TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Genotype | Reference or source |
|---|---|---|
| E. coli strains | ||
| DH5α | Δ(argF-lac)169 ϕ80dlacZ58(M15) ΔphoA8 glnV44 deoR481 gyrA96 recA1 endA1 hsdR17 | Lab stock |
| EC1000 | Emr; MC1000 derivative carrying single copy of pWV01repA in glgB | Lab stock |
| E. faecalis strains | ||
| FA2-2 | Rifampin/fusidic acid-resistant mutant derived from plasmid-free strain JH2 | 30 |
| FAZL1 | ΔfabI of E. faecalis FA2-2 | 18 |
| BHK260 | Spontaneous RBS mutant S1 of FAZL1, C-12 to G | This work |
| BHK261 | Spontaneous RBS mutant S2 of FAZL1, C-12 deletion | This work |
| BHK262 | Spontaneous RBS mutant S3 of FAZL1, A-9 to G | This work |
| BHK263 | Spontaneous RBS mutant S4 of FAZL1, T-19 to G | This work |
| FAZL6 | FA2-2 fabK (disruption) | This work |
| FAZL7 | FAZL1 fabK (disruption) | This work |
| FAZL8 | S1 fabK (disruption) | This work |
| FAZL9 | S2 fabK (disruption) | This work |
| FAZL10 | S3 fabK (disruption) | This work |
| FAZL11 | S4 fabK (disruption) | This work |
| Plasmids | ||
| pTRKL2 | Ermr, low-copy-no. E. coli/Lactococcus shuttle vector | 42 |
| pCR2.1 | Ampr Kanr, TOPO TA cloning vector | Invitrogen |
| pZL52 | Ampr Kanr, pCR2.1 carrying E. faecalis FAZL1 fabK plus 275 bp upstream and 288 bp downstream | This work |
| pZL53 | Ampr Kanr, pCR2.1 carrying E. faecalis mutant S1 (strain BHK260) fabK plus 275 bp upstream and 288 bp downstream | This work |
| pZL54 | Ampr Kanr, pCR2.1 carrying E. faecalis mutant S2 (strain BHK261) fabK plus 275 bp upstream and 288 bp downstream | This work |
| pZL55 | Ampr Kanr, pCR2.1 carrying E. faecalis mutant S3 (strain BHK262) fabK plus 275 bp upstream and 288 bp downstream | This work |
| pZL56 | Ampr Kanr, pCR2.1 carrying E. faecalis mutant S4 (strain BHK263) fabK plus 275 bp upstream and 288 bp downstream | This work |
| pZL157 | Ermr, EcoRV and BamHI-digested fabK fragment of pZL52 inserted into same sites of pTRKL2 | This work |
| pZL158 | Ermr, EcoRV- and BamHI-digested fabK fragment of pZL53 inserted into same sites of pTRKL2, C-12 to G | This work |
| pZL159 | Ermr, EcoRV- and BamHI-digested fab fragment of pZL54 inserted into same sites of pTRKL2, C-12 deletion | This work |
| pZL160 | Ermr, EcoRV- and BamHI-digested fabK fragment of pZL55 inserted into same sites of pTRKL2, A-9 to G | This work |
| pZL161 | Ermr, EcoRV- and BamHI-digested fabK fragment of pZL56 inserted into same sites of pTRKL2, T-19 to G | This work |
| pBHK368 | Ermr, fabK A-9T of pZL157 | This work |
| pBHK369 | Ermr, fabK A-9C of pZL157 | This work |
| pBHK370 | Ermr, fabK C-12A of pZL157 | This work |