Table 1.
Bacterial strains and plasmids
| Bacterial strain or plasmid | Relevant characteristic(s) | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | λ−ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK−) supE44 thi-1 gyrA relA1 | Gibco-BRL |
| A. tumefaciens | ||
| A348 | Strain C58 containing octopine-type Ti plasmid pTiA6NC | 23 |
| C58C1RS | Strain C58, Strr Rifr | 24 |
| KE1 | ΔvirE operon | 25 |
| At12516 | virE2− | 26 |
| Mx219 | A348 with a virF::Tn3HoHo1 mutation | 27 |
| Mx311 | A348 with a virD2::Tn3HoHo1 mutation | 27 |
| LBA4404 | T-DNA deletion strain | 28 |
| KA2000 | A348 with a ΔvirD4 mutation | 18 |
| KA2002 | LBA4404 with a ΔvirD4 mutation generated by marker exchange-eviction mutagenesis | This study |
| PC1006 | A348 with a nonpolar ΔvirB6 mutation | 29 |
| PC1010 | A348 with a nonpolar ΔvirB10 mutation | 29 |
| PC1011 | A348 with a nonpolar ΔvirB11 mutation | 29 |
| Plasmids | ||
| Vectors | ||
| pXZ153 | Kanr; IncP plasmid with Kanr gene from pUC4K | 30 |
| pBin19 | Kanr; IncP plasmid with T-DNA border repeats | 31 |
| pBSBBR | Carbr Kanr; pBSIISK+ (Crbr) ligated to pBBR1MCS2 (Kanr) | 32 |
| pBSIISK+NdeI | Carbr; pBSIISK+ containing an NdeI site at the translational start site of lacZ | 29 |
| pPC914KS | 29 | |
| pPC914KS.NcoI | 33 | |
| IncQ/CloDF plasmids | ||
| pML122ΔKanr | Genr; mobilizable RSF1010 (IncQ) derivative | 34 |
| pJB31 | Spcr; RSF1010 derivative | 35 |
| pAJ1 | pJB31 lacking mobA | 36 |
| pAJ6 | pJB31 lacking oriT | 36 |
| pClo-Leu | pBin19 derivative carrying the mob region of pCloDF13; Kanr | Gift from J. Escudero, A. Vergunst, and P. J. J. Hooykaas |
| pΔClo-LEU | pJClo deleted of CloDF13 mob region | 37 |
| pCloΔEB-LEU | pJClo deleted of nonessential mobE and mobB T4CP gene | 37 |
| pCloΔEC-LEU | pJClo deleted of nonessential mobE and mobC relaxase gene | 37 |
| virD2 plasmids | ||
| pMY1153 | pUC18CM expressing the virD operon from the Ptac promoter | 38 |
| pKA171 | Carbr; pPC914KS expressing PvirB-virD2 | This study |
| pKA172 | Carbr; pPC914KS expressing PvirB-virD2ΔC35 | This study |
| pKA250 | Kanr Carbr; pXZ153 expressing PvirB-virD4 and PvirB-virD2 | This study |
| pKA251 | Kanr Carbr; pXZ153 expressing PvirB-virD4 and PvirB-virD2ΔC35 | This study |
| virD4 plasmids | ||
| pKA9 | Carbr; pPC914KS expressing PvirB-virD4 | 18 |
| pKA21 | Kanr; pXZ153 expressing PvirB-virD4 | 18 |
| pKA42 | Kanr; pBBR1MCS2 expressing PvirB-virD4 | 18 |
| pKA84 | Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4Δ2–10 | This study |
| pKA85 | Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4Δ2–67 | This study |
| pKA43 | Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4Δ2-86 | 39 |
| pKA155 | Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4Δ42-48 | This study |
| pKA158 | Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4Δ54-60 | This study |
| pKA157 | Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4F49C | This study |
| pKA156 | Kanr Carbr; pBBR1MCS2 expressing PvirB-virD4Y51A | This study |
| virB6 plasmids | ||
| pSJB964 | Kanr Carbr; pXZ151 expressing PvirB-virB6 | 40 |
| pSJB5XXX | Kanr Carbr; pXZ151 expressing virB6.i4 alleles; XXX is the position of the residue relative to the beginning of the protein that immediately precedes the i4 mutation | 40 |
| virB11 plasmids | ||
| pXZB100 | Kanr Carbr; pXZ151 expressing PvirB-virB11 | 30 |
| pXZB102 | I265T | 30 |
| pXZB105 | L75F | 30 |
| pXZB108 | L11P/D56G/E335G | 30 |
| pXZB101 | I88T/I103T | 30 |
| pXZB103 | H269R | 30 |
| pXZB109 | I103T/M301T | 30 |
| pJCB8XXX | Kanr Carbr; pXZ151 expressing virB11.i4 alleles; XXX is the position of the residue relative to the beginning of the protein that immediately precedes the i4 mutation | 41 |