TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Description or sequence | Source or reference | Plasmid(s) generateda |
|---|---|---|---|
| Strains | |||
| E. faecalis | |||
| JH2-2 | rif fus | 39 | |
| FA2-2 | rif fus | 25 | |
| OG1X | str gel | 36 | |
| FA3333 | FA2-2 defective in cAD1 | 2 | |
| E. coli | |||
| DH5α | endA1 recA1 gyrA96 thi-1 hsdR17 supE44 relA1 Δ(argF-lacZYA)U169 φ80lacZΔM15 | BRL | |
| BL21(DE3)pLysS | F−ompT rB− mB− DE3 | Invitrogen | |
| Plasmids | |||
| pAM714 | pAD1::Tn917; erm; Agg Tra | 35 | |
| pAM330 | pAD1::pAD2; erm km str | 12 | |
| pAM330Δ9 | pAM330 with deletion repAΔ9 | This study | |
| pET30a | Expression vector | Novagen | |
| pASK60 | Expression vector | Biometra | |
| pSU18 | E. coli cloning vector, cm, p15A | 4 | |
| pAM88 | Suicide vector; cm, p15A, cm+ | 22 | |
| pDAK246ΔE | pAD1 minireplicon; erm par | 53 | |
| pAM3314 | 504-bp internal repA cloned in pAM401 | 3 | |
| pAM3316 | 209-bp internal repA cloned in pAM401 | 3 | |
| pAM3318 | 158-bp internal repA cloned in pAM401 | 3 | |
| pAM88A* | repA (frameshift) cloned in pAM88 | This study | |
| pAM88It | It region cloned in pAM88 | This study | |
| pAM88-3314 | 504-bp internal repA cloned in pAM88 | This study | |
| pAM88-3316 | 209-bp internal repA cloned in pAM88 | This study | |
| pAM88-3318 | 158-bp internal repA cloned in pAM88 | This study | |
| pAM88oriV | MfeI-RsaI 170-bp repA cloned in pAM88 | This study | |
| pSU18b | bac promoter cloned in pSU18 | This study | |
| pSU18b* | bac* promoter cloned in pSU18 | This study | |
| pAM434b* | bac* promoter cloned in pAM434 | This study | |
| pAM434brepA | bacrepA cloned in pAM434 | This study | |
| pAM434brepAΔ9 | bacrepAΔ9 cloned in pAM434 | This study | |
| pAM88ABC | pAD1 rep region cloned in pAM88 | This study | |
| pAM88AΔ9BC | pAD1 rep(Δ9) region cloned in pAM88 | This study | |
| pET30aRepA | repA gene cloned in pET30a | This study | |
| pET30aRepA5′ | 5′ repA gene cloned in pET30a | This study | |
| pET30aRepA3′ | 3′ repA gene cloned in pET30a | This study | |
| pASK60RepA | repA gene cloned in pASK60 | This study | |
| pASK60RepB | repB gene cloned in pASK60 | This study | |
| pAM2603 | 7.5-kb pAD1 replication proficient region | 1 | |
| pBluescript | E. coli cloning vector; ap | Stratagene | |
| pTAd | E. coli cloning vector; ap, km | Clontech | |
| pTAdA | repA cloned in pTAd | This study | |
| pTAdA* | repA (frameshift) cloned in pTAd | This study | |
| pTAdAΔ9 | repAΔ9 cloned in pTAd | This study | |
| pTAdIt | It region in pTAd | This study | |
| pTAdIt5′ | 5′ Iteron repeats (see Fig. 1A) in pTAd | This study | |
