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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 rEPclB.(A) Internal structure of a small aggregate of rEPclB molecules. The micrograph suggests multiple flexible molecules, reminiscent of those observed for rEPclA, with dark globular structures (presumably globular domains of PfN, PfC and Pf2), and poorly defined linear structures (presumably stalks containing the PCoil and Col domains). The rEPclB molecules seem to aggregate heavily through one of the globular domains. (B) Possible examples of individual rEPclB molecules observed, isolated from the large aggregates, in some electron micrographs. (C) Interpretation of the observed in terms of three globular domains (PfN, Pf2 and PfC) connected by two stalk regions. Approximate molecular dimensions are shown for comparison purposes with rEPclA.
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pone-0037872-g011: Rotary shadowing electron microscopy of rEPclB.(A) Internal structure of a small aggregate of rEPclB molecules. The micrograph suggests multiple flexible molecules, reminiscent of those observed for rEPclA, with dark globular structures (presumably globular domains of PfN, PfC and Pf2), and poorly defined linear structures (presumably stalks containing the PCoil and Col domains). The rEPclB molecules seem to aggregate heavily through one of the globular domains. (B) Possible examples of individual rEPclB molecules observed, isolated from the large aggregates, in some electron micrographs. (C) Interpretation of the observed in terms of three globular domains (PfN, Pf2 and PfC) connected by two stalk regions. Approximate molecular dimensions are shown for comparison purposes with rEPclA.

Mentions: To investigate a possible structural organization of these aggregates, a sample of IPTG-induced rEPclB was used for rotary shadowing electron microscopy. The sample contained exclusively high-molecular weight aggregates that appeared in the electron micrographs as large masses of protein that, nevertheless, appeared to have a relatively narrow size distribution (300–500 nm in diameter, data not shown). Close inspection of the smallest aggregates (probably at an early stage of formation) revealed an internal structure that could be reconciled with entangled, multiple flexible linear beaded molecules (Figure 11). In the vicinity of these aggregates it was possible to discover individual features reminiscent of the rEPclA molecular morphology, but with three globular “domains” instead of two, connected by two flexible stalks (Figure 11). The terminal globular structures would correspond to the PfN and PfC domains, and the internal one would include the Pf2 domains. The flexible stalks would correspond to the two Col domains and the PCoil domain predicted in the rEPclB sequence. All these structures (and rEPclB) are presumed to be trimeric due to the presence of the PfC, Col and PCoil domains, all shown to trimerize in rEPclA. The structural organization for rEPclB would therefore be similar to that seen for rEPclA. However, an effective protocol to increase the amount of non-aggregated protein will be necessary to demonstrate these assumptions and to properly characterize the molecular architecture of rEPclB (work in progress).


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 rEPclB.(A) Internal structure of a small aggregate of rEPclB molecules. The micrograph suggests multiple flexible molecules, reminiscent of those observed for rEPclA, with dark globular structures (presumably globular domains of PfN, PfC and Pf2), and poorly defined linear structures (presumably stalks containing the PCoil and Col domains). The rEPclB molecules seem to aggregate heavily through one of the globular domains. (B) Possible examples of individual rEPclB molecules observed, isolated from the large aggregates, in some electron micrographs. (C) Interpretation of the observed in terms of three globular domains (PfN, Pf2 and PfC) connected by two stalk regions. Approximate molecular dimensions are shown for comparison purposes with rEPclA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037872-g011: Rotary shadowing electron microscopy of rEPclB.(A) Internal structure of a small aggregate of rEPclB molecules. The micrograph suggests multiple flexible molecules, reminiscent of those observed for rEPclA, with dark globular structures (presumably globular domains of PfN, PfC and Pf2), and poorly defined linear structures (presumably stalks containing the PCoil and Col domains). The rEPclB molecules seem to aggregate heavily through one of the globular domains. (B) Possible examples of individual rEPclB molecules observed, isolated from the large aggregates, in some electron micrographs. (C) Interpretation of the observed in terms of three globular domains (PfN, Pf2 and PfC) connected by two stalk regions. Approximate molecular dimensions are shown for comparison purposes with rEPclA.
Mentions: To investigate a possible structural organization of these aggregates, a sample of IPTG-induced rEPclB was used for rotary shadowing electron microscopy. The sample contained exclusively high-molecular weight aggregates that appeared in the electron micrographs as large masses of protein that, nevertheless, appeared to have a relatively narrow size distribution (300–500 nm in diameter, data not shown). Close inspection of the smallest aggregates (probably at an early stage of formation) revealed an internal structure that could be reconciled with entangled, multiple flexible linear beaded molecules (Figure 11). In the vicinity of these aggregates it was possible to discover individual features reminiscent of the rEPclA molecular morphology, but with three globular “domains” instead of two, connected by two flexible stalks (Figure 11). The terminal globular structures would correspond to the PfN and PfC domains, and the internal one would include the Pf2 domains. The flexible stalks would correspond to the two Col domains and the PCoil domain predicted in the rEPclB sequence. All these structures (and rEPclB) are presumed to be trimeric due to the presence of the PfC, Col and PCoil domains, all shown to trimerize in rEPclA. The structural organization for rEPclB would therefore be similar to that seen for rEPclA. However, an effective protocol to increase the amount of non-aggregated protein will be necessary to demonstrate these assumptions and to properly characterize the molecular architecture of rEPclB (work in progress).

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