TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid(s) | Descriptiona | Source and/or reference |
|---|---|---|
| Strains | ||
| WCF5 | R10 traR traR-lacZ Kmr | 6 |
| R10 | Octopine type strain; pTiR10 | S. K. Farrand |
| C58 | Nopaline type strain; pTiC58 | S. K. Farrand |
| C58C1RS | Ti plasmid-less derivative of C58; Rifr Smr | S. K. Farrand |
| HC158 | C58 containing pHC320 inserted into the nopaline-type Ti plasmid pTiC58 by Campbell-type integration; traR traR-lacZ Kmr | This study |
| HC159(pYDH902) | R10 cured of pTiR10 and containing cosmid pYDH902 | This study |
| HC161 | Strain with polar mutation of trbD by the insertion of pHC327 | This study |
| HC162 | Strain with polar mutation of trbJ by the insertion of pHC328 | This study |
| HC163 | Strain with polar mutation of trbK by the insertion of pHC329 | This study |
| HC164 | Strain with polar mutation of trbF by the insertion of pHC330 | This study |
| Plasmids | ||
| pCF218 | PtetR-traR Tcrrep-RP4 | 6 |
| pBBR1MCS5 | Broad-host-range vector; rep-pBRR1 Gmr | 14 |
| pVIK111 | Carries promoterless lacZ; oriR6K Kmr | 12 |
| pKNG101 | sacB+ Smrrep-R6K oriT-RP4 | 13 |
| pPR1068 | pMAL2 derivative with NdeI site at ATG codon of malE; Ptac-MBP-lacZα lacIq Apr ori-ColE1 | Paul Riggs (20) |
| pPZP200 and pPZP201 | Broad-host-range vectors; rep-pVS1 Spr | 10 |
| pJZ335 | traR from pTiA6NC cloned into pPZP201 | 26 |
| pJZ381 | EcoRI fragment containing traR cloned into pBBR1MCS5 | 2 |
| pYDH902 | Cosmid containing rep and traI-trb operons; rep-RP4 Tcr | 4 |
| pHC011 | pPR1068 digested with NdeI and SacI, with replacement by a linker containing NdeI-KpnI-SacI sites to delete the malE gene; Ptac is fused to NdeI-KpnI-SacI-AvaI-XmnI-EcoRI-BamHI-XbaI-SalI-PstI-HindIII; rep-ColE1 Apr | This study |
| pHC012 | pHC011 digested with EcoRV and KpnI and ligated to pBBRMCS5 after digestion with SphI and KpnI, with 3′-end fill-in of the SphI site with the Klenow fragment of DNA polymerase I; Ptac is fused to NdeI-KpnI-ApaI-XhoI-SalI-Bsp106I-ClaI-HindIII-EcoRI-PstI-SmaI- BamHI-SpeI-XbaI-BstXI-SacI; rep-pBBR1 Gmr | This study |
| pHC320 | pVIK111 containing an EcoRI-XbaI fragment including the 5′ end of traR and upstream sequences; rep-R6K Kmr | This study |
| pHC327 | PCR fragment of trbD made using oligonucleotides TDF and TDR and cloned into pKNG101 for Campbell recombination mutagenesis; rep-R6K Smr | This study |
| pHC328 | PCR fragment of trbJ made using oligonucleotides TJF and TJR and cloned into pKNG101 for Campbell recombination mutagenesis; rep-R6K Smr | This study |
| pHC329 | PCR fragment of trbK made using oligonucleotides TKF and TKR and cloned into pKNG101 for Campbell recombination mutagenesis; rep-R6K Smr | This study |
| pHC330 | PCR fragment of trbF made using oligonucleotides TFF and TFR and cloned into pKNG101 for Campbell recombination mutagenesis; rep-R6K Smr | This study |
| pHC335 | pJZ335 digested with BamHI and ligated to remove a small BamHI fragment between Plac and traR; carries traR cloned into pPZP201; rep-pVS1 Spr | This study |
| pHC361 | PCR fragment containing trbJK made using oligonucleotides TrbJKF-N and R10-trbKJK3, digested with BamHI, and cloned into pHC012; Ptac-trbJK rep-pBBR1 Gmr | This study |
| pHC364 | trbBCDEJK cloned as a HindIII-BamHI fragment into pHC012; Ptac-trbBCDEJK rep-pBBR1 Gmr | This study |
| pHC368 | PCR fragment containing trbK made using oligonucleotides R10-trbKF-N and R10-trbKJK3, digested with BamHI, and cloned into pHC012; Ptac-trbK rep-pBBR1 Gmr | This study |
| pUP200 | 1,236-nucleotide DNA fragment made by PCR amplification using pHC012 as the template and oligonucleotides MfeI-For and NsiI-Rev as primers, cloned into the EcoRI-PstI gap of pPZP200, with the Ptac promoter upstream of the multiple-cloning site of pHC012; rep-pVS1 Gmr | This study |
| pUP402 | PCR fragment containing trbJ made using oligonucleotides TrbJKF-N and TrbJR-N and cloned into pHC012; Ptac-trbJ rep-pVS1 Gmr | This study |
| pUP403 | PCR fragment containing trbJ made using oligonucleotides TrbJKF-N and TrbJR-N and cloned into pUP200; Ptac-trbJ rep-pVS1 Gmr | This study |
| pUP404 | trbK from pHC368 cloned into pUP200; Ptac-trbK rep-pVS1 Gmr | This study |
| pUP405 | Derivative of pHC368 lacking the 3′ codon of trbK | This study |
| pUP406 | Derivative of pUP404 lacking the 3′ codon of trbK | This study |
| pUP407 | Derivative of pUP402 lacking the 3′ codon of trbJ | This study |
| pUP408 | Derivative of pUP403 lacking the 3′ codon of trbJ | This study |
a
MBP, maltose-binding protein.