TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Genotype or description | Reference |
|---|---|---|
| Strains | ||
| DH1 | F−recA1 gyrA96 | 17 |
| RR1023 | pOX38 recA56 | 10 |
| NG135 | F−recA56 rpsL | 10 |
| Plasmids | ||
| pKD100 | pBR322 derivative carrying a model IS903 transposon; Cmr Apr Kmr | 10 |
| pKD1 | pBR322 derivative carrying a Kmr IS903 derivative | 39 |
| pOX38 | A 55-kb F derivative that is conjugative | 14 |
| pUCF1 | 1.1-kb PCR fragment (pOX38 tra region from nt 1700–2846)a inserted into pUC118 HindIII site; fragment amplified with primers oKD94 and oKD95 | This work |
| pUCF2 | pUCF1 with MluI-BstEII (end-filled) deletion of the traY promoter | This work |
| pUCF9 | Point mutations at the −35 (BamHI site created) and −10 (SphI site created) hexamers of traY promoter in pUCF1 | This work |
| RK2 | 60-kb naturally occurring conjugative plasmid; Tcr Apr Kmr | 29 |
| pRK21761 | RK2 derivative with E. coli lac operon in tetA gene and four point mutations in oriT which creates an XbaI site; Apr Kmr | 34 |
| pUB307 | RK2 derivative with ∼5.5 kb deletion of Apr Tn1; Tcr Kmr | 5 |
| pUBF4 | 0.4-kb PCR fragment (pOX38 tra region from nt 2056–2471)a cloned into the HindIII site of pUB307; fragment amplified with primers oKD96 and oKD97 | This work |
| pUBF12 | 1.1-kb HindIII fragment from pUCF1 inserted into HindIII site of pUB307 | This work |
| pUBF20 | Same as for pUBF4 except the fragment is cloned in the opposite orientation | This work |
| pUBF28 | 0.8-kb PCR fragment (pOX38 tra region from nt 1700–2471)a cloned into the HindIII site of pUB307; fragment amplified with primers oKD94 and oKD97 | This work |
| pUBF30 | Same as for pUBF28 except the fragment is cloned in the opposite orientation | This work |
| pUBF31 | 0.8-kb PCR fragment (pOX38 tra region from nt 2056–2846)a cloned into the HindIII site of pUB307; fragment amplified with primers oKD95 and oKD96 | This work |
| pUBF33 | Same as for pUBF31 except the fragment is cloned in the opposite orientation | This work |
| pUBF36 | 1.0-kb HindIII fragment from pUCF2 inserted into HindIII site of pUB307 | This work |
| pUBF38 | 1.1-kb HindIII fragment from pUCF9 inserted into HindIII site of pUB307 | This work |
| pUBF42 | Same as for pUBF12 except the fragment is cloned in the opposite orientation | This work |
| pUBF46 | 1.1-kb HindIII fragment from pUCF1 inserted into HindIII site of pUBT10 | This work |
| pUBF51 | 1.1-kb HindIII fragment from pUCF1 inserted into HindIII site of pUBT13 | This work |
| pRKF2 | Replacement of the 7.1-kb AseI-BglII fragment of RK2 with a 947-bp PCR fragment (nt 12044–12990)b containing oriV; the direction of DNA replication is opposite that of RK2 and pUB307 | This work |
| pRKF6 | Same as for pRKF2 except the oriV fragment is inserted in the native orientation | This work |
| pUBT2 | 16-kb EcoRI-AvrII fragment of pRK21761 replaced with the EcoRI-AvrII fragment of pUB307; Tcr Kmr | This work |
| pUBT10 | 273-bp PCR fragment (RK2 oriT region from nt 51123–51395)b cloned into the XbaI site of pUBT2; fragment amplified with primers oKD156 and oKD157 | This work |
| pUBT13 | Same as for pUBT10 except the oriT fragment is cloned in the opposite orientation | This work |
| pHP45Ω | Ω fragment encoding Smr/Spr gene flanked by transcription and translation termination signals in pBR322 | 30 |
| pUBF38Ω1 | HindIII Ω fragment from pHP45Ω is inserted at the HindIII site of pUBF38 between the Kmr promoter and the pOX38 fragment | This work |