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

Mohammadi T, van Dam V, Sijbrandi R, Vernet T, Zapun A, Bouhss A, Diepeveen-de Bruin M, Nguyen-Dist├Ęche M, de Kruijff B, Breukink E - EMBO J. (2011)

Bottom Line: The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II.The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Organic Chemistry, Institute of Biomembranes, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Padualaan, Utrecht, The Netherlands.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3102273&req=5

f2: 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.

Mentions: To address the role of FtsW in the flipping process, we developed a novel fluorescence resonance energy transfer (FRET)-based assay to study the translocation of the membrane-anchored cell wall precursor Lipid II in bacterial membrane vesicles. This assay makes use of the specific recognition of Lipid II by vancomycin (Breukink and de Kruijff, 2006) and the inability of this antibiotic to cross the membrane. Using NBD-labelled Lipid II as a donor and tetramethylrhodamine cadaverine (TMR)-labelled vancomycin as an acceptor of the fluorescence, a strong FRET signal (a fluorescence signal yielded by the energy transfer between NBD and TMR fluorophores when they are in close proximity) is detectable only when Lipid II is present (Figure 2). This FRET signal is absent when the fluorescence of NBD-Lipid II that is located in the outer leaflet of the vesicles has been quenched beforehand by the membrane impermeant reductant dithionite (Supplementary Figure S1).


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

Mohammadi T, van Dam V, Sijbrandi R, Vernet T, Zapun A, Bouhss A, Diepeveen-de Bruin M, Nguyen-Dist├Ęche M, de Kruijff B, Breukink E - EMBO J. (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102273&req=5

f2: 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.
Mentions: To address the role of FtsW in the flipping process, we developed a novel fluorescence resonance energy transfer (FRET)-based assay to study the translocation of the membrane-anchored cell wall precursor Lipid II in bacterial membrane vesicles. This assay makes use of the specific recognition of Lipid II by vancomycin (Breukink and de Kruijff, 2006) and the inability of this antibiotic to cross the membrane. Using NBD-labelled Lipid II as a donor and tetramethylrhodamine cadaverine (TMR)-labelled vancomycin as an acceptor of the fluorescence, a strong FRET signal (a fluorescence signal yielded by the energy transfer between NBD and TMR fluorophores when they are in close proximity) is detectable only when Lipid II is present (Figure 2). This FRET signal is absent when the fluorescence of NBD-Lipid II that is located in the outer leaflet of the vesicles has been quenched beforehand by the membrane impermeant reductant dithionite (Supplementary Figure S1).

Bottom Line: The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II.The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Biology and Organic Chemistry, Institute of Biomembranes, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Padualaan, Utrecht, The Netherlands.

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.

Show MeSH
Related in: MedlinePlus