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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

Rotary shadowing electron microscopy of rEPclA.(A, B) Different micrographs showing dumbbell-shaped structures corresponding to rEPclA trimers. The globular shapes correspond to the PfN and PfC terminal domains, presumably forming trimeric structures themselves. Flexible stalks connecting these globular structures contain the trimeric collagen triple-helical region (Col) and the trimeric α-helical coiled-coil region (PCoil) of rEPclA. The concentration of rEPclA was 5 µg/ml. Scale bar  = 100 nm. (C) Detailed view of an rEPclA trimer. The arrows indicate the globular terminal domains and the thin (Col) and thick (PCoil) regions of the stalk, respectively. The globular domains can be identified as N- or C-terminal by their position with respect to the two stalk zones.
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pone-0037872-g008: Rotary shadowing electron microscopy of rEPclA.(A, B) Different micrographs showing dumbbell-shaped structures corresponding to rEPclA trimers. The globular shapes correspond to the PfN and PfC terminal domains, presumably forming trimeric structures themselves. Flexible stalks connecting these globular structures contain the trimeric collagen triple-helical region (Col) and the trimeric α-helical coiled-coil region (PCoil) of rEPclA. The concentration of rEPclA was 5 µg/ml. Scale bar  = 100 nm. (C) Detailed view of an rEPclA trimer. The arrows indicate the globular terminal domains and the thin (Col) and thick (PCoil) regions of the stalk, respectively. The globular domains can be identified as N- or C-terminal by their position with respect to the two stalk zones.

Mentions: Full-length rEPclA was analyzed by rotary shadowing electron microscopy. Examination of the electron micrographs of rEPclA showed a “dumbbell-shaped” structure with two globular particles joined by a semi-flexible stalk, in which it is possible to distinguish two regions of different thickness (Figures 8 and 9). Sequence analysis of the PCoil region and the CD spectrum of PfN–PCoil both suggest that the PCoil domain is a trimeric α-helical coiled-coil structure, and the observed thicker region of the stalk is consistent with such coiled-coil helical structure, which is known to have a larger cross-section than a collagen triple helix [45]. The remaining thinner region corresponds to the collagen triple-helical domain.


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)

Rotary shadowing electron microscopy of rEPclA.(A, B) Different micrographs showing dumbbell-shaped structures corresponding to rEPclA trimers. The globular shapes correspond to the PfN and PfC terminal domains, presumably forming trimeric structures themselves. Flexible stalks connecting these globular structures contain the trimeric collagen triple-helical region (Col) and the trimeric α-helical coiled-coil region (PCoil) of rEPclA. The concentration of rEPclA was 5 µg/ml. Scale bar  = 100 nm. (C) Detailed view of an rEPclA trimer. The arrows indicate the globular terminal domains and the thin (Col) and thick (PCoil) regions of the stalk, respectively. The globular domains can be identified as N- or C-terminal by their position with respect to the two stalk zones.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037872-g008: Rotary shadowing electron microscopy of rEPclA.(A, B) Different micrographs showing dumbbell-shaped structures corresponding to rEPclA trimers. The globular shapes correspond to the PfN and PfC terminal domains, presumably forming trimeric structures themselves. Flexible stalks connecting these globular structures contain the trimeric collagen triple-helical region (Col) and the trimeric α-helical coiled-coil region (PCoil) of rEPclA. The concentration of rEPclA was 5 µg/ml. Scale bar  = 100 nm. (C) Detailed view of an rEPclA trimer. The arrows indicate the globular terminal domains and the thin (Col) and thick (PCoil) regions of the stalk, respectively. The globular domains can be identified as N- or C-terminal by their position with respect to the two stalk zones.
Mentions: Full-length rEPclA was analyzed by rotary shadowing electron microscopy. Examination of the electron micrographs of rEPclA showed a “dumbbell-shaped” structure with two globular particles joined by a semi-flexible stalk, in which it is possible to distinguish two regions of different thickness (Figures 8 and 9). Sequence analysis of the PCoil region and the CD spectrum of PfN–PCoil both suggest that the PCoil domain is a trimeric α-helical coiled-coil structure, and the observed thicker region of the stalk is consistent with such coiled-coil helical structure, which is known to have a larger cross-section than a collagen triple helix [45]. The remaining thinner region corresponds to the collagen triple-helical domain.

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