TABLE 2.
Bacterial plasmids constructed in this work
| Plasmid | Description | Constructiona |
||
|---|---|---|---|---|
| Vector | Insert | Digestion/oligonucleotides (5′→3′) | ||
| Plasmids expressing PtrwA-trwA-trwB | ||||
| pDEL010 | trwB(K275A) | pSU4633 | PCR on pSU4633 | TGAAGCGAGCGCGGCGCTGAC |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCTGGATCCGATGCATCCAGACGATCA | ||||
| pMTX520 | trwB(D158A) | pSU4633 | PCR on pSU4633 | CCGCATGGTAATTGTGGCGCCGAATGGCGATATG |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX521 | trwB(D356A) | pSU4633 | PCR on pSU4633 | GGCTGTTCATCGCCGAGCTCGCTTCGCTGGAAAAG |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX524 | trwB(Q386A) | pSU4633 | PCR on pSU4633 | GTGGCGGGCCTGGCGTCGACCTCGCAG |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX525 | trwB(Q390A) | pSU4633 | PCR on pSU4633 | CTGCAATCGACCTCAGCGCTTGATGACGTGT |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX527 | trwB(R240A E241A) | pSU4633 | PCR on pSU4633 | ACGCCTTCCATGGCAGCGCTGTTCCACTGGA |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX528 | trwB(H244A W245A) | pSU4633 | PCR on pSU4633 | ATGCGCGAATTGTTCGCCGCGACAACGATCGCCAC |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX530 | trwB(R375A) | pSU4633 | PCR on pSU4633 | GCACTCACCAAAGGCGCCAAGGCAGGGCTTCGG |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX533 | trwB(D252A D253A) | pSU4633 | PCR on pSU4633 | TCGCCACGTTTGCAGCGCTGCGGGGGTTTC |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX546b | trwB(D252R D253R E259R) | pSU4633 | PCR on pSU1443 | CATGCGCGAATTGTTCCACTGGACAACCATCGCCACGTTTCGTCGACCCCGGATGAATGTCA |
| CCAGCAAACAAAGATTCGGCCAAAGTTCCTCGCAGAAACCCCCGCAGTCGACGGATTTGCACTGCC | ||||
| pMTX549 | trwB(N271D) | pSU4633 | PCR on pSU4633 | TGCTGGGTCGGATGAAGCGAG |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCTGGATCCGATGCATCCAGACGATCA | ||||
| pMTX552 | trwB(K421A D425A) | pSU4633 | PCR on pSU4633 | CCGACCCGGCAACCAATGAGGCCATGAGTTTGA |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCTGGATCCGATGCATCCAGACGATCA | ||||
| pMTX553c | trwB(K398A) | pSU4633 | PCR on pSU4633 | ACGGCGTGGCAGAGGCGCAG |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCTGGATCCGATGCATCCAGACGATCA | ||||
| pMTX554 | trwB(Δ17C) | pSU4633 | PCR on pSU4633 | CCAGGATCCTCGGACAAGGCGAATTTG |
| CCAGAATTCAAAGCGGAACCTTGGCAA | ||||
| pMTX558 | trwB(R124A) | pSU4633 | PCR on pMTX518 | CCACATATGAATAGCGTCGGACAAGG |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| pMTX559 | trwB(E357A) | pSU4633 | PCR on pSU4633 | CTGTTCATCGACGCGTTGGCTTCGCTG |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX560 | trwB(Δ12C) | pSU4633 | PCR on pSU4633 | CCAGAATTCAAAACTGCTTGATTTCAAG |
| CCAGGATCCTCGGACAAGGCGAATTTG | ||||
