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Appendix S1. The supplementary material includes structural and functional data on the PulG pilus and control data on the failure of the Pul secreton to assemble the TcpA pilus. Table S1. Similarity of atomic structures of type IV pilins and PulG. The structures were superimposed with the program lsqman as described in Fig. S2. Blue numbers (top right) show the rms. deviation for Ca atoms in a 5 ? cut-off. Red numbers (down left) correspond to the number of matched (and identical) residues for each protein pair. The number in boldface on the diagonal is the total number of amino acids being compared. The structures are ordered according to their similarity to PulG. Table S2. Oligonucleotide primers used to amplify pilE and tcpA genes. Table S3. Oligonucleotide primers used to construct pilin/pseudopilin chimeras in which the N-terminal domains were interchanged. Fig. S1. The metal binding site in the dimeric crystal form of PulG25-134(His)6. The metal ion is tetrahedrally coordinated between E44 residues in the middle of the N-terminal a-helices of each PulG monomer and by two water molecules. N123 at the end of S3 stabilizes the complex via H-bonds to the water molecules and the non-coordinating e-oxygens of the E44 residues. Dashed black lines indicate coordinating bonds and dashed red lines the H-bonds. Fig. S2. Processing of preTcpA in E. coli strain carrying the prepilin peptidase structural gene pulO. proteins were separated by SDS-PAGE and electroblotted onto a nitrocellulose membrane that was probed with antibodies against TcpA (a gift from R. Taylor). Fig. S3. Analysis of pellet (P) and sheared fractions (SF) of E. coli stains PAP7000BG, PAP7501 (pCHAP1216) and PAP9001 (pCHAP1216) carrying pCHAP3100 (ppdD), pCHAP7023 and pCHAP7019 (encoding G17-D and G21-D, respectively) by immunoblotting. The relative amounts of secreton components present in the cells are indicated by numbers (chromosomal expression level = 1, expression from plasmid pBR322 = 20). Whether genes under lacZp control are repressed or not is determined by the presence or absence, respectively, of the lacq1 repressor gene (+ or -). Membranes were probed with anti-PpdD and anti-LamB antibodies. P; pellet fraction after shearing; SF, sheared fraction. Fig. S4. Multiple sequence alignment of the first 23 N-terminal amino acids of PulG, PilE, PilAPAK and PpdD. Positions 11, 14, 17 and 20 of the PulG, PilE and PilAPAK sequences are occupied by amino acids of equal characteristics (+), whereas the same positions in PpdD are occupied by amino acids of opposite characteristics (-). Sequence alignment was done using the MultiAlign software (http://prodes.toulouse.inra.fr/multalin/multalin.html). Fig. S5. Western blot analysis of bacterial pellet (p), sheared fractions (SF) and concentrated sheared fractions (cSF) from E. coli K-12 strain PAP7500BG(?pulG) harbouring pCHAP3100 (encoding PpdD) (lanes 1-3) and pCHAP7028 (encoding G21(modified)-D) (lanes 4-6), respectively. The blotted membrane was probed with antisera against PpdD. P; pellet fraction after shearing; SF, sheared fraction; cSF, concentrated sheared fraction. Fig. S6. A structural alignment of the C-terminal globular domain of the pseudopilin PulG with known type IV pilin structures (the type IVb pilin TcpA from V. cholerae and the type IVa pilins PilA(PilA1) from P. aeruginosa strain PAK, PilE from N. gonorrhoeae strain MS11 and PilA (PilA2) from P. aeruginosa strain k122-4. The superposition was performed by the brute force fit option in LSQMAN (Kleywegt, G.J. and Jones, T.A. 1995. A super position. CCP4/ESF-EACBM Newsletter on Protein Crystallography 31:9-14) using a cut-off distance of 5 ?. The transfer matrix was calculated for the first 100 amino acids of the PulG model since amino acids N128-G132 forming S4 are exchanged between both monomers in the asymmetric unit. These residues were taken from the other monomer. I126 and G127 (in italics) were excluded from the alignment since they form a putative flexible hinge region. Matched residues within the cut-off distance are printed in capital letters. Secondary structure elements calculated with DSSP (Kabsch, W and Sander, C. 1983. Dictionary of protein secondary structure: pattern recognition of hydrogen bond and geometrical features. Biopolymers, 22:2577-2637.) are highlighted with colours: blue, _-helix; light blue, 310-helix; green, _-strand. Crosses indicate positions that are occupied by structurally related amino acids in all 5 structures and stars that these residues are similar.

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