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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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Thermal denaturation and renaturation of recombinant PfN–PCoil (orange) and PfN (red) monitored by CD at 222 nm (corresponding to a minimum in both CD spectra).The CD was measured in a 1 mm path length cell as a function of increasing temperature between 5°C and 95°C (left) and then decreasing temperature between 95°C and 5°C (right). The temperature was changed at a rate of 1°C per minute. Both PfN–PCoil (0.35 mg/ml) and PfN (0.1 mg/ml) were in 10 mM Tris, 150 mM NaCl, pH 7.4. PfN–PCoil showed a sharp transition at around 49°C corresponding to the loss of α-helical coiled-coil structure. The CD signal was almost completely recovered upon cooling, with a sharp transition about 45°C. This behaviour is indicative of a reversible structural transition for the α-helical coiled-coil. The PfN fragment gradually lost its CD signal with a transition midpoint of about ∼52°C. The gradual nature of this transition suggests denaturation rather than a cooperative unfolding. The PfN CD signal was not regained upon cooling.
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pone-0037872-g007: Thermal denaturation and renaturation of recombinant PfN–PCoil (orange) and PfN (red) monitored by CD at 222 nm (corresponding to a minimum in both CD spectra).The CD was measured in a 1 mm path length cell as a function of increasing temperature between 5°C and 95°C (left) and then decreasing temperature between 95°C and 5°C (right). The temperature was changed at a rate of 1°C per minute. Both PfN–PCoil (0.35 mg/ml) and PfN (0.1 mg/ml) were in 10 mM Tris, 150 mM NaCl, pH 7.4. PfN–PCoil showed a sharp transition at around 49°C corresponding to the loss of α-helical coiled-coil structure. The CD signal was almost completely recovered upon cooling, with a sharp transition about 45°C. This behaviour is indicative of a reversible structural transition for the α-helical coiled-coil. The PfN fragment gradually lost its CD signal with a transition midpoint of about ∼52°C. The gradual nature of this transition suggests denaturation rather than a cooperative unfolding. The PfN CD signal was not regained upon cooling.

Mentions: To examine the thermal transitions of the PfN–PCoil domain, the CD of two more samples of purified recombinant PfN–PCoil (0.35 mg/ml concentration) and PfN (0.1 mg/ml) were monitored at 222 nm as a function of continuously increasing temperature, from 5°C to 95°C and then cooling back to 5°C at the same speed (1°C per minute). The heating thermal curve showed a single sharp transition at around 49°C (Figure 7), corresponding to the loss of the strong α-helical CD spectrum. The cooling thermal curve also showed a single sharp transition at around 45°C indicating partial re-gaining of the α-helical structure (the baseline in Figure 7 does not recover its initial value). This data suggests reversibility for the thermal transition of PfN–PCoil, mainly for the formation of the trimeric α-helical coiled-coil structure in the PCoil domain; the PfN domain does not recover completely after thermal denaturation and slow cooling. The actual value of the temperature of the transition is sensitive to the heating or cooling rate. A repeat of the heating experiment with a slower speed (0.33°C per minute) gave a transition temperature of 52°C for the PfN–PCoil fragment (data not shown).


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)

Thermal denaturation and renaturation of recombinant PfN–PCoil (orange) and PfN (red) monitored by CD at 222 nm (corresponding to a minimum in both CD spectra).The CD was measured in a 1 mm path length cell as a function of increasing temperature between 5°C and 95°C (left) and then decreasing temperature between 95°C and 5°C (right). The temperature was changed at a rate of 1°C per minute. Both PfN–PCoil (0.35 mg/ml) and PfN (0.1 mg/ml) were in 10 mM Tris, 150 mM NaCl, pH 7.4. PfN–PCoil showed a sharp transition at around 49°C corresponding to the loss of α-helical coiled-coil structure. The CD signal was almost completely recovered upon cooling, with a sharp transition about 45°C. This behaviour is indicative of a reversible structural transition for the α-helical coiled-coil. The PfN fragment gradually lost its CD signal with a transition midpoint of about ∼52°C. The gradual nature of this transition suggests denaturation rather than a cooperative unfolding. The PfN CD signal was not regained upon cooling.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037872-g007: Thermal denaturation and renaturation of recombinant PfN–PCoil (orange) and PfN (red) monitored by CD at 222 nm (corresponding to a minimum in both CD spectra).The CD was measured in a 1 mm path length cell as a function of increasing temperature between 5°C and 95°C (left) and then decreasing temperature between 95°C and 5°C (right). The temperature was changed at a rate of 1°C per minute. Both PfN–PCoil (0.35 mg/ml) and PfN (0.1 mg/ml) were in 10 mM Tris, 150 mM NaCl, pH 7.4. PfN–PCoil showed a sharp transition at around 49°C corresponding to the loss of α-helical coiled-coil structure. The CD signal was almost completely recovered upon cooling, with a sharp transition about 45°C. This behaviour is indicative of a reversible structural transition for the α-helical coiled-coil. The PfN fragment gradually lost its CD signal with a transition midpoint of about ∼52°C. The gradual nature of this transition suggests denaturation rather than a cooperative unfolding. The PfN CD signal was not regained upon cooling.
Mentions: To examine the thermal transitions of the PfN–PCoil domain, the CD of two more samples of purified recombinant PfN–PCoil (0.35 mg/ml concentration) and PfN (0.1 mg/ml) were monitored at 222 nm as a function of continuously increasing temperature, from 5°C to 95°C and then cooling back to 5°C at the same speed (1°C per minute). The heating thermal curve showed a single sharp transition at around 49°C (Figure 7), corresponding to the loss of the strong α-helical CD spectrum. The cooling thermal curve also showed a single sharp transition at around 45°C indicating partial re-gaining of the α-helical structure (the baseline in Figure 7 does not recover its initial value). This data suggests reversibility for the thermal transition of PfN–PCoil, mainly for the formation of the trimeric α-helical coiled-coil structure in the PCoil domain; the PfN domain does not recover completely after thermal denaturation and slow cooling. The actual value of the temperature of the transition is sensitive to the heating or cooling rate. A repeat of the heating experiment with a slower speed (0.33°C per minute) gave a transition temperature of 52°C for the PfN–PCoil fragment (data not shown).

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