TABLE 1.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Genotype or description | Source or reference |
|---|---|---|
| Strains | ||
| DH5α | F′/endA1 hsdR17(rK− mK+) supE44 thi-1 reacA1 gyrA(Nalr) relA1 Δ(lacZYA-argF)U169 (φ80dlacΔ(lacZ)M15) | Promega Corp., Madison, Wis. |
| S17.1 | recA pro hsdR (RP4-2 Tc::Mu Km::Tn7) | 23 |
| CSH56 | F−ara Δ(lac pro) supD nalA thi | Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| HB101 | F− Δ(mcrC-mrr) hsdS20 recA13 ara-14 proA2 lacY1 λ−galK2 rpsL20(Smr) xyl-5 mtl-1 supE44 | 3 |
| Plasmids | ||
| pUC19 | AmprlacZ′; ColE1 replicon, cloning vector | 28 |
| pACYC184 | Tcr Cmr; p15A replicon, cloning vector | 5 |
| pBR322 | Ampr Tcr; ColE1 replicon, cloning vector | 2 |
| pKK223-3 | Ampr; ColE1 replicon, expression vector | 4 |
| pTC-F14Cm | Cmr; natural pTC-F14 plasmid with a chloramphenicol resistance gene inserted at the single BamHI site | 9 |
| pTC-F14Km | Kmr; pTC-F14Cm with the chloramphenicol resistance gene replaced by the kanamycin resistance gene from Tn5 | This study |
| pDER412 | Cmr; natural pTF-FC2 plasmid with chloramphenicol resistance gene cloned into the Tn5467 transposon | 16 |
| pMmob | Ampr, 5,554-bp BamHI-XbaI fragment of pTC-F14 containing all mobilization genes and the repB primase, cloned into pUC19 | This study |
| pMmob1-pMmob9 | Ampr; PCR-based deletions of the mobilization region of pTC-F14 cloned into pUC19; refer to Fig. 1 | This study |
| pMmob1184 | Cmr; minimum mobilization region one of pTC-F14 cloned into the tetracycline resistance marker of pACYC184 | This study |
| pMmob1322 | Ampr; minimum mobilization region one of pTC-F14 cloned into pBR322 | This study |
| pmobE | Ampr; PCR product of mobE gene of pTF-FC2 cloned into pKK223-3 | This study |
| pmobDE | Ampr; PCR product of mobDE genes of pTF-FC2 cloned into pKK223-3 | This study |
| pmobCDE | Ampr; PCR product of mobCDE genes of pTF-FC2 cloned into pKK223-3 | This study |
| pAC105 | Cmr; exonuclease III shortening of the pTF-FC2 mobilization region containing mobC, -D, and -E cloned into pACYC184 | 20 |
| pAC209 | Cmr; exonuclease III shortening of the pTF-FC2 mobilization region containing mobA, -B, -C, and -D and a truncated mobE cloned into pACYC184 | 20 |
| pAC218 | Cmr; exonuclease III shortening of the pTF-FC2 mobilization region containing mobA, -B, and -C with mobD and -E removed also in pACYC184 | 20 |
| pAC221 | Cmr; exonuclease III shortening of the pDER412 mobilization region containing mobA and -B and the oriT cloned into pACYC184 | 20 |
| pOriTF14 | Ampr; a 203-bp HindIII-NcoI fragment of pTC-F14 containing the oriT cloned into pUC19 | This study |
| pOriTFC2 | Ampr; the oriT of pTF-FC2 cloned into pUC19 | This study |
| Primers | ||
| mobEF2 | (EcoRI) 5′-TACAGAATTCAGCAAGCGCATGAGC-3′ | This study |
| mobDEF2 | (EcoRI) 5′-TACAGAATTCCCAAAACCCGACAGC-3′ | This study |
| mobCDEF2 | (EcoRI) 5′-TATAGAATTCCCACGTGGCGAAGCC-3′ | This study |
| mobER2 | (XbaI) 5′-TACATCTAGAATGTTGAGCGCGTCG-3′ | This study |
| mobAR14 | (EcoRI) 5′-TACAGAATTCCGGTCCATGTCGTCG-3′ | This study |
| repBR14 | (EcoRI) 5′-TACAGAATTCGGGTAATCGGATGGC-3′ | This study |
| mobC′R14 | (PstI) 5′-TATACTGCAGCTTTCCCGCCTTTGC-3′ | This study |
| mobCR14 | (PstI) 5′-TATACTGCAGTTGCCACCACCGACG-3′ | This study |
| mobDR14 | (PstI) 5′-TATACTGCAGTCGGGTGTCGGTTCC-3′ | This study |
| mobER14 | (PstI) 5′-TACACTGCAGCTGTCCGAAAGTAGG-3′ | This study |
| mobAR14#2 | 5′-TGGCGTCGCTTGTTTGGTTC-3′ | 10 |