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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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Rotary shadowing electron microscopy of Col–PfC and PfN–PCoil fragments.(A) Micrograph showing the morphology of Col–PfC fragments (concentration 1 µg/ml). (B) Detailed view of one Col–PfC fragment. The globular shape corresponds to a trimer of PfC domains and the stalk corresponds to the trimeric collagen triple-helical domain (Col). The N-terminal end of the stalk shows a short, unravelled tail, where the PfN and PCoil domains have been removed. (C) Micrograph showing the morphology of PfN–PCoil fragments (concentration 5 µg/ml). (D) Detailed view of three PfN–PCoil fragments. The globular shapes correspond to trimers of PfN domains and the short tails correspond to the PCoil domains.
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pone-0037872-g010: Rotary shadowing electron microscopy of Col–PfC and PfN–PCoil fragments.(A) Micrograph showing the morphology of Col–PfC fragments (concentration 1 µg/ml). (B) Detailed view of one Col–PfC fragment. The globular shape corresponds to a trimer of PfC domains and the stalk corresponds to the trimeric collagen triple-helical domain (Col). The N-terminal end of the stalk shows a short, unravelled tail, where the PfN and PCoil domains have been removed. (C) Micrograph showing the morphology of PfN–PCoil fragments (concentration 5 µg/ml). (D) Detailed view of three PfN–PCoil fragments. The globular shapes correspond to trimers of PfN domains and the short tails correspond to the PCoil domains.

Mentions: (A) Magnified view of a representative rEPclA molecule from a rotary shadowing electron micrograph. (B) The same molecule with the background masked out showing the different molecular dimensions analyzed below. (C) Average dimensions obtained from multiple measures on electron micrographs: N···C and <Coil···Col are averages of 224 measures on eight rEPclA micrographs; DC, LCol and TCol are averages of 76, 35 and 74 measures, respectively, on three Col–PfC micrographs (Figure 10A); DN, LCoil and TCoil are averages of 200 measures on one PfN–PCoil micrograph (Figure 10B). (D) Histograms showing the distribution of N···C and <Coil···Col values on the sample of 224 rEPclA molecules.


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 Col–PfC and PfN–PCoil fragments.(A) Micrograph showing the morphology of Col–PfC fragments (concentration 1 µg/ml). (B) Detailed view of one Col–PfC fragment. The globular shape corresponds to a trimer of PfC domains and the stalk corresponds to the trimeric collagen triple-helical domain (Col). The N-terminal end of the stalk shows a short, unravelled tail, where the PfN and PCoil domains have been removed. (C) Micrograph showing the morphology of PfN–PCoil fragments (concentration 5 µg/ml). (D) Detailed view of three PfN–PCoil fragments. The globular shapes correspond to trimers of PfN domains and the short tails correspond to the PCoil domains.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037872-g010: Rotary shadowing electron microscopy of Col–PfC and PfN–PCoil fragments.(A) Micrograph showing the morphology of Col–PfC fragments (concentration 1 µg/ml). (B) Detailed view of one Col–PfC fragment. The globular shape corresponds to a trimer of PfC domains and the stalk corresponds to the trimeric collagen triple-helical domain (Col). The N-terminal end of the stalk shows a short, unravelled tail, where the PfN and PCoil domains have been removed. (C) Micrograph showing the morphology of PfN–PCoil fragments (concentration 5 µg/ml). (D) Detailed view of three PfN–PCoil fragments. The globular shapes correspond to trimers of PfN domains and the short tails correspond to the PCoil domains.
Mentions: (A) Magnified view of a representative rEPclA molecule from a rotary shadowing electron micrograph. (B) The same molecule with the background masked out showing the different molecular dimensions analyzed below. (C) Average dimensions obtained from multiple measures on electron micrographs: N···C and <Coil···Col are averages of 224 measures on eight rEPclA micrographs; DC, LCol and TCol are averages of 76, 35 and 74 measures, respectively, on three Col–PfC micrographs (Figure 10A); DN, LCoil and TCoil are averages of 200 measures on one PfN–PCoil micrograph (Figure 10B). (D) Histograms showing the distribution of N···C and <Coil···Col values on the sample of 224 rEPclA molecules.

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