TABLE 1.
Strains and plasmids used in this study
| Strains or plasmids | Genotype or phenotype | Reference or source |
|---|---|---|
| E. coli strains | ||
| DH5α | F− ϕ80d lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1hsdR17 (rk− mk +) supE44 ë−thi-1 gyrA relA1 | (49) |
| S17-1 | 294 (recA pro res mod+) Tpr Smr (pRP4–2-Tc::Mu-Km::Tn7) | (50) |
| SM10 | Kmr supE44 thi-1 thr-1 recA leu B6 lacY1 ton A21 RP4–2-Tc::Mu-Km::Tn7 | (50) |
| BL21 (DE3) | F− ompT hsdSB (rB−mB−) gal dcm met(DE3) | Novagen |
| C41 (DE3) | Derived from BL21 (DE3), at least one uncharacterized mutation | (51) |
| C43 (DE3) | Derived from C41 (DE3), at least two uncharacterized mutations | (51) |
| A. vinosum strains | ||
| Rif50 | Rifr; spontaneous rifampicin-resistant mutant of A. vinosum DSM 180T | (31) |
| rhd_2599:: ΩSm | Rifr; insertion of ΩSm in rhd_2599 of Rif50 | This study |
| ΔtusA | Rifr; Rif50 with in-frame deletion of tusA | This study |
| Δrtd | Rifr; Rif50 with in-frame deletion of rhd_2599, tusA, and dsrE2 | This study |
| Δrtd::ΩKm | Rifr Kmr; Rif50 with in-frame deletion of rhd_2599, tusA, dsrE2, and ΩKm in center of deleted sequence | This study |
| Plasmids | ||
| pHP45-Ω | Apr Smr/Spcr | (52) |
| pHP45Ω-Km | Apr Kmr | (53) |
| pGEM-5Zf | Apr; lacZ f1 ori | Promega |
| pGEM5-tusA | Apr; rhd_2599-tusA-dsrE2 amplicon cloned into SalI restriction site of pGEM-5Zf | This study |
| pGEM5-rhd_2599::ΩSm | Apr Smr; SmaI fragment from pHP45-Ω ligated into Bsp68I restriction site of pGEM 5-tusA | This study |
| pK18mobsacB | Kmr, lacZ', sacB, Mob+ | (35) |
| pK18mobsacBΔtusA | Kmr; 1,21 kb amplicon with deleted tusA cloned into XbaI restriction site of pK18mobsacB | This study |
| pK18mobsacBΔrtd | Kmr; 1,21-kb amplicon with deleted rhd_2599-tusA-dsrE2 cloned into BamHI-restriction site of pK18mobsacB | This study |
| pSUP202 | Apr Cmr Tcr; Mob+ RP4 oriT | (50) |
| pSUP202-rhd_2599::ΩSm | SalI fragment from pGEM5-rhd_2599::ΩSm cloned into SalI of pSUP202 | This study |
| pSUP301 | Apr Kmr; RP4 oriT p15A ori Mob+ | (50) |
| pSUP301Δrtd | Apr; 1,21 kb amplicon with deleted rhd_2599-tusA-dsrE2 cloned into HindIII-restriction site of pSUP301 | This study |
| pSUP301Δrtd::ΩKm | Apr Kmr; pHP45Ω-Km kanamycin cassette inserted into pSUP301Δrtd using EcoRI restriction sites | This study |
| pET-15b | Apr, T7 promoter, His tag (N terminus) | Novagen |
| pET15bRhd | Apr, NdeI/XhoI fragment of amplified rhd_2599 in NdeI/XhoI of pET-15b | This study |
| pET15bRhd-C64S | Apr, NdeI/BamHI fragment of amplified rhd_2599-C64S in NdeI/BamHI of pET-15b | This study |
| pET15bRhd-C74S | Apr, NdeI/BamHI fragment of amplified rhd_2599-C74S in NdeI/BamHI of pET-15b | This study |
| pET15bTusA | Apr, NdeI/BamHI fragment of amplified tusA in NdeI/BamHI of pET-15b | This study |
| pET15bTusA-C15S | Apr; NdeI/BamHI fragment of amplified tusA-C15S in NdeI/BamHI of pET-15b | This study |
| pET15bEcTusA | Apr, NdeI/BamHI fragment of amplified E. coli tusA in NdeI/BamHI of pET-15b | This study |
| pASK-IBA5plus | Apr, Tet promoter, Strep-Tag (N-terminal) | IBA |
| pIBADsrE2 | Apr; BsaI fragment of amplified dsrE2 in BsaI of pASK-IBA5plus | This study |
| pIBADsrE2-C110S | Apr; BsaI fragment of amplified dsrE2-Cys110Ser in BsaI of pASK-IBA5plus | This study |
| pIBADsrE2-C120S | BsaI fragment of amplified dsrE2-C120S in BsaI of pASK-IBA5plus | This study |
| pIBADsrE2-C156S | Apr; BsaI fragment of amplified dsrE2-Cys156Ser in BsaI of pASK-IBA5plus | This study |
| pETEFH | Apr; NdeI/BamHI fragment of amplified dsrEFH in pET-15b | (54) |
| pETE78FH | Apr; NdeI/BamHI fragment of amplified dsrE78FH in pET-15b | (6) |
| pETCEX | Apr; NdeI/BamHI fragment of amplified dsrC in pET-15b | (5) |
| pETCEXSer-100 | Apr; NdeI/BamHI fragment of amplified dsrC100 in pET-15b | (5) |
| pETCEXSer-111 | Apr; NdeI/BamHI fragment of amplified dsrC111 in pET-15b | (5) |