Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane

Abstract

Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.

Figure 1

Schematic representation of the chemical structure of Lipid II (A) and NBD-labelled Lipid II (B). This peptidoglycan precursor consists of an undecaprenyl chain, phosphate (Pi), MurNAc (M) and GlcNAc (G). The pentapeptide moiety of MurNAc is symbolized by circles. NBD fluorophore is attached at the lysine of the pentapeptide moiety residue.

Figure 2

The interaction between fluorescently labelled vancomycin and Lipid II leads to FRET. In this assay, NBD-Lipid II (labelled at the amino group of the lysine at position 3 of the pentapeptide) and vancomycin-TMR (labelled at its C terminus with tetramethylrhodamine cadaverine) are used as a FRET pair. Fluorescence spectra of NBD-Lipid II (in LUVs prepared as described in Materials and methods) and vancomycin-TMR separately and together are presented (while being exited at the wavelength of the NBD group). The energy transfer between NBD and TMR fluorophores yields a fluorescence signal when they are in close proximity of each other. This is generally reflected by a decrease in fluorescence of NBD-Lipid II and increase in fluorescence of vancomycin-TMR. A.U.: arbitrary units.

Figure 3

Expression of FtsW increases the translocation of NBD-Lipid II from the inner to the outer leaflet of the bacterial membrane. Generation of a FRET signal was monitored over time in RSO vesicles prepared from wild-type TOP10F’ strain (A), and TOP10F’ harbouring a plasmid encoding His-tagged FtsW, where the expression of the ftsW gene is under the control of an IPTG-inducible promoter (B). The time course of fluorescence was monitored during 30 min. Assaying the wild-type strain yielded a gradual increase in the FRET signal in time (A). Overexpression of the ftsW gene causes a significant increase in the FRET signal (B). Background signals obtained from RSO vesicles, where neither NBD-UDP-MurNAc-pentapeptide nor UDP-GlcNAc were incorporated and were subtracted from each measurement. Synthesis of NBD-Lipid II in the vesicles was monitored using TLC. All measurements are representative of at least three independent experiments. A.U.: arbitrary units. (C) The time course of the FRET signal in (A) and (B) displayed as a 578/534 ratio, where 578 nm is the emission maximum of vancomycin-TMR and 534 nm is the emission maximum of NBD-Lipid II. Error bars represent s.d. of the mean value of the ratios measured at 578±5 and 534±5 nm.

Figure 4

The translocation of NBD-Lipid II across the bacterial membrane is affected by the depletion of FtsW. The LMC1436 strain (FtsW depletion strain) was grown in the presence of arabinose to induce the expression of FtsW, and RSO vesicles were prepared as described before. These behaved similarly as the wild-type E. coli strains in yielding a moderate augmentation in fluorescence in time (A). When this strain was grown in the presence of glucose (inducing the depletion of FtsW), a reduced FRET signal (reflecting a reduced Lipid II translocation) is obtained (B) compared with that where the expression of FtsW was induced. The fluorescence spectra in (B) were normalized to the same scale as in (A). All measurements are representative of at least three independent experiments. A.U: arbitrary units. (C) The time course of the FRET signal in (A) and (B) displayed as a 578/534 ratio, where 578 nm is the emission maximum of vancomycin-TMR and 534 nm is the emission maximum of NBD-Lipid II. Error bars represent s.d. of the mean value of the ratios measured at 578±5 and 534±5 nm.

Figure 5

FtsW induces transbilayer movement of NBD-Lipid II in proteoliposomes. (A) Coomassie-stained SDS–PAGE gel analysis of purified FtsW. The arrow at ∼37 kDa points to the FtsW band. (B) LUVs containing NBD-Lipid II symmetrically distributed between the inner and outer leaflets of the bilayer and solubilized with Triton X-100 were reconstituted with no protein, the control protein KcsA or FtsW following the procedure detailed in Materials and methods. After addition of dithionite (1), a reduction of almost 50% of the fluorescence signal is displayed by protein-free vesicles and proteoliposomes containing KscA. In contrast, ∼70% quenching is obtained when FtsW-containing proteoliposomes were employed. When 0.1% Triton X-100 was added (2) to permeabilize the vesicles, a complete quenching of all the fluorescence is achieved. All measurements were carried out at 20°C and are representative of at least three independent experiments. A.U.: arbitrary units.

Figure 6

The effect of FtsW in facilitating the transbilayer movement of Lipid II in model membranes is concentration dependent. Proteoliposomes were reconstituted in the presence of FtsW at the following protein/phospholipid molar ratio: 1:40 000 (1), 1:20 000 (2), 1:10 000 (3) and 1:5000 (4). The assay was performed as delineated under Figure 5. The percentage of quenching of fluorescence is dependent on the concentration of FtsW used in the reconstitution procedure. All measurements were carried out at 20°C and are representative of at least three independent experiments. A.U.: arbitrary units.

Figure 7

Increased accessibility of Lipid II to vancomycin in proteoliposomes containing FtsW. Vesicles without any protein or reconstituted with the control protein KcsA or with FtsW were prepared according to the procedure described before. FRET measurements were then carried out at 14°C after addition of vancomycin-TMR. FtsW-containing vesicles display a much higher FRET signal than the protein-free and KcsA-containing vesicles. All measurements are representative of at least three independent experiments. A.U.: arbitrary units.