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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-Distèche 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.

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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.
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f3: 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.

Mentions: We then assessed if this FRET approach would be suitable to assay Lipid II transport in right-side-out (RSO) membrane vesicles prepared from E. coli cells. The assay is based on the synthesis of NBD-labelled Lipid II at the inner leaflet of RSOs, which will be translocated across the membrane to appear at the outer leaflet rendering it accessible to vancomycin (see the hypothetical plot in Supplementary Figure S2, right panel). The appearance of Lipid II at the outer leaflet and concomitant binding to TMR-labelled vancomycin will lead to a decrease in the NBD fluorescence, which will be accompanied by an increase of TMR fluorescence (Supplementary Figure S2, left panel). In the RSOs, synthesis of fluorescently labelled Lipid II was enabled by following a freeze–thaw procedure to introduce the precursors NBD-UDP-MurNAc-pentapeptide and UDP-GlcNAc into the lumen of the vesicles. When RSO vesicles derived from wild-type E. coli prepared in this way were incubated at 14°C (All FRET measurements were carried out at 14°C to prevent the decrease in the fluorescence of NBD-labelled Lipid II in time at elevated temperatures resulting from transglycosylase activity, most likely of PBPs as reported on earlier in van Dam et al (2007). This decrease leads to a reduction in the total fluorescence of the FRET signal when measurements are performed at temperatures around 25°C.) in the presence of vancomycin-TMR, an increase in the fluorescence of the latter accompanied by a decrease in the NBD fluorescence was detected (Figure 3A). This gradual increase in the FRET signal reflects the appearance of Lipid II on the outside of the vesicles, demonstrating that the assay is capable of measuring Lipid II transport in bacterial membranes. Upon overexpression of FtsW in the same E. coli strain, the translocation of Lipid II was considerably increased (Figure 3B). This indicates that FtsW is involved in the transit of Lipid II from the inner to the outer leaflet of the membrane. Enhanced translocation of Lipid II was also detectable when transport of NBD-Lipid II was monitored using membrane vesicles derived from cells overexpressing Streptococcus pneumoniae FtsW (Supplementary Figure S3), which signifies that the effect of FtsW is species independent. Overexpression of other (control) proteins did not result in an augmentation of Lipid II translocation (Supplementary Figure S4A–C).


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-Distèche M, de Kruijff B, Breukink E - EMBO J. (2011)

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

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

f3: 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.
Mentions: We then assessed if this FRET approach would be suitable to assay Lipid II transport in right-side-out (RSO) membrane vesicles prepared from E. coli cells. The assay is based on the synthesis of NBD-labelled Lipid II at the inner leaflet of RSOs, which will be translocated across the membrane to appear at the outer leaflet rendering it accessible to vancomycin (see the hypothetical plot in Supplementary Figure S2, right panel). The appearance of Lipid II at the outer leaflet and concomitant binding to TMR-labelled vancomycin will lead to a decrease in the NBD fluorescence, which will be accompanied by an increase of TMR fluorescence (Supplementary Figure S2, left panel). In the RSOs, synthesis of fluorescently labelled Lipid II was enabled by following a freeze–thaw procedure to introduce the precursors NBD-UDP-MurNAc-pentapeptide and UDP-GlcNAc into the lumen of the vesicles. When RSO vesicles derived from wild-type E. coli prepared in this way were incubated at 14°C (All FRET measurements were carried out at 14°C to prevent the decrease in the fluorescence of NBD-labelled Lipid II in time at elevated temperatures resulting from transglycosylase activity, most likely of PBPs as reported on earlier in van Dam et al (2007). This decrease leads to a reduction in the total fluorescence of the FRET signal when measurements are performed at temperatures around 25°C.) in the presence of vancomycin-TMR, an increase in the fluorescence of the latter accompanied by a decrease in the NBD fluorescence was detected (Figure 3A). This gradual increase in the FRET signal reflects the appearance of Lipid II on the outside of the vesicles, demonstrating that the assay is capable of measuring Lipid II transport in bacterial membranes. Upon overexpression of FtsW in the same E. coli strain, the translocation of Lipid II was considerably increased (Figure 3B). This indicates that FtsW is involved in the transit of Lipid II from the inner to the outer leaflet of the membrane. Enhanced translocation of Lipid II was also detectable when transport of NBD-Lipid II was monitored using membrane vesicles derived from cells overexpressing Streptococcus pneumoniae FtsW (Supplementary Figure S3), which signifies that the effect of FtsW is species independent. Overexpression of other (control) proteins did not result in an augmentation of Lipid II translocation (Supplementary Figure S4A–C).

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