Cytoplasmic ATP hydrolysis powers transport of lipopolysaccharide across the periplasm in E. coli

Abstract

Millions of molecules of lipopolysaccharide (LPS) must be assembled on the Escherichia coli cell surface each time the cell divides. The biogenesis of LPS requires seven essential lipopolysaccharide transport (Lpt) proteins to move LPS from the inner membrane through the periplasm to the cell surface. However, no intermediate transport states have been observed. We developed methods to observe intermediate LPS molecules bound to Lpt proteins in the process of being transported in vivo. Movement of individual LPS molecules along these binding sites required multiple rounds of adenosine triphosphate (ATP) hydrolysis in vitro, which suggests that ATP is used to push a continuous stream of LPS through a transenvelope bridge in discrete steps against a concentration gradient.

Fig. 1. LPS crosslinks to the inner surface of LptC and LptA in vivo

(A) Cartoon of LPS transport in E. coli. MsbA flips LPS across the IM, and seven Lpt proteins transport it to the cell surface. (B) Specific amino acid positions in LptC crosslinked to LPS. In His-tagged LptC, eight residues on the inside surface, 11 residues on the outside surface, and four residues in the disordered region (shown as a dashed line) were mutated to incorporate pBPA. Four positions (red), three on the inside surface and one in the disordered region, crosslinked to LPS upon UV-irradiation. Crosslinking adducts (LPS X LptC) were detected by nickel affinity chromatography followed by immunoblotting with anti-LPS antibodies. Non-crosslinked LPS was also detected. (C) Specific amino acid positions in LptA crosslinked to LPS. As in (B), six and eight positions on the inside and outside surfaces, respectively, of the β-jellyroll were mutated to pBPA and evaluated following UV-irradiation by immunoblotting. Five specific positions (red) on the inside surface of LptA crosslinked to LPS.

Fig. 2. LPS cross-linking in LptA and LptC depends on the IM components of the Lpt machine

(A) LPS accumulates near residue T47 of LptC in LptBFGC complexes. LptC mutants that crosslink to LPS were overexpressed with or without co-overexpression of LptBFG. Crosslinking was detected similarly to Fig. 1B. Crosslinking at position 47 significantly increases when LptBFG are co-overexpressed. (B) LPS accumulates at several positions of LptA in LptBFGC-dependent manner. In analogous method to that in (A), LptA mutants that crosslink to LPS were overexpressed with or without co-overexpression of LptBFGC. Crosslinking at all five positions increases when LptBFGC are co-overexpressed.

Fig. 3. LPS transport can be reconstituted in vitro

(A) LPS accumulates in LptC in membrane vesicles over time in an LptBFG and ATP-dependent manner. RSO membrane vesicles containing overexpressed LptC(T47pBPA) with or without co-overexpression of LptBFG were prepared in the presence or absence of ATP. After incubation at 30°C for time indicated, the vesicles were UV-irradiated. Crosslinking was detected as in Fig. 1B. (B) LPS is released to LptA in an LptBFGC, time, and ATP-dependent manner. RSO membrane vesicles were prepared from wild-type cells or cells overexpressing LptC, LptBFG, or LptBFGC with or without added ATP. Purified LptA(I36pBPA or H37pBPA) was added to these vesicles, incubated at 30°C for time indicated, and then UV-irradiated. (C) Model of the stacked crystal structures of LptC (green) and LptA (purple). Residues I36 and H37 in LptA, which interact with LPS and LptC, respectively, are depicted as stick structures. (D) Periplasmic bridge components properly assemble in vitro. Purified LptA(I36pBPA or H37pBPA) was added to LptC- or LptBFGC-enriched RSO membrane vesicles. Samples were incubated at 30°C, and UV-irradiated. LptA(H37pBPA) crosslinked to LptC in an ATP, time, and LptBFG-independent manner.

Fig. 4. LPS is pushed in a continuous stream through the Lpt bridge

(A) Inhibiting ATP hydrolysis inhibits transfer of LPS from LptC to LptA. RSO membrane vesicles overexpressing LptBFG-LptC(T47pBPA) were incubated at 30°C for 30 min in the presence of ATP to accumulate LPS in LptC. Purified LptA(I36pBPA) was then added with or without vanadate, and the samples were incubated for an additional 60 min. Crosslinking was detected as in Fig. 1B. (B) Model of LPS biogenesis. LptBFG extracts LPS from the IM and transports it to LptC using ATP hydrolysis energy. ATP hydrolysis is used again to push LPS from LptC to LptA.