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Fig. 4. Specificity of anti-VirB antibodies. Antibody specificity was determined by Western blot assays (1). Proteins were transferred to nitrocellulose after SDS/PAGE and probed with purified rabbit anti-VirBn antibodies (except for chicken anti-VirB8 antibodies) followed by peroxidase-conjugated secondary antibodies. Samples analyzed were uninduced A. tumefaciens A348 (odd lanes) and induced bacteria (even lanes). The VirB-specific band is indicated by an arrowhead. Numbers indicate the mass and position of protein markers. The molecular masses of the cross-reacting bands agreed well with those of the cognate VirB proteins. No other cross-reacting band was observed in either induced or uninduced bacteria devoid of the VirB proteins.

1. Judd, P., Kumar, R. & Das, A. (2005) Mol. Microbiol. 55, 115–124.

All antibodies were raised in rabbit except anti-VirB8 antibodies were raised in both rabbit and chicken. All Trp fusions contain the N-terminal 323 residues of the E. coli TrpE except for the VirB6 and VirB7 fusions, which contain 90 N-terminal residues.

1. Judd, P., Kumar, R. & Das, A. (2005) Mol. Microbiol. 55, 115-124.

2. Kumar, R., Xie, Y.-H. & Das, A. (2000) Mol. Microbiol. 36, 608-617.

3. Kumar, R. & Das, A. (2002) Mol. Microbiol. 43, 1523-1532.

To express virB gene subsets the gene was placed under the control of virD promoter by molecular cloning. Plasmids pAD1758, pAD1536, pLA17, and pAD1746 express virB3, virB8, virB7-virB10, and virB6-virB11, respectively. A 0.4-kb fragment encompassing the virB3 coding region (base pairs 1833–2232 of ref. 1) was obtained by PCR amplification and cloned downstream of the virD_p of plasmid pAD1417 (2). The virD_p-virB3 gene was isolated as a 0.8-kb KpnI fragment and cloned into the IncP plasmid pAD1412 to construct pAD1758 (2). Plasmid pAD1536 was constructed by cloning a 1-kb fragment containing the virB8 coding region (base pairs 6299–7197) into plasmid pAD1416 (3) and fusing the resulting plasmid to pAD1412. Plasmid pLA17 is a fusion of plasmid pLA13 containing a chimeric virD_p-virB7-virB10 gene (3) and the IncP plasmid pTJS75. To construct plasmid pAD1746, virB6-virB11 was cloned as a 5.3-kb SacI fragment into plasmid pAD1336 (4). The resultant plasmid pAD1745 was fused to the IncP plasmid pAD1708 that contains the virG mutant virGN54D (4). A deletion of virB8 (codons 3–225) was introduced in pAD1745 by site-specific mutagenesis with the deoxy-oligonucleotide dCTGAATATGCCATGCTCGAGGAAGATACCGTTTC as the mutagenic primer. The virB11 deletion mutant was constructed by deleting the terminal EcoRI-SacI region in pAD1745. Plasmids were introduced into A. tumefaciens A348DB by electroporation (4).

The VirB segments used as antigens and plasmids used for the overproduction of the fusion proteins are listed in Table 4. Plasmids pAD1273, pAD1275, and pAD1279 contain a 1.4-kb BamHI-XhoI fragment containing virB11 in the TrpE fusion vector pATH2 (5), a 5.1-kb BamHI fragment containing virB4 in pATH3, and a 1.1-kb PstI fragment containing virB5 in pATH2, respectively. Plasmid pTJ15 contains virB1 as a 0.8-kb XhoI-HindIII fragment in plasmid pRSETB (Invitrogen). A XhoI site was introduced at codon 18 of virB1 by site-specific mutagenesis. Plasmid pSC1 contains a 0.34-kb EcoRI-NcoI fragment in the plasmid vector pAD1427 (6).

1. Ward, J. E., Akiyoshi, D. E., Regier, D., Datta, A., Gordon, M. P. & Nester, E. (1988) J. Biol. Chem. 263, 5804–5814.

2. Das, A. & Xie, Y.-H. (1998) Mol. Microbiol. 27, 405–414.

3. Das, A., Anderson, L. & Xie, Y.-H. (1997) J. Bacteriol. 179, 3404–3409.

4. Anderson, L. B., Hertzel, A. V. & Das, A. (1996) Proc. Natl. Acad. Sci. USA 93, 8889–8894.

5. Koerner, T., Hill, J., Myers, A. & Tzagoloff, A. (1991) Methods Enzymol. 194, 477–490.

6. Judd, P., Kumar, R. & Das, A. (2005) Mol. Microbiol. 55, 115–124.