TABLE 1.

Bacterial strains and plasmids

Bacterial strain or plasmidRelevant characteristicsSource or reference
E. coli
    DH5αλ φ80d/lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK mK+) supE44 thi-1 gyrA relA1Gibco-BRL
    CJ236dut ung thi relA; pCJ105 (Camr)Bio-Rad
    BL21(DE3)F ompT hsdsB(rB mB) dcm gal λ (DE3)Novagen
    S17-1Tra genes from RP4 integrated into chromosome for plasmid mobilization39
A. tumefaciens
    A136Strain C58 cured of pTiC5848
    A348A136 containing octopine-type Ti plasmid pTiA6NC22
    A348SpcrA348 with Spcr by spontaneous mutagenesis39
    PC1000A348 derivative with deletion of the virB operon from Ti plasmid pTiA6NC19
PC1006-PC1010A348 with deletions of virB6 to virB10, respectively, from Ti plasmid pTiA6NC7
    Mx358A348 carrying a Tn3HoHo1 insertion in virE243
    LBA4404T-DNA deletion strain35
Plasmidsa
    pBSIISK+Crbr; cloning vectorStratagene
    pBSIIKS+Crbr; cloning vectorStratagene
    pBSIISK+NdeICrbr; pBSIISK+ containing an NdeI site at the translational start site of lacZ7
    pBSIIKS+NdeICrbr; pBSIIKS+ containing an NdeI site at the translational start site of lacZ7
    pBSIIKS+NcoICrbr; pBSIIKS+ containing an NcoI site at the translational start site of lacZ7
    pGEX2TCrbr; vector for construction of GST gene fusionsPharmacia
    pPC914KS+Crbr; pBSIIKS+ derivative expressing PvirB-virB1; vector for expression from PvirB by substitution of other genes for virB119
    pSW172Tetr; broad-host-range IncP plasmid containing Plac and polylinker sequence from pIC1911
    pXZ151Kanr; pSW172 derivative broad-host-range IncP plasmid with the Kanr gene from pUC4K inserted at the unique StuI site51
    pML122ΔKmGenr; mobilizable RSF1010 (IncQ) derivative21
    pXZ61Crbr; pBSIISK+NdeI expressing Plac-virB6This study
    pSJ964Crbr; pPC914KS+ expressing PvirB-virB6This study
    pSJ6300Crbr; pGEX2T carrying ∼300-bp BamHI-EcoRI fragment from pSJ6192; expresses Plac-GST-′virB6This study
    pZZ11Crbr; pPC914KS expressing PvirB-GSTbThis study
    pVK8Crbr; pZZ11 expressing PvirB-GST-virB8 by introduction of an ∼1-kb NdeI-XhoI fragment from pPC9104This study
    pES8Crbr; pZZ11 expressing PvirB-GST-virB10 by introduction of an ∼1-kb NdeI-XhoI fragment from pPC9104This study
    pAS8Crbr Kanr; pES8 ligated to the IncP plasmid pXZ151This study
    pPC988Camr; pBCKS+ expressing Plac-virB87
    pPC9108Camr; pBCKS+ expressing Plac-virB107
    pSJ610Crbr; pSJ964 carrying virB6-virB11 by insertion of an ∼4-kb ClaI-EcoRV fragment from pPC801 into similarly digested pSJ964This study
    pSJ6xxx seriesCrbr; pXZ61 carrying in-frame tandem NcoI-BamHI or NdeI-BamHI restriction sites in virB6 at positions following the residue indicated; virB6.i4 alleles are expressed from PlacThis study
    pSJ5xxx seriesCrbr; pSJ964 with virB6.i4 alleles from pSJ6xxx plasmid series substituted for wild-type virB6; alleles are expressed from PvirBThis study
  • a Except where indicated, ColE1 plasmids expressing virB constructs were ligated to IncP plasmid pXZ151 for introduction into A. tumefaciens; the cointegrate plasmid in A. tumefaciens is given the ColE1 plasmid name plus a “B” (for ligation to a broad-host-range replicon).

  • b See the text for details.