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The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein.

Johnson CL, Ridley H, Marchetti R, Silipo A, Griffin DC, Crawford L, Bonev B, Molinaro A, Lakey JH - Mol. Microbiol. (2014)

Bottom Line: Delipidated LPS (LPS(Δ) (LIPID) ) shows weaker binding; and thus full affinity requires the lipid component.The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF.ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK.

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High‐resolution 31P MAS‐NMR.A and B. (A) Rc LPS in DOPC membranes in the absence (top) and in the presence (bottom) of ColN‐R; and, from Rd LPS in DOPC membranes (B) in the absence (top) and in the presence (bottom) of ColN‐R. Spectra were acquired at 20°C with 5 kHz sample spinning. The spectral changes due to ColN‐R binding are shaded (see also Table 1).C. The structure of lipid A and core region of LPS is shown for reference with the Rc LPS‐specific sugar residues shaded. Note the dotted line bond connecting the non‐stoichiometric glucosamine to Hep(III). Most assignments described in the text refer to peaks resolved by spectral deconvolutions which are not evident here and are listed in Table 1. The clear peaks in the Rd LPS spectrum (B) are the pyrophosphates on Hep (I) with the phosphate proximal to the pyranose ring at −13 ppm and the proximal one at −9.9 ppm. The extra complexity in this region of the Rc LPS spectrum indicates additional unassigned pyrophosphates probably on the additional t‐Hep, glucose and glucosamine moieties which bind to ColN‐R (see Figs 4C and 5C).
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mmi12568-fig-0005: High‐resolution 31P MAS‐NMR.A and B. (A) Rc LPS in DOPC membranes in the absence (top) and in the presence (bottom) of ColN‐R; and, from Rd LPS in DOPC membranes (B) in the absence (top) and in the presence (bottom) of ColN‐R. Spectra were acquired at 20°C with 5 kHz sample spinning. The spectral changes due to ColN‐R binding are shaded (see also Table 1).C. The structure of lipid A and core region of LPS is shown for reference with the Rc LPS‐specific sugar residues shaded. Note the dotted line bond connecting the non‐stoichiometric glucosamine to Hep(III). Most assignments described in the text refer to peaks resolved by spectral deconvolutions which are not evident here and are listed in Table 1. The clear peaks in the Rd LPS spectrum (B) are the pyrophosphates on Hep (I) with the phosphate proximal to the pyranose ring at −13 ppm and the proximal one at −9.9 ppm. The extra complexity in this region of the Rc LPS spectrum indicates additional unassigned pyrophosphates probably on the additional t‐Hep, glucose and glucosamine moieties which bind to ColN‐R (see Figs 4C and 5C).

Mentions: We used high‐resolution, solid‐state 31P MAS‐NMR to investigate sites of natural phosphorylation and pyrophosphorylation in Rc and Rd LPS and to follow changes in 31P dynamics following addition of ColN‐R. The data were collected in a membrane environment by incorporating Rc LPS (Fig. 5A) and Rd LPS (Fig. 5B) in DOPC membranes (dominant resonance at −1.09 ppm). Phosphate resonances at −0.2 and −0.5 ppm were attributed to Rd LPS, as these were common to both Rc and Rd LPS spectra. These correspond to the single phosphate groups shown in Fig. 5C. The Rc LPS spectra reveal additional phosphorylation sites between −3 and 1 ppm (0.5, −0.2 and −2.3 ppm). Since these are unique to Rc LPS and are absent from Rd LPS, it suggests that pyranose sites on Glc‐(Hep‐II)‐Hep‐GlcN, are available for phosphorylation (Fig. 5C).


The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein.

Johnson CL, Ridley H, Marchetti R, Silipo A, Griffin DC, Crawford L, Bonev B, Molinaro A, Lakey JH - Mol. Microbiol. (2014)

High‐resolution 31P MAS‐NMR.A and B. (A) Rc LPS in DOPC membranes in the absence (top) and in the presence (bottom) of ColN‐R; and, from Rd LPS in DOPC membranes (B) in the absence (top) and in the presence (bottom) of ColN‐R. Spectra were acquired at 20°C with 5 kHz sample spinning. The spectral changes due to ColN‐R binding are shaded (see also Table 1).C. The structure of lipid A and core region of LPS is shown for reference with the Rc LPS‐specific sugar residues shaded. Note the dotted line bond connecting the non‐stoichiometric glucosamine to Hep(III). Most assignments described in the text refer to peaks resolved by spectral deconvolutions which are not evident here and are listed in Table 1. The clear peaks in the Rd LPS spectrum (B) are the pyrophosphates on Hep (I) with the phosphate proximal to the pyranose ring at −13 ppm and the proximal one at −9.9 ppm. The extra complexity in this region of the Rc LPS spectrum indicates additional unassigned pyrophosphates probably on the additional t‐Hep, glucose and glucosamine moieties which bind to ColN‐R (see Figs 4C and 5C).
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mmi12568-fig-0005: High‐resolution 31P MAS‐NMR.A and B. (A) Rc LPS in DOPC membranes in the absence (top) and in the presence (bottom) of ColN‐R; and, from Rd LPS in DOPC membranes (B) in the absence (top) and in the presence (bottom) of ColN‐R. Spectra were acquired at 20°C with 5 kHz sample spinning. The spectral changes due to ColN‐R binding are shaded (see also Table 1).C. The structure of lipid A and core region of LPS is shown for reference with the Rc LPS‐specific sugar residues shaded. Note the dotted line bond connecting the non‐stoichiometric glucosamine to Hep(III). Most assignments described in the text refer to peaks resolved by spectral deconvolutions which are not evident here and are listed in Table 1. The clear peaks in the Rd LPS spectrum (B) are the pyrophosphates on Hep (I) with the phosphate proximal to the pyranose ring at −13 ppm and the proximal one at −9.9 ppm. The extra complexity in this region of the Rc LPS spectrum indicates additional unassigned pyrophosphates probably on the additional t‐Hep, glucose and glucosamine moieties which bind to ColN‐R (see Figs 4C and 5C).
Mentions: We used high‐resolution, solid‐state 31P MAS‐NMR to investigate sites of natural phosphorylation and pyrophosphorylation in Rc and Rd LPS and to follow changes in 31P dynamics following addition of ColN‐R. The data were collected in a membrane environment by incorporating Rc LPS (Fig. 5A) and Rd LPS (Fig. 5B) in DOPC membranes (dominant resonance at −1.09 ppm). Phosphate resonances at −0.2 and −0.5 ppm were attributed to Rd LPS, as these were common to both Rc and Rd LPS spectra. These correspond to the single phosphate groups shown in Fig. 5C. The Rc LPS spectra reveal additional phosphorylation sites between −3 and 1 ppm (0.5, −0.2 and −2.3 ppm). Since these are unique to Rc LPS and are absent from Rd LPS, it suggests that pyranose sites on Glc‐(Hep‐II)‐Hep‐GlcN, are available for phosphorylation (Fig. 5C).

Bottom Line: Delipidated LPS (LPS(Δ) (LIPID) ) shows weaker binding; and thus full affinity requires the lipid component.The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF.ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.

View Article: PubMed Central - PubMed

Affiliation: Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, UK.

Show MeSH
Related in: MedlinePlus