| pTAdIt3′ | 3′ Iteron repeats (see Fig. 1A) in pTAd | This study | |
| pBlueScriptoriV | oriV region (see Fig. 1) in pBlueScript | This study | |
| pTAdIR1 | IR1 repeats (see Fig. 5A) in pTAd | This study | |
| pTAdIR1* | IR1* repeats (see Fig. 5A) in pTAd | This study | |
| pTAdItC | ItC repeats (see Fig. 1A) in pTAd | This study | |
| pAM88A*-IR3 + 4 | pAM88A* with mutated IR-1 repeats 3 and 4 | This study | |
| pAM88A*-IR1 + 2 | pAM88A* with mutated IR-1 repeats 1 and 2 | This study | |
| pAM88A*-IR1 + 2 + 3 + 4 | pAM88A* with mutated IR-1 repeats 1, 2, 3, and 4 | This study | |
| pAM88A*-5×IR | pAM88A* with all five IR-1 repeats mutated | This study | |
| Oligonucleotides | |||
| RepA | TTCATTGTAAATACGGTT | pAM88It | |
| RepB | CTTCCCAACGCCGCC | pAM88It, pTAdIt3′ | |
| It1 | TTAAGAATACCAAAACATTATT | pTAdIt5′ | |
| It2 | CCTTTCTACAAAAGGATT | pTAdIt5′ | |
| It3 | CCTTTTGTAGAAAGGTT | pTAdIt3′ | |
| 401A | GAGCAAGAGATTACGCGCAG | pAM88/3314, 6, 8 | |
| 401B | TGCCGGCCACGATGCGTCC | pAM88/3314, 6, 8 | |
| ETrepA.1 | CCCAGATCTGAACAACTTCAAATTTTTTAATGT | pAM88A*, pET30aRepA, and pET30aRepA5′ | |
| ETrepA.2 | CCCAAGCTTATTGATTTTCAACCCAGTT | pAM88A*, pET30aRepA, and pET30aRepA3′ | |
| ETrepA.3 | CCCAAGCTTAACCAAGGGATTCAGCGTT | pET30aRepA5′ | |
| ETrepA.4 | CCCAGATCTAACAAACGCTGAATCCC | pET30aRepA3′ | |
| ASK60repB.1 | TTTAACGGCCGGCATGGTTAAAAAAATTGTATT | pASK60repB | |
| ASK60repB.2 | ATTTTTCGCGACTCATTAGCAGTCGTCCCTTC | pASK60repB | |
| ASK60repA.1 | TTTAACGGCCGGCATGAACAACTTCAAATTTTTTAATGT | pAM434brepA, Δ9, pASK60repA | |
| ASK60repA.2 | ATTTTTCGCGATTGATTTTCAACCCAGTT | pAM434brepA, Δ9, pASK60repA | |
| Bac1 | AGAGCGTCGACTGATTGAA | pAM434b* | |
| Bac2 | GGGGTACCGTCGATCTTATCGCGATT | pAM434b* | |
| ItCs | TTTTTTACTATCTTACTATTTTACTAC | pTAdItC | |
| ItCas | ATTTTTTGTAGTAAAATAGTAAGATAG | pTAdItC | |
| IR1s | AATTGAATCAAGAGGGTATGAAAATCATACCCTGCCAAAAC | pTAdIR1 | |
| IR1as | AATTGTTTTGGCAGGGTATGATTTTCATACCCTCTTGATTC | pTAdIR1 | |
| IR1*s | AATTGAATCAAGAGCCTTTCAAAATGAAAGGCTGCCAAAAC | pTAdIR1* | |
| IR1*as | AATTGTTTTGGCAGCCTTTCATTTTGAAAGGCTCTTGATTC | pTAdIR1* | |
| TraB | CAAGATAATACGTTTTATTAGACAC | ||
| M1s | CAAAACGTTGATAAATCAGAGCCTTTGAAAATCAGT | pAM88A*-5×IR | |
| M1as | ACTGATTTTCAAAGGCTCTGATTTATCAACGTTTTG | pAM88A*-5×IR | |
| M2 | GAGCCTTTCAAAATGAAAGGCTGCCAAAACGTTGATAAATCAG | pAM88A*-IR3 + 4 | |
| M3 | GCAGCCTTTCATTTTGAAAGGCTCTTGATTACCAAGGGATTC | pAM88A*-IR3 + 4 | |
| M4 | GAGCCTTTCAAAATGAAAGGCTCCCCCCAAAGAAAAACAAA | pAM88A*-IR1 + 2, pAM88A*-IR1 + 2 + 3 + 4 | |
| M5 | GGAGCCTTTCATTTTGAAAGGCTCTGATTTATCAACGTTTTC | pAM88A*-IR1 + 2, pAM88A*-IR1 + 2 + 3 + 4 |
a
For clarity, only relevant plasmids generated by using the specific oligonucleotides are indicated.