Figure 1.
Schematic of the C. difficile eut operon and enzymatic pathway. A.
The genomic position of the operon is labelled above the encoded genes, which are colour coded according to their proposed function; Purple boxes represent genes with putative enzymatic activities; orange boxes, genes encoding regulatory proteins; blue boxes, genes with homology to bacterial microcompartment proteins; green box, gene encoding a trans-membrane transporter; and the white box, gene with unknown function. Intergenic regions are shown as large gaps between identified coding sequences, putative promoter elements and transcriptional terminator loops are shown as arrows and loops respectively. B. Proposed pathway and roles of specific enzymes in ethanolamine utilisation by C. difficile.
Table 1.
C. difficile eut operon organisation.
Figure 2.
X-ray crystal structure of CD1908. A.
Cartoon representation of the CD1908 monomer with secondary structure elements coloured and labelled from N- to C-termini. B. Hexameric arrangement of CD1908 showing the asymmetric unit contents in blue and symmetry related molecules in grey, with the three-fold symmetry axis generating this arrangement shown as a grey triangle.
Table 2.
Crystal parameters and data collection statistics for C. difficile Eut proteins.
Figure 3.
Alignment of CD1908 and its homologues.
A. Ribbon view of a structural alignment of CD1908 (blue), PduU (green) and EutS(G39V) (red). B. Orthogonal view of A, viewed down the hexameric axis. C. Alignment of CD1908 with the bent EutS hexamer, the orientation of the central axes are shown with grey lines. D. Orthogonal view of C, viewed down the multimeric symmetry axis. Rather than being a symmetric hexamer, EutS displays a split arrangement of two trimers.
Figure 4.
A. Cartoon representation of the CD1918 monomer with secondary structure elements coloured and labelled from N- to C-termini. B. Hexameric arrangement of CD1918 showing the asymmetric unit contents in blue and symmetry related molecules in grey; the two-fold crystallographic symmetry axis that generates this arrangement is shown as a grey ellipse.
Figure 5.
Electrostatic surfaces and characteristics of CD1908 and CD1918 hexamers.
A. The electrostatic potential of the solvent accessible surface of CD1908 was calculated using the default parameters in PDB2PQR [67] and APBS [68] and mapped to the molecular surface in PyMol and displayed over a cartoon representation of the molecule. Blue indicates regions of positive potential (> +5 kT/e) and red indicates negative potential (< −5 kT/e). Both faces of the structure are shown. B. Cross-section view of the CD1908 pore with the electrostatic surface and stick view of the protein molecule shown. The diameter of the pore at the top and bottom of the hexamer is shown. C. Enlarged view of the top of the pore from panel D showing the positions of the conformationally flexible Gln11 residue with arrows indicating the two conformations seen in the crystal structure. D. Electrostatic potential of the surface of the CD1918 hexamer displayed as described for panel A. E. Cross-section of the CD1918 hexamer displayed in the same manner as panel A, in this molecule the pore is open and contains a sulphate ion with ordered solvent molecules visible in the pore. F. Enlarged view of the top of the pore showing the peptide nitrogen of Gly41 and side-chain of Lys13, which form the boundary of this opening.
Figure 6.
A. Cartoon view of the asymmetric unit contents in the CD1925 crystal. A dimer is formed between the two cupin-barrels present in the asymmetric unit, coloured green and cyan, with secondary structure elements labelled. The loop between residues 56 and 64 in chain A that adopts two conformations is highlighted with a black circle. Orthogonal views are shown at left and right with the molecule rotated into the plane of the figure. B. Electrostatic potential of CD1925 mapped onto the molecular surface of the protein, calculated and displayed as for Fig. 3 with positive potential shown in blue ( > + 5 kT/e) and negative potential shown in red (< −5 kT/e). A surface cleft is visible in the view at left, highlighted by the dotted white oval. An acetate ion has been modelled into this cleft to illustrate its scale.
Figure 7.
Potential active site of CD1925.
A. Ribbon view of the structure of CD1925 with homologous structures superimposed. CD1925 is shown in cyan, the structures of the homologous cupins, EutQ from Salmonella typhimurium (PDBID: 2PYT, red) and metal binding cupin from Thermotoga maritima (PDBID: 1VJ2, orange) and Bacillus subtilis (PDBID: 2Y0O, green), are shown superimposed on CD1925. Despite low sequence homology within the cupin family, the core architecture of the fold is almost identical. B. Metal coordinating residues from 1VJ2 and 2Y0O are shown with their equivalent residues from CD1925 and 2PYT, molecule and residue colours are the same as for A. Where the metal binding cupins have histidine residues, the EutQ homologues possess aromatic and hydrophobic residues and share a conserved glutamic acid residue with the catalytic cupin 2Y0O. C. Sequence alignment of CD1925 and homologues from Listeria monocytogenes (LMO1187), Enterococcus faecalis (EF1617), 2PYT and 1VJ2. Residues lining the proposed active site are well conserved among EutQ type proteins, shown with stars, and distinct from the equivalent positions in the metal binding cupins represented by 1VJ2. D. Stereo view of the proposed active site of CD1925. Residues lining the cleft shown in Fig. 6B are displayed as sticks with their final 2mFo-dFc electron density shown as a grey mesh and contoured at 1.5σ.
Figure 8.
Thin-section transmission electron micrographs of E. coli cells over-expressing C. difficile Eut proteins.
A. All cells display a normal cellular morphology in E. coli producing CD1908, image taken at a microscope magnification of 34,000 x. B. E. coli cells over producing CD1925 also display normal cellular morphology, image taken at a microscope magnification of 34,000 x. C. Transverse views of E. coli cells over-expressing CD1918, around 90% of cells display significant internal morphological changes, 10 nm wide lamellar structures are visible as rose-shaped arrangements, image taken at 92,000 x magnification. D. Longitudinal views of E. coli cells overexpressing CD1918 show that the structures formed extend throughout the cytoplasm in bunches, where they appear to interfere with cell division in around 90% of cells seen in this view, image taken at 64,000 x magnification.
Figure 9.
Crystal contacts of CD1918 and its homologues.
A. Views of the crystal packing of CD1918 along the a,c (top) and b,c (bottom) planes with contact regions highlighted as roman numerals: i, ii, and iii. The crystallographic 2-fold generating the hexamer is shown as a black ellipse for a single multimer. B. crystal packing for EutM (PDBID: 3MPW), this protein packs into a regular two-dimensional lattice in the a,c plane (top) with a single conserved crystallographic interface, i. The b,c plane has widely spaced alternating layers distinct to CD1918, due to interactions between the C-terminal his-tag added to the construct for purification. C. Residues mediating CD1918 crystal contacts, panels i, ii and iii correspond to the interfaces marked in A. Interface i is conserved between CD1908 and its homologues, EutM and PduA, and in the crystal structures includes a coordinated sulphate ion; while interface ii is formed by solvent mediated contacts between chains. Interface iii is formed by non-conserved residues and forms a tight offset-layer packing between layers of hexamers. D. Sequence alignment of CD1918, PduA (PDBID: 3NGK) and EutM (PDBID: 3MPW). The secondary structure assignment for CD1918 is shown above the alignment. Conserved residues are shown in red, with strict conservation highlighted with a red background. Residues participating in crystal contacts shown in C are highlighted with i: stars; ii: triangles; iii: open circles.
Table 3.
Primers.