Table 1.
Strains, plasmids and primers used in this study
| Strain, plasmid, or primer | Description or sequencea (5′ to 3′) | Source, reference, or restriction enzyme |
|---|---|---|
| E. faecalis | ||
| JH2-2 | Fusr Rifr; plasmid-free wild-type strain | 13 |
| JHB1 | JH2-2 isogenic derivative citO mutant | 9 |
| JHA | JH2-2 isogenic derivative oadA mutant | This work |
| JHB | JH2-2 isogenic derivative oadB mutant | This work |
| JHD | JH2-2 isogenic derivative oadD mutant | This work |
| E. coli | ||
| DH5α | fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80dlacZΔM15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17; used as an intermediate host for cloning | 12 |
| EC101 | supE thi Δ(lac-proAB) (F′ traD36 proAB lacIqZΔM15) repA; Kanr; used as the host for pBV-Gh constructs | 14 |
| BL21(DE3) | F− ompT hsdSB(rB− mB−) gal dcm (DE3) | 15 |
| Plasmids | ||
| pGEM-T Easy | Plasmid for intermediate cloning | Promega |
| pBV-Gh | Plasmid used for the construction of deletion mutants | 10 |
| pBV-GhA | pBV-Gh derivative carrying 500 bp upstream and downstream portions of oadA gene | This work |
| pBV-GhB | pBV-Gh derivative carrying 530 bp upstream and downstream portions of oadB gene | This work |
| pBV-GhD | pBV-Gh derivative carrying 520 bp upstream and downstream portions of oadD gene | This work |
| pET28a | His-tagged protein expression plasmid | Novagen |
| pET-A | pET28a derivative expressing His6-OadA | This work |
| pBAD24 | Arabinose-inducible plasmid | 11 |
| pBAD-D | pBAD24-His derivative expressing His6-OadD | This work |
| pMal-c2X | NEB | |
| pMal-H | pMal-c2X derivative expressing MBP-OadH | This work |
| pET-ADB | pET28a derivative expressing His6-OadA, OadD, and OadB | This work |
| pET-AHDB | pET28a derivative expressing OAD complex subunits | This work |
| Primers | ||
| OadAUp-For | CACACACCATGGGTTGTTAGAAAC | NcoI |
| OadAUp-Rev | CCCGTAAAGCTTTTCTGTAAAACG | HindIII |
| OadALo-For | AAAGATAAGCTTCGCGGAGAATATG | HindIII |
| OadALo-Rev | GCTAAACCATGGTGGTTTAACACCG | NcoI |
| OadBUp-For | AAACCATGGATGTTACCCAAGGCC | NcoI |
| OadBUp-Rev | CGGGGATCCTAACAATGGACCG | BamHI |
| OadBLo-For | CAAGGATCCGTGCTGCGGGTATTTC | BamHI |
| OadBLo-Rev | TTACCATGGCAGATTCTTTTCATTG | NcoI |
| OadDUp-For | CGGGGTACCAATGCTTATATACCAATG | KpnI |
| OadDUp-Rev | ACTGAATTCTCTTTCCCATCAATTG | EcoRI |
| OadDLo-For | CAGGAATTCATGTTACCCAAGGC | EcoRI |
| OadDLo-Rev | CCAAGGTACCCAATCGAAGCAGC | KpnI |
| OadA1 | GACACGAAAGGAGCAGCCATATGAG | NdeI |
| OadA2 | CGGAAAGCTTGCCCGTTCTATTCTG | HindIII |
| OadB1 | GTATTCGTTCTCGAGTCCGTCCTATG | XhoI |
| OadD1 | GCATCCATGGGATTACGCAAATTTAAAATTTC | NcoI |
| OadD2 | GCTAGGATCCCTAGTTAATTGTTATTAATGGTTCC | BamHI |
| OadD3 | GGGCTGTCAGAAGAAGCTTAGCTAGTTG | HindIII |
| OadH1 | AGAGAATTCATGGGCCTATTTAAGTC | EcoRI |
| OadH2 | CCCAAGCTTTTAAATTTGCGTAAC | HindIII |
| OadH3 | CTATAATGAAGCTTTACCAAGAATTAGTG | HindIII |
a
Fus, fusidic acid; Rif, rifampin; Tet, tetracycline; Em, erythromycin.