TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant genotype | Reference or source |
|---|---|---|
| Strains | ||
| MC4100 | F−araD139 Δ(argF-lac)U169 rpsL150 relA1 flbB5301 deoC ptsF25 rbsR | 5 |
| MG1655 | F− λ−rph−1 | GSC7740 |
| JM109 | F′ traD36 proA+ proB+ lacIqlacZΔM15 recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 Δ(lac-proAB) | 2 |
| FB8 | Wild-type E. coli K-12, UTH1038 | 17 |
| MC4100gadX | MC4100 gadX::Cmr | This study |
| MC4100gadW | MC4100 gadW::Cmr | This study |
| MC4100gadE | MC4100 gadE::Kanr | This study |
| MG1655gadW | MG1655 gadW::Cmr | This study |
| Plasmids | ||
| pBs | (pBluescriptSK) multicopy phagemid vector; ColE1 replicon, lacZα bla | Stratagene |
| pBsW | 1,080-bp EcoRI-BamHI fragment encompassing the gadW gene | This study |
| pBsX | 2.1-kb EcoRI-ClaI genomic fragment encompassing the gadX gene, the intergenic gadXW region, and first 162 nt of the gadW ORF in pBs | 37 |
| pBsX′ | 1.7-kb EcoRI-EcoRV fragment encompassing the gadX gene in pBs | This study |
| pTgadW | gadW ORF (735 bp) cloned in pBs-derived T/A vector | 22 |
| pBsPgadA | Promoter region of gadA (from position −176 to position 77) in pBs | 37 |
| pBsPgadBC | Promoter region of gadB (from position −173 to position 77) in pBs | 37 |
| pBsPgadB-307 | Promoter region of gadB (from position −307 to position 77) in pBs | This study |
| pMAL-c2 | Phagemid vector; ColE1 replicon, Ptac malE::lacZα lacIqbla | NEB |
| pmalE::gadW | gadW ORF and part of the multiple-cloning site of pBs (764 bp) ligated to the EcoRI-HindIII sites of pMAL-c2 | This study |
| pmalE::gadX | gadX ORF and part of the multiple-cloning site of pBs (832 bp) ligated to the EcoRI-HindIII sites of pMAL-c2 | 37 |
| pUC18 | Multicopy vector; ColE1 replicon, lacZα bla | NEB |
| pUCgadW | 761-bp gadW PCR fragment ligated to the EcoRI-BamHI sites of pUC18 | This study |
| pQE60 | Expression vector; ColE1 replicon, PT5-lacO RBSII | QIAGEN |
| pQEgadW | 785-bp EcoRI-HindIII gadW fragment from pUCgadW in pQE60 | This study |
| pRS415 | Operon fusion vector; ColE1 replicon lacZYA bla | 34 |
| pKgadB-244 | Promoter region of gadB (from position −244 to position 77) in pKK232.8 | 14 |