TABLE 2.
Primers used in this study
| Primer | Sequence (5′-3′)a | Purpose |
|---|---|---|
| msrA-F | CGCGGATCCATGGCATGGTTTTTCGCACCCG (BamHI) | For cloning msrA wild type and mutants into pXMJ19 or pET28a |
| msrA-R | ACGCGTCGACTTAGATAGGGAGATTCTCCTC (SalI) | |
| DmsrA-F1 | CGCGGATCCGTTGAAGACTCCAAAAAGGGCATC (HindIII) | To generate pK18mobsacB-ΔmsrA |
| DmsrA-R1 | GCGAAAAACCATGCCATAACAGG | |
| DmsrA-F2 | CCTGTTATGGCATGGTTTTTCGCACAAGAATCCCGATGGCTACTG | |
| DmsrA-R2 | ACATGCATGCGGTCACCTGGTAGTTCGATGTCC (SphI) | |
| PmsrA-F1 | TCCCCCGGGCTGCCCGGCAACAGTGTG (SmaI) | To generate pK18mobsacB-PmsrA::lacZY |
| PmsrA-R | TCTAGAGAAAAACCATGCCATAACAGG (XbaI) | |
| PmsrA-F2 | GCGCCTGCAACGTAGGTTGCG | To gain 400-bp msrA promoter DNA segment |
| lacZY-F | CCTGTTATGGCATGGTTTTTCTCTAGAACTAGTATGACCATGATTACGGATTC(SpeI) | To generate lacZY fragment |
| lacZY-R | AAAACTGCAGTTAAGCGACTTCATTCACCTG (PstI) | |
| msrA-C56S-F | TGGATTGGCCTTGGTAGTTTCTGGGGTGTGG | To generate pXMJ19-msrA:C56S and pET28a-msrA:C56S |
| msrA-C56S-R | CCACACCCCAGAAACTACCAAGGCCAATCCA | |
| msrA-C91S-F | CACTTATCGCGAGGTGAGTTCTGGGCGCACCG | To generate pXMJ19-msrA:C91S and pET28a-msrA:C91S |
| msrA-C91S-R | CGGTGCGCCCAGAACTCACCTCGCGATAAGTG | |
| msrA-C204S-F | GAATCCCGATGGCTACAGCCCTCATCACTCCAC | To generate pXMJ19-msrA:C204S and pET28a-msrA:C204S |
| msrA-C204S-R | GTGGAGTGATGAGGGCTGTAGCCATCGGGATTC | |
| msrA-C213S-F | CCACGGGCATCCCGAGCGGGGTAGAAGCTT | To generate pXMJ19-msrA:C213S and pET28a-msrA:C213S |
| msrA-C213S-R | AAGCTTCTACCCCGCTCGGGATGCCCGTGG | |
| trx-F | CGCGGATCCATGAGCAATGTTGTTGCAGTAAC (BamHI) | To generate pET28a-trx and pET28a-trx:C35S |
| trx-R | CCGCTCGAGCTAGAGGTGCTTCTCTAGTTTTTC (XhoI) | |
| DmetE-F1 | GGAAGATCTATCGATGCGGAGAGCATCAG (BglII) | To generate pK18mobsacB-ΔmetE |
| DmetE-R1 | CCATTCCAGTAGCCTTCGAGCGC | |
| DmetE-F2 | GCGCTCGAAGGCTACTGGAATGGGCTAAGCAGGCTCGTGAGAAAATCG | |
| DmetE-R2 | ACGCGTCGACGTTCTGGTGGGGTGGATCAG (SalI) | |
| DmetH-F1 | CGCGGATCCGCTATTTGCCACTCCGATGTTTG (BamHI) | To generate pK18mobsacB-ΔmetH |
| DmetH-R1 | TGTTGTGGGCTGGTGAAGTAAC | |
| DmetH-F2 | GTTACTTCACCAGCCCACAACATCTACCACCCAGAGGCAAAG | |
| DmetH-R2 | ACGCGTCGACATACTCGGCTGCAATCCAGAC (SalI) | |
| Dtrx-F1 | CGCGGATCC GCTGACCTCAACCCCATCATGTTC (BamHI) | To generate pET28a-trx:C35S |
| Dtrx-R2 | CCCAAGCTT GCTGCTGGGATCATCCATGTGC (HindIII) | |
| trx-C35S-F | GAATGGTGTGGCCCCAGCAAGAAGCTCAGCC | |
| trx-C35S-R | GGCTGAGCTTCTTGCTGGGGCCACACCATTC | |
| mrx1-F | CCGGGATCCATGAGCAACGTAACCATTTACGCC (BamHI) | To generate pET28a-mrx1 and pET28a-mrx1:C15S |
| mrx1-R | CCCGTCGACTTAGGCTAATGCTTCGATTTTGG (SalI) | |
| Dmrx1-F1 | CGCGGATCCATTGTCTCGGCGTGGAATGTTTCC (BamHI) | To generate pK18mobsacB-Δmrx1 and pET28a-mrx1:C15S |
| Dmrx1-R1 | GATTACTTCCGCAGCTAGGGGATTGAGGAGGGATCGGCAGTAAG | |
| Dmrx1-F2 | ATCCCCTAGCTGCGGAAGTAATC | |
| Dmrx1-R2 | ACGCGTCGACACCCATTTGTATTCGCCCGTG (SalI) | |
| mrx1-C15S-F | GATTGGTGCCCTTACAGCCGATCCCTCCTC | To generate pET28a-mrx1:C15S |
| mrx1-C15S-R | GAGGAGGGATCGGCTGTAAGGGCACCAATC | |
| Control-F | ATGACCACCAGCAACCCC | To generate control DNA for EMSA |
| Control-R | CCCTCGGGGGAACTTCCCGG |
a
Underlining indicates restriction enzyme cutting sites added for cloning. Italics denote the mutation sites in overlap PCR for site-directed mutagenesis.