Figure 1.
Collagen-like proteins from prophages embedded in the genomes of E. coli O157:H7 and other EHEC strains, referred here as EPclA to EPclD (EHEC Prophage collagen-like A to D).
(A) Domain architectures. The collagen triple helical domains are labelled “Col”, and domains predicted to adopt an α-helical coiled-coil conformation (see text) are labelled “PCoil” (for phage coiled-coils). Key to other domain labels (Table 1): PfN, phage fibre N-terminal domain; PfC, phage fibre C-terminal domain; PfC2, phage fibre C-terminal domain, variant 2; Pf2, phage fibre repeat 2. (B) Sequence of a representative collagen-like protein with EPclA architecture (ECs2717), from the genome of E. coli O157:H7 Sakai. (C) Sequence of a representative collagen-like protein with EPclB architecture (Z1483), from the genome of E. coli O157:H7 EDL933. Amino acid sequences corresponding to the different predicted domains are colour-coded as in (A).
Table 1.
Domains observed in collagen-like proteins from the genomes of EHEC strains.
Table 2.
Collagen-like proteins from the genomes of E. coli O157:H7 EDL933 and Sakai strains, and their corresponding prophage locations.
Table 3.
Examples of collagen-like proteins from other E. coli and Shigella strains, and their prophage locations.
Table 4.
Position-specific amino acid preferences in collagen triple-helical domains of EPclPs, human collagens, and collagen-like proteins from different groups of organisms.
Figure 2.
Coiled-coiled predictions for the amino acid sequences of (A) EPclA (ECs2717) and (B) EPclB (ECs1228), using the PCoils [70] and Marcoil [71] algorithms.
The graphs indicate regions of high probability for α-helical coiled-coil formation. Three different sequence window sizes were used with the PCoils algorithm: 14, 21 and 28 residues. Two different matrices were used in Marcoil: 9FAM, and MTK-based.
Figure 3.
Analysis by analytical ultracentrifugation of the average molar mass of a sample of purified rEPclA as a function of increasing concentration of guanidinium chloride (GuHCl).
Weight-averaged molar mass was determined using a single ideal species model (see Methods). Mean value masses for the upper and lower plateaux were 138±6 kDa and 43±1 kDa respectively (averages of three measures). The molecular mass of native rEPclA (0 M GuHCl) is consistent with three times that of denatured rEPclA (see text). The transition midpoint concentration is 2.38±0.09 M GuHCl.
Table 5.
Average molecular weights of different recombinant fragments, calculated from the SEC/MALLS data.
Figure 4.
Far-UV CD analysis of the Col–PfC fragment after purification from rEPclA by SEC.
(A) CD spectra at 4°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 0.2 mg/ml 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 the Col–PfC fragment, monitored by CD at 220 nm as a function of increasing temperature between 4°C and 60°C, with a protein concentration of 0.2 mg/ml in 10 mM Tris, 150 mM NaCl, pH 7.4, and a heating rate of 0.33°C/min.
Figure 5.
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.
Figure 6.
Far-UV CD spectra of the PfN–PCoil and PfN fragments after purification by SEC:
(A) PfN and PfN–PCoil at 20°C; (B) PfN–PCoil at 20°C and 60°C; (C) PfN at 20°C and 60°C. In all panels the vertical axis measures mean residue ellipticity Θ in degrees cm2 dmol-1. The CD data was collected between 195 and 260 nm, with protein concentrations of 0.2 mg/ml (PfN–PCoil) or 0.3 mg/ml (PfN), in 20 mM phosphate buffer (Na2HPO4/NaH2PO4), 100 mM NaCl, pH 7.4. Measurements were taken in a 0.5 mm path length cell.
Figure 7.
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.
Figure 8.
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.
Figure 9.
Molecular dimensions of rEPclA and its domains.
(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.
Figure 10.
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.
Figure 11.
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.
Figure 12.
Typical morphologies of podoviridae and siphoviridae particles (reproduced with permission from ViralZone, Swiss Institute of Bioinformatics:
www.expasy.org/viralzone, [79]). The representative 933W phage and most field isolates show a podoviridae morphology, with isometric capsids of about 60–70 nm in diameter and short tails of 10–30 nm in length [12], [56]. EPclPs would be the main components on the side tail fibres.