| pMTX590 | trwB(S270P) | pSU4633 | None | None (random mutagenesis) |
| pMTX593 | trwB(R318H) | pSU4633 | None | None (random mutagenesis) |
| Plasmids expressing trwB under control of lactose promoter | ||||
| pDEL003 | pSU19::trwA-trwB | pHP139 | PCR on pSU4622 | TGACATATGGCACTAGGCGAC |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| pDEL009 | pSU19::trwB(K275A) | pHP139 | pDEL010 | KpnI + EcoRI |
| pHP138d | pET29c::oriT-trwA-trwC | pET29:trwAC | PCR on pSU2007 | TTACTCTAGACTCATTTTCTGCATCATTGT |
| TTACTCTAGATTGTAGTGGCATAACACTA | ||||
| pHP139 | pSU19::trwB | pSU19 | PCR on pMTX601 | TTACAAGCTTAGGAGGATCCATATGCATCCAGACGATCAAA |
| ATGCGGCATCAGAGCAGATTG | ||||
| pHP140 | pSU19::trwB(Q390A) | pHP139 | pMTX525 | KpnI + EcoRI |
| pHP141 | pSU19::trwB(R240A E241A) | pHP139 | pMTX527 | KpnI + EcoRI |
| pHP142 | pSU19::trwB(H244A W245A) | pHP139 | pMTX528 | KpnI + EcoRI |
| pHP143 | pSU19::trwB(D252A D253A) | pHP139 | pMTX533 | KpnI + EcoRI |
| pHP145 | pSU19::trwB(N271D) | pHP139 | pMTX549 | KpnI + EcoRI |
| pHP149 | pSU19::trwB(V74I) | pHP139 | pHP106 | BamHI + KpnI |
| pHP150 | pSU19::trwB(P18S S95N) | pHP139 | pHP107 | BamHI + KpnI |
| pHP169 | pSU19::trwB(K421A D425A) | pHP139 | pMTX552 | KpnI + EcoRI |
| pHP170 | pSU19::trwB(K398A) | pHP139 | pMTX553 | KpnI + EcoRI |
| Constructs in vectors for bacterial 2-hybrid assay | ||||
| pHP106 | pUT18C::trwB(V74I) | pMTX601 | PCRe on pMTX601 | GAAGTTCTCGCCGGATGT |
| BamHI + KpnI | ATGCGGCATCAGAGCAGATTG | |||
| pHP107 | pUT18C::trwB(P18S S95N) | pMTX601 | PCRe on pMTX601 | GAAGTTCTCGCCGGATGT |
| BamHI + KpnI | ATGCGGCATCAGAGCAGATTG | |||
| pHP108 | pUT18C::trwB(P18S) | pMTX601 | PCRe on pMTX601 | GAAGTTCTCGCCGGATGT |
| BamHI + KpnI | ATGCGGCATCAGAGCAGATTG | |||
| pHP119 | pUT18C::trwEBt(T107S) | pMTX697 | PCRe on pMTX697 | GAAGTTCTCGCCGGATGT |
| BamHI + NdeI | ATGCGGCATCAGAGCAGATTG | |||
| pHP120 | pUT18C::trwEBt(P57A) | pMTX697 | PCRe on pMTX697 | GAAGTTCTCGCCGGATGT |
| BamHI + NdeI | ATGCGGCATCAGAGCAGATTG | |||
| pHP121 | pUT18C::trwEBt(P57L T107A) | pMTX697 | PCRe on pMTX697 | GAAGTTCTCGCCGGATGT |
| BamHI + NdeI | ATGCGGCATCAGAGCAGATTG | |||
| pHP122 | pUT18C::trwEBt(P57S) | pMTX697 | PCRe on pMTX697 | GAAGTTCTCGCCGGATGT |
| BamHI + NdeI | ATGCGGCATCAGAGCAGATTG | |||
| pHP123 | pUT18C::trwEBt(V54L T126A) | pMTX697 | PCRe on pMTX697 | GAAGTTCTCGCCGGATGT |
| BamHI + NdeI | ATGCGGCATCAGAGCAGATTG | |||
| pHP124 | pUT18C::trwEBt(V52L) | pMTX697 | PCRe on pMTX697 | GAAGTTCTCGCCGGATGT |
| BamHI + NdeI | ATGCGGCATCAGAGCAGATTG | |||
| pHP126 | pT25::trwB(V74I) | pT25 | pHP106 | BamHI + EcoRI |
| pHP127 | pT25::trwB(P18S) | pT25 | pHP108 | BamHI + EcoRI |
| pMTX544 | pT25::trwB(K136T) | pT25 | PCR on pSU4632 | CCTGGATCCGATGCATCCAGACGATCA |
| CCAGGATCCTAGATAGTCCCCTCAACAA | ||||
