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Collagen-like proteins in pathogenic E. coli strains.

Ghosh N, McKillop TJ, Jowitt TA, Howard M, Davies H, Holmes DF, Roberts IS, Bella J - PLoS ONE (2012)

Bottom Line: Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk.This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins.Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom.

ABSTRACT
The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.

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Related in: MedlinePlus

Far-UV CD spectra of the PfN–PCoil and PfN fragments after purification by SEC:(A) PfN and PfN–PCoil at 20°C; (B) PfN–PCoil at 20°C and 60°C; (C) PfN at 20°C and 60°C. In all panels the vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with protein concentrations of 0.2 mg/ml (PfN–PCoil) or 0.3 mg/ml (PfN), in 20 mM phosphate buffer (Na2HPO4/NaH2PO4), 100 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell.
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pone-0037872-g006: Far-UV CD spectra of the PfN–PCoil and PfN fragments after purification by SEC:(A) PfN and PfN–PCoil at 20°C; (B) PfN–PCoil at 20°C and 60°C; (C) PfN at 20°C and 60°C. In all panels the vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with protein concentrations of 0.2 mg/ml (PfN–PCoil) or 0.3 mg/ml (PfN), in 20 mM phosphate buffer (Na2HPO4/NaH2PO4), 100 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell.

Mentions: The secondary structures of the recombinant fragments PfN–PCoil and PfN were also studied by CD spectroscopy. Recombinant PfN–PCoil and PfN fragments were each purified with nickel-affinity and size-exclusion chromatographies. Concentrations of the PfN–PCoil and PfN samples were measured as 0.2 mg/ml and 0.3 mg/ml respectively, from their absorption at 280 nm and molar extinction coefficients ε = 7000 M-1 cm-1, initially calculated from the amino acid sequences of the PfN–PCoil and PfN recombinant fragments and adjusted using the observed UV absorption of samples in 8 M urea (see Materials and Methods). The CD spectrum of PfN–PCoil at 4°C in phosphate buffer (Figure 6A) shows the characteristic features of an α-helical protein, with two minima at 208 nm and 222 nm and a maximum at 195 nm. This spectrum is consistent with the prediction of an α-helical coiled-coil conformation for the PCoil region. The α-helical features were still present in a spectrum measured at 45°C, although the signal intensities at the two minima started to decrease (data not shown). These features disappeared when reaching 60°C (Figure 6B), and were mostly recovered upon cooling the sample back to 20°C (data not shown). The spectrum of the PfN domain (Figure 6A) was also consistent with an α-helical structure but the intensity of the two minima at 208 nm and 222 nm was much lower than in the PfN–PCoil spectrum, indicating a lower α-helical content in this domain. This spectrum also vanished at 60°C (Figure 6C), but it was not recovered upon cooling back to 20°C (data not shown). Thus, the main contribution to the CD signal comes from the PCoil domain, most likely through the formation of a trimeric α-helical coiled-coil structure that disappears at a temperature of 60°C but is regained when the temperature is lowered again.


Collagen-like proteins in pathogenic E. coli strains.

Ghosh N, McKillop TJ, Jowitt TA, Howard M, Davies H, Holmes DF, Roberts IS, Bella J - PLoS ONE (2012)

Far-UV CD spectra of the PfN–PCoil and PfN fragments after purification by SEC:(A) PfN and PfN–PCoil at 20°C; (B) PfN–PCoil at 20°C and 60°C; (C) PfN at 20°C and 60°C. In all panels the vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with protein concentrations of 0.2 mg/ml (PfN–PCoil) or 0.3 mg/ml (PfN), in 20 mM phosphate buffer (Na2HPO4/NaH2PO4), 100 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368898&req=5

pone-0037872-g006: Far-UV CD spectra of the PfN–PCoil and PfN fragments after purification by SEC:(A) PfN and PfN–PCoil at 20°C; (B) PfN–PCoil at 20°C and 60°C; (C) PfN at 20°C and 60°C. In all panels the vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with protein concentrations of 0.2 mg/ml (PfN–PCoil) or 0.3 mg/ml (PfN), in 20 mM phosphate buffer (Na2HPO4/NaH2PO4), 100 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell.
Mentions: The secondary structures of the recombinant fragments PfN–PCoil and PfN were also studied by CD spectroscopy. Recombinant PfN–PCoil and PfN fragments were each purified with nickel-affinity and size-exclusion chromatographies. Concentrations of the PfN–PCoil and PfN samples were measured as 0.2 mg/ml and 0.3 mg/ml respectively, from their absorption at 280 nm and molar extinction coefficients ε = 7000 M-1 cm-1, initially calculated from the amino acid sequences of the PfN–PCoil and PfN recombinant fragments and adjusted using the observed UV absorption of samples in 8 M urea (see Materials and Methods). The CD spectrum of PfN–PCoil at 4°C in phosphate buffer (Figure 6A) shows the characteristic features of an α-helical protein, with two minima at 208 nm and 222 nm and a maximum at 195 nm. This spectrum is consistent with the prediction of an α-helical coiled-coil conformation for the PCoil region. The α-helical features were still present in a spectrum measured at 45°C, although the signal intensities at the two minima started to decrease (data not shown). These features disappeared when reaching 60°C (Figure 6B), and were mostly recovered upon cooling the sample back to 20°C (data not shown). The spectrum of the PfN domain (Figure 6A) was also consistent with an α-helical structure but the intensity of the two minima at 208 nm and 222 nm was much lower than in the PfN–PCoil spectrum, indicating a lower α-helical content in this domain. This spectrum also vanished at 60°C (Figure 6C), but it was not recovered upon cooling back to 20°C (data not shown). Thus, the main contribution to the CD signal comes from the PCoil domain, most likely through the formation of a trimeric α-helical coiled-coil structure that disappears at a temperature of 60°C but is regained when the temperature is lowered again.

Bottom Line: Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk.This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins.Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, United Kingdom.

ABSTRACT
The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.

Show MeSH
Related in: MedlinePlus