TABLE 1.
Strains, plasmids, and primers used
| Strain, plasmid, or primer | Description | Background | Reference or source |
|---|---|---|---|
| Strains | |||
| S. coelicolor | |||
| M145 | Prototroph SCP1− SCP2− | Lab stock | |
| N373 | ramC::acc(3)IV SCP1− SCP2− | 7 | |
| E. coli | |||
| BL21 | F−dcm ompT hsdS(rB− mB−) gal met | Novagen | |
| BL21(DE3)plysS | F−dcm ompT hsdS(rB− mB−) gal λ(DE3) (pLysS Camr) | Novagen | |
| XL-1 Blue | recA1 endA1 gyrA96 thi-1 glnV44 hsdR17 supE44 relA1 lac [F′ proAB lac1q ZΔM15 Tn10(Tetr)] | Stratagene | |
| Er2-1 | F′ lacI leuB6 thi-1 fhuA31 lacY1 tsx-78 galK2 galT22 supE44 hisG4 rpsL136(Strr) xyl-5 mtl-1 dam-13::Tn9(Camr) dcm-6 mcrB1 mcrA hsdR2(rk− mk+) | J. McCormick | |
| Plasmids | |||
| E. coli | |||
| pET21a | T7 promoter based expression plasmid (Ampr) | Novagen | |
| pGEX-5x-1 | N-terminal GST fusion expression vector (Ampr) | Amersham Pharmacia | |
| pEMH-3 | RamC N terminus-GST fusion (Ampr) | pGEX-5X-1 | This study |
| pEMH-4 | RamC N terminus-His6 fusion (Ampr) | pET21a | This study |
| S. coelicolor | |||
| pSETΩ | aac(3)IV aadA+ (Spcr) | pSET152 | 7 |
| pTO8 | ramC (Spcr) | pSETΩ | 7 |
| pTO8-K259A | ramC K259A (Spcr) | pTO8 | This study |
| pTO8-D369A | ramC D369A (Spcr) | pTO8 | This study |
| pTO8-D387A | ramC D387A (Spcr) | pTO8 | This study |
| pTO8-D421A | ramC D421A (Spcr) | pTO8 | This study |
| Oligonucleotides | |||
| JJ5 | 5′-GGAGCACATATGAACAAGGGCTACGCCGTG-3′ | ||
| JJ31 | 5′-ACACGACAATTCTCACCGGTCCACGGCCACCCC-3′ | ||
| JJ32 | 5′-TGTGCTGAATTCACGGCTCCTGGTGGGGCC-3′ | ||
| K259ATop | 5′-ACCGGTGCGCGGGTCGTGCTAGCGGAGGCCCGT-3′ | ||
| K259ABot | 5′-ACGGGCCTCCGCTAGCACGACCCGCGCACCGGT-3′ | ||
| D369ATop | 5′-CCGGGGTGTGGTCTTCAACGCGTTGCACCTGTTC-3′ | ||
| D369ABot | 5′-GAACAGGTGCAACGCGTTGAAGACCACACCCCGG-3′ | ||
| D387ATop | 5′-ACGACAGCGTCGCCCTGCTAGCCTTCGAG-3′ | ||
| D387ABot | 5′-CTCGAAGGCTAGCAGGGCGACGCTGTCGT-3′ | ||
| D421ATop | 5′-TTCGTGGCGCCTCCCGCGCGCCGCGGG-3′ | ||
| D421ABot | 5′-CCCGCGGCGCGCGGGAGGCGCCACGAA-3′ |