica plated on agar plates supplemented (+ ara) or not supplemented (− ara) with arabinose. The log of the serial dilutions is indicated on the right of the panel.

FIG 5

Expression levels of LptA, LptB, and LptC or LptC^Δ139-191 proteins in FL905 upon depletion of the chromosomally encoded LptC. FL905 cells transformed with pGS100 (−) or pGS100 derivatives harboring wild-type LptC (C, carried by pGS103) or LptC^Δ139-191 (C^Δ, carried by pGS417) as indicated on the left of panels A and B, respectively, coexpressed with LptA (A, carried by pGS404 and pGS418, respectively), LptB (B, carried by pGS429 and pGS431, respectively), or LptAB (AB, carried by pGS415 and pGS419, respectively), as indicated on the top of the lanes, were grown in LD in the presence (+ ara) or absence (− ara) of arabinose and chloramphenicol, as described in Materials and Methods. Samples collected 4 h after a shift to the nonpermissive condition were analyzed by Western blotting using anti-LptA, anti-LptB, anti-LptC, and anti-LptE antibodies. An equal amount of cells (OD₆₀₀, 0.6) was loaded onto each lane. The migrations of full-length LptC and LptC^Δ are indicated on the left side of panel B. The last lane of each image in panel B (C^Δ + IPTG) was loaded with 10 μl of cell extract of FL905 harboring pGS417 (LptC^Δ139-191) arabinose depleted for 3 h and further incubated 1 h with 0.1 mM IPTG to induce expression of the truncated LptC protein. LptE level was used as a sample loading control.

FIG 6

Rescue of FL905 growth by overexpression of truncated Ec-lptC or Pa-lptC alleles. FL905 cells transformed with pGS100 (−) or pGS100 derivatives harboring wild-type LptC (pGS103), LptC^Δ139-191 (pGS417), LptC190N (pGS408), or Pa-LptC (pGS111), as indicated on top of the lanes, were grown in LD-chloramphenicol-arabinose, serially diluted 1:10 in microtiter wells, and replica plated on LD-agar plates with chloramphenicol and with (+ ara) or without (− ara) arabinose and with IPTG (+ IPTG) to induce the lptC allele on the plasmids, as described in Materials and Methods. The log of the serial dilutions is indicated on the right.

FIG 7

Suppression of LptC defects by lptB mutants. FL905 cells transformed with pGS100 (−) or pGS100 derivatives harboring wild-type LptC (C, carried by pGS103) or LptC^Δ139-191 (C^Δ, pGS417) coexpressed with LptB (B, carried by pGS429 and pGS431, respectively), LptB^F90A (B^F90A carried by pGS429-LptB^F90A and pGS431-LptB^F90A, respectively), LptB^F90Y (B^F90Y, carried by pGS429-LptB^F90Y and pGS431-LptB^F90Y, respectively), or LptB^E163Q (B^E163Q, carried by pGS429-LptB^E163Q and pGS431-LptB^E163Q, respectively), as indicated on top of the lanes, were grown in LD-chloramphenicol-arabinose, serially diluted 1:10 in microtiter wells, and replica plated on LD-agar plates with chloramphenicol in the presence (+ ara) or in the absence (− ara) of arabinose, as described in Materials and Methods. The log of the serial dilutions is indicated on the right.