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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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Far-UV CD analysis of rEPclA after purification by SEC.(A) CD spectra at 4°C, 45°C, 55°C and 4°C after immediately cooling back the sample (see text). The vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with a protein concentration of 0.04 mg/ml (4°C) or 0.3 mg/ml (the rest) in 10 mM Tris, 150 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell. (B) Thermal denaturation of rEPclA monitored by CD at 216 nm (the maximum between the two minima at 208 and 224 nm). The CD was measured as a function of increasing temperature between 20°C and 75°C, with a protein concentration of 0.3 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.
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pone-0037872-g005: Far-UV CD analysis of rEPclA after purification by SEC.(A) CD spectra at 4°C, 45°C, 55°C and 4°C after immediately cooling back the sample (see text). The vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with a protein concentration of 0.04 mg/ml (4°C) or 0.3 mg/ml (the rest) in 10 mM Tris, 150 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell. (B) Thermal denaturation of rEPclA monitored by CD at 216 nm (the maximum between the two minima at 208 and 224 nm). The CD was measured as a function of increasing temperature between 20°C and 75°C, with a protein concentration of 0.3 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.

Mentions: The CD spectrum of rEPclA is different. A diluted sample of rEPclA was purified by nickel-affinity and size exclusion chromatographies and its CD spectrum was measured between 195 and 260 nm at 4°C (Figure 5A). The concentration of the rEPclA sample was calculated as 0.04 mg/ml from its UV absorption at 280 nm and a molar extinction coefficient ε = 17000 M-1 cm-1, deduced from the amino acid sequence of rEPclA. The CD spectrum showed two minima of negative ellipticity at 205 nm and 224 nm, the first one being deeper, and a small local maximum between the two minima, at 216 nm. To investigate this region in more detail a second sample with higher concentration, 0.3 mg/ml, was analyzed at different temperatures. When the sample was heated to 45°C, the height of the 216 nm maximum decreased significantly, the intensities of the two minima became more similar to each other, and their positions shifted to 210 nm and 222 nm, respectively (Figure 5A). This spectrum resembled more that of an α-helical coiled-coil conformation. Upon further increase of the temperature the overall ellipticity became less negative, and the two minima started to disappear and vanished completely when reaching 55°C (Figure 5A). The spectrum did not change upon further increase of temperature to 65°C. The slight decrease in ellipticity at 216 nm around 45°C and the more similar intensities of the two minima at that temperature suggest changes in the secondary structure that are consistent with the loss of the triple helical conformation in the Col domain while maintaining an α-helical conformation (Figures 4 and 5). Such α-helical structure appears to be more stable and does not disappear completely until 55°C. Immediate cooling of the same sample from 65°C back to 4°C recovered approximately half of the initial CD spectrum (Figure 5A).


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 analysis of rEPclA after purification by SEC.(A) CD spectra at 4°C, 45°C, 55°C and 4°C after immediately cooling back the sample (see text). The vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with a protein concentration of 0.04 mg/ml (4°C) or 0.3 mg/ml (the rest) in 10 mM Tris, 150 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell. (B) Thermal denaturation of rEPclA monitored by CD at 216 nm (the maximum between the two minima at 208 and 224 nm). The CD was measured as a function of increasing temperature between 20°C and 75°C, with a protein concentration of 0.3 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368898&req=5

pone-0037872-g005: Far-UV CD analysis of rEPclA after purification by SEC.(A) CD spectra at 4°C, 45°C, 55°C and 4°C after immediately cooling back the sample (see text). The vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with a protein concentration of 0.04 mg/ml (4°C) or 0.3 mg/ml (the rest) in 10 mM Tris, 150 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell. (B) Thermal denaturation of rEPclA monitored by CD at 216 nm (the maximum between the two minima at 208 and 224 nm). The CD was measured as a function of increasing temperature between 20°C and 75°C, with a protein concentration of 0.3 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.
Mentions: The CD spectrum of rEPclA is different. A diluted sample of rEPclA was purified by nickel-affinity and size exclusion chromatographies and its CD spectrum was measured between 195 and 260 nm at 4°C (Figure 5A). The concentration of the rEPclA sample was calculated as 0.04 mg/ml from its UV absorption at 280 nm and a molar extinction coefficient ε = 17000 M-1 cm-1, deduced from the amino acid sequence of rEPclA. The CD spectrum showed two minima of negative ellipticity at 205 nm and 224 nm, the first one being deeper, and a small local maximum between the two minima, at 216 nm. To investigate this region in more detail a second sample with higher concentration, 0.3 mg/ml, was analyzed at different temperatures. When the sample was heated to 45°C, the height of the 216 nm maximum decreased significantly, the intensities of the two minima became more similar to each other, and their positions shifted to 210 nm and 222 nm, respectively (Figure 5A). This spectrum resembled more that of an α-helical coiled-coil conformation. Upon further increase of the temperature the overall ellipticity became less negative, and the two minima started to disappear and vanished completely when reaching 55°C (Figure 5A). The spectrum did not change upon further increase of temperature to 65°C. The slight decrease in ellipticity at 216 nm around 45°C and the more similar intensities of the two minima at that temperature suggest changes in the secondary structure that are consistent with the loss of the triple helical conformation in the Col domain while maintaining an α-helical conformation (Figures 4 and 5). Such α-helical structure appears to be more stable and does not disappear completely until 55°C. Immediate cooling of the same sample from 65°C back to 4°C recovered approximately half of the initial CD spectrum (Figure 5A).

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