MsbA transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner membrane: topography of francisella novicida LpxE expressed in Escherichia coli

Abstract

The lipid A anchor of Francisella tularensis lipopolysaccharide (LPS) lacks both phosphate groups present in Escherichia coli lipid A. Membranes of Francisella novicida (an environmental strain related to F. tularensis) contain enzymes that dephosphorylate lipid A and its precursors at the 1- and 4'-positions. We now report the cloning and characterization of a membrane-bound phosphatase of F. novicida that selectively dephosphorylates the 1-position. By transferring an F. novicida genomic DNA library into E. coli and selecting for low level polymyxin resistance, we isolated FnlpxE as the structural gene for the 1-phosphatase, an inner membrane enzyme of 239 amino acid residues. Expression of FnlpxE in a heptose-deficient mutant of E. coli caused massive accumulation of a previously uncharacterized LPS molecule, identified by mass spectrometry as 1-dephospho-Kdo2-lipid A. The predicted periplasmic orientation of the FnLpxE active site suggested that LPS export might be required for 1-dephosphorylation of lipid A. LPS and phospholipid export depend on the activity of MsbA, an essential inner membrane ABC transporter. Expression of FnlpxE in the msbA temperature-sensitive E. coli mutant WD2 resulted in 90% 1-dephosphorylation of lipid A at the permissive temperature (30 degrees C). However, the 1-phosphate group of newly synthesized lipid A was not cleaved at the nonpermissive temperature (44 degrees C). Our findings provide the first direct evidence that lipid A 1-dephosphorylation catalyzed by LpxE occurs on the periplasmic surface of the inner membrane.

Fig. 1. Structures of the lipid A and Kdo regions of LPS in E. coli and F. tularensis

Evidence for these structures has been presented elsewhere (3, 4, 36). A, the LPS molecules made by the heptose-deficient E. coli mutant WBB06; B, the Kdo-lipid A region of F. tularensis LPS; C, structure of the Kdo₂-[4′-³²P]lipid A substrate for the 1-phosphatase; D, structure of the Kdo₂-[4′-³²P]lipid IV_A used to detect both the 4′-phosphatase and the 1-phosphatase.

Fig. 2. Detection of lipid A 1-phosphatase and 4′-phosphatase activities in F. novicida U112 membranes

Membranes (0.5 mg/ml) of F. novicida U112 (lanes 2–5) or E. coli Novablue/pLpxE (10), which expresses the 1-phosphatase of R. leguminosarum (lanes 6 and 7), were assayed for the indicated times at 30 °C with 5 μMKdo₂-[4′-³²P]lipid IV_A (20,000 cpm/nmol) as the substrate (structure in Fig. 1D). The products were separated by TLC in the solvent chloroform, pyridine, 88% formic acid/water (30:70:16:10, v/v/v/v) and were detected with a PhosphorImager. The R_F of the 1-dephospho-Kdo₂-[4′-³²P]lipid IV_A was confirmed by comparison with the product generated with the previously characterized LpxE 1-phosphatase of R. leguminosarum, expressed in E. coli Novablue (lanes 6 and 7).

Fig. 3. Lipid A 1-phosphatase and Kdo trimming activities in membranes of F. novicida

A, membranes of different strains were assayed for 1-phosphatase activity with substrate Kdo₂-[4′-³²P]lipid A as the substrate. Lane 1, no enzyme; lane 2, F. novicida U112; lane 3, XW1/pXYW-1; lane 4, XL1-Blue/pACYC184; lane 5, Novablue/pET28b-FnLpxE; lane 6, Novablue/pET28b. The reaction was performed at 30 °C for 30 min with 0.2 mg/ml membranes. The TLC solvent was CHCl₃/methanol/acetic acid/water (25:25:4:4, v/v/v/v). B, the lag in the formation of 1-dephospho-Kdo lipid A by F. novicida membranes demonstrates that the Kdo-trimming enzyme prefers a substrate lacking the 1-phosphate group. The 1-dephospho-Kdo lipid A was identified by mass spectrometry based on its [M − H]⁻ at m/z 1938.9 and by its conversion to 1-dephospho-Kdo₂ lipid A in the presence of E. coli KdtA (65).

Fig. 4. Alignment of LpxE sequences from F. novicida and R. leguminosarum

The conserved residues are in boldface type. The predicted FnLpxE protein contains all three motifs of the lipid phosphatase superfamily (25), which are underlined. The proteins are 44% identical and 57% similar over 122 residues with one gap. The E value in a two-sequence comparison is 6 × 10–20. The F. novicida and F. tularensis proteins (not shown) are 98% identical over 239 amino acid residues.

Fig. 5. Lipid A 1-dephosphorylation in E. coli XL1-Blue expressing FnLpxE

A, SDS-PAGE analysis of 25-μg protein samples of the indicated membranes: XL1-Blue/pWSK29 (lane 1); XL1-Blue/pWSK29-FnLpxE (lane 2). The molecular mass of FnLpxE is about 27.5 kDa. B, cells expressing FnLpxE dephosphorylate over 90% of their endogenous lipid A, as judged by steady state ³²P-labeling and TLC of the lipid A species in the solvent CHCl₃, pyridine, 88% formic acid/water (50:50:16:5, v/v/v/v). Lipids were detected with a PhosphorImager. Lane 1, XL1-Blue/pWSK29; lane 2, XL1-Blue/pWSK29-FnLpxE.

Fig. 6. Lipid A 1-dephosphorylation by FnLpxE in a heptose-deficient mutant

The lipids were labeled uniformly with ³² and Pi extracted with a Bligh-Dyer mixture. The crude ³²P-labeled lipids were separated by TLC in chloroform/methanol/acetic acid/water (25:15:4:4, v/v/v/v) and detected with a PhosphorImager. Lane 1, a Kdo₂-[4′-³²P]lipid A standard; lane 2, lipids from WBB06; lane 3, lipids from WBB06/pWSK29-FnLpxE.

Fig. 7. MALDI-TOF mass spectrometry of purified 1-dephos-pho-Kdo₂-lipid A

The spectra were acquired in the negative mode (A) and the positive mode (B ). The molecular weight of 1-dephospho-Kdo₂-lipid A is 2157.4.

Fig. 8. Lipid A 1-dephosphorylation by FnLpxE is MsbA-dependent

FnLpxE expressed in the temperature-sensitive MsbA mutant WD2 (19) cannot dephosphorylate newly synthesized lipid A when MsbA is inactivated at 44 °C for 30 min. Lane 1, W3110A/pWSK29, 44 °C; lane 2, W3110A/pWSK29-FnLpxE, 44 °C; lane 3, WD2/pWSK29-FnLpxE, 44 °C; lane 4, WD2/pWSK29-FnLpxE, 30 °C; lane 5, WD2/pWSK29, 30 °C. The TLC of the lipid A species was carried out with the solvent CHCl₃, pyridine, 88% formic acid/water (50:50:16:5, v/v/v/v). The small amount of 1-dephosphorylated lipid A present in lanes 1, 3, and 5 is not present in living cells. It is an artifact that arises due to the partial loss of the anomeric phosphate during pH 4.5 hydrolysis at 100 °C of LPS, which releases the lipid A from LPS by cleaving the Kdo-lipid A linkage.

Fig. 9. Proposed model for 1-dephosphorylation of lipid A in the inner membrane of Gram-negative bacteria

The lipid A and core portions of LPS are synthesized on the inner surface of the inner membrane (3). MsbA, an essential ABC transporter, exports the nascent LPS to the outer surface of the inner membrane (19, 43), where FnLpxE 1-dephosphorylates the lipid A portion of the molecule.