| pMTX545 | pUT18C::trwB(K136T) | pUT18C | PCR on pSU4632 | CCTGGATCCGATGCATCCAGACGATCA |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| pMTX601 | pUT18C::trwB with KpnI | pUT18C | PCR on pSU4623 | CCTGGATCCGATGCATCCAGACGATCA |
| CCAGAATTCTAGATAGTCCCCTCAACAA | ||||
| pMTX615 | pUT18C::trwB(Δ12C) | pMTX513 | pMTX560 | BsmI + EcoRI |
| pMTX616 | pUT18C::trwB(R375A) | pMTX513 | pMTX530 | BsmI + StyI |
| pMTX617 | pUT18C::trwB(D356A) | pMTX513 | pMTX521 | BsmI + StyI |
| pMTX625 | pUT18C::trwB(R318H) | pMTX513 | pMTX593 | BsmI + EcoRI |
| pMTX627 | pT25::trwB(R375A) | pT25 | pMTX616 | BamHI + KpnI |
| pMTX628 | pT25::trwB(D356A) | pT25 | pMTX617 | BamHI + KpnI |
| pMTX629 | pUT18C::trwB(Q386A) | pMTX601 | pMTX524 | BsmI + StyI |
| pMTX630 | pT25::trwB(R318H) | pT25 | pMTX625 | BamHI |
| pMTX633 | pUT18C::trwB(D158A) | pMTX601 | pMTX520 | KpnI + StyI |
| pMTX645 | pUT18C::trwB(E357A) | pMTX601 | pMTX559 | KpnI + StyI |
| pMTX647 | pT25::trwB(Δ12C) | pT25 | pMTX615 | BamHI + EcoRI |
| pMTX648 | pT25::trwB(D158A) | pT25 | pMTX633 | BamHI + EcoRI |
| pMTX652 | pT25::trwB(Q386A) | pT25 | pMTX629 | BamHI + EcoRI |
| pMTX656 | pT25::trwB(E357A) | pT25 | pMTX645 | BamHI + EcoRI |
For construction, the first column lists the vector plasmids, the second column lists the plasmids from which the inserts were obtained and the method, and the third column indicates either the restriction enzymes used for cloning or the oligonucleotides used for PCR amplification of the desired fragment, with the restriction sites underlined. When three oligonucleotides are shown, the mutant was generated by the megaprimer method. The first two oligonucleotides were used for the first PCR (the first oligonucleotide codes for the mutation, and if a restriction site was introduced it is underlined), and this PCR was used as a megaprimer together with the third oligonucleotide in a second PCR. The final PCR products were cloned either as a KpnI-EcoRI fragment in pSU4633 (except in pMTX558, which was digested with NdeI plus EcoRI).
This plasmid was obtained by Red mutagenesis as described in Materials and Methods.
This mutant was found to contain the additional mutation R417S, which will be omitted for clarity.
The oriT fragment from plasmid pSU2007 was PCR amplified and inserted into the XbaI site of plasmid pET29c::trwAC, lying 5′ to the start of trwA; the orientation was selected to reconstruct the same oriT-trwA arrangement as in R388.
PCRs were performed with the GeneMorph system to introduce random mutations (see Materials and Methods). The oligonucleotides flank the inserts in vector pUT18C. After the mutagenic PCR, the fragment with the desired mutated region was obtained with the indicated restriction endonucleases.