Table 1.
Bacterial strains, plasmids, and oligonucleotides used in the study
| Strain, plasmid, or oligonucleotide | Relevant features | Reference or source |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | λ− ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rK− mK−) supE44 thi-1 gyrA relA1 | Gibco-BRL |
| BL21(DE3) | F− ompT hsdSB(rB− mB−) gal dcm (DE3) | Novagen |
| EC1000 | E. coli cloning host; provides RepA in trans | 32 |
| E. faecalis | ||
| CK104 | OG1RFΔupp2 | 31 |
| OG1ES | Ermr Strr | 43 |
| OG1SSp | Strr Spcr | 19 |
| Plasmids | ||
| Expression plasmids/vectors | ||
| pET-28 b(+) | Kanr; expression vector for His tagging | Novagen |
| pAP1 | Kanr; pET-28a+ derivative | 6 |
| pGEX6P-1 | Crbr; expression vector for GST tagging | Amersham |
| pGEM-T Easy | Crbr; TA cloning vector | Promega |
| pDL278p23 | Spcr; pDL278 with Lactococcus lactis constitutive promoter P23 | 8 |
| pCJK47 | Carries oriTpCF10, lacZ, and P-pheS* cassette | 30 |
| pCJK21 | Ermr Spcr; shuttle vector; inducible promoter Pnis | 31 |
| pCF10 derivatives | ||
| pCF10 | Pheromone-inducible conjugative plasmid | 17 |
| pCF10ΔprgJ | pCF10 with prgJ deleted | This study |
| pCF10ΔprgH | pCF10 with prgH deleted | This study |
| pCF10ΔpcfC | pCF10 with pcfC deleted | 9 |
| pCF10ΔpcfF | pCF10 with pcfF deleted | 8 |
| pCF10ΔpcfG | pCF10 with pcfG inactivated by Ll.ltrBΔORF-Kan insertion | 43 |
| pCF10ΔoriT | pCF10 with oriT deleted | 43 |
| prgJ expression plasmids | ||
| pCY51 | pCJK21 expressing Pnis-prgJ-short | This study |
| pCM62 | pDL278p23 expressing P23-prgJ-short | This study |
| pCY81 | pCJK47 with prgJ flanking sequences for construction of pCF10ΔprgJ | This study |
| pCM58 | pGEM-T Easy containing prgJ.K471E for cloning into pCJK21 | This study |
| pCM59 | pGEM-T Easy containing prgJ.K471E for cloning into pET-28b(+) | This study |
| pCM64 | pCJK21 expressing Pnis-prgJ | This study |
| pCM69 | pCJK21 expressing Pnis-prgJ.K471E | This study |
| pINY1807 | pWM401 containing an ∼4.2-kb EcoRI fragment of pCF10 that includes prgIΔN5, full-length prgJ, and prgKΔC379 | 10 |
| pCY50 | pAP1 expressing prgJ-short-His6 | This study |
| pCM67 | pET28b(+) expressing prgJ-His6 | This study |
| pCM70 | pET28b(+) expressing prgJ.K471E-His6 | This study |
| pcfC, pcfF, pcfG, and pcfH expression plasmids | ||
| pCY26 | pGEX2T expressing Ptac-GST-pcfCΔN103 | 9 |
| pCY34 | pGEX6p-1 expressing GST-pcfF | 9 |
| pCY52 | pGEX6p-1 expressing GST-pcfG | This study |
| pCY82 | pCJK47 with prgH flanking sequences for construction of pCF10ΔprgH | This study |
| Oligonucleotides | ||
| Oligonucleotides for cloning | ||
| prgJ5F | 5′ GCTCTAGACATGGCCTATGCGCTTCAAAG 3′ | |
| prgJ5R | 5′ TCCCCCCGGGATCCTCTACAATGTGCATGAG 3′ | |
| prgJ3F | 5′ TCCCCCCGGGATTATGAACACCGACGCTTAG 3′ | |
| prgJ3R | 5′ ACATGCATGCCAAGCCAATACCTTGTTC 3′ | |
| PrgJ-pET5 | 5′ CATCCATGGCGCACATTGTAGAGGATGAGTTT 3′ | |
| PrgJ-pET3 | 5′ CCGCTCGAGGGTACCTTAGTGGTGGTGGTGGTGGTGCCTAGGAGCGTCGGTGTTCATAATTTCAAA 3′ | |
| PrgJ-XbaI | 5′ GCTCTAGACTAAGCGTCGGTGTTCAT 3′ | |
| PrgJ_rev_Xho | 5′ AATACTCGAGCTAAGCGTCGGTGTTCAT 3′ | |
| PrgJ_for_Nde | 5′ AATACATATGCACATTGTAGAGGAT 3′ | |
| KE_for | 5′ TCTGGTTCTGGTGAGGGAATGGCGACCAAAT 3′ | |
| KE_rev | 5′ ATTTGGTCGCCATTCCCTCACCAGAACCAGA 3′ | |
| PrgJ-pET6 | 5′ TAACCATGGGCAATTTGTTCAAAAAGAAACAATC 3′ | |
| pcfG-NdeI | 5′ GGAATTCCATATGGTGTATACAAAACATTTTGTT 3′ | |
| pcfG-XhoI | 5′ CCGCTCGAGTTATAGTTTGGGCTTAATGTC 3′ | |
| prgH5F | 5′ GCTCTAGAGATGTCTATATTCAGGTTGAA 3′ | |
| prgH5R | 5′ TCCCCCCGGGTCCTTTTGGATTTCTCATTGC 3′ | |
| prgH3F | 5′ TCCCCCCGGGGCCAAACGATTAGTAGGAGCG 3′ | |
| prgH3R | 5′ ACATGCATGCCGCTCGTCTTGTTCATACTGC 3′ | |
| Oligonucleotides for TrIP | ||
| TrIP-ChromF | 5′ TTACCAGTTTTGCAGTAGGG 3′ | |
| TrIP-ChromR | 5′ TTCAGCCACTGTCATAGCTTG 3′ | |
| TrIP-pcfE-F | 5′ ATGGACCAATTAACGGACG 3′ | |
| TrIP-pcfE-R | 5′ CCCATCGCTACACTTTGACAT 3′ | |
| Oligonucleotides for EMSA | ||
| pCF10oriTF3 | 5′ GGTAAGTCGAAACGTCAAT 3′ | |
| pCF10oriTR1 | 5′ CTCCTTAGTTTCGACAATTG 3′ | |
| 50-mer oriTS | 5′ ATATCGCAACATGCTAGCATGTTGCTCCGCTTGCAAAAAGAAAGCCTACC 3′ | |
| 50-mer oriTAS | 5′ GGTAGGCTTTCTTTTTGCAAGCGGAGCAACATGCTAGCATGTTGCGATAT 3′ | |
| 50-mer non-oriT | 5′ ATGCAGACGTACAAAAAAGAACTGAAACCGTATCTCTACCTTGGTGTTTT 3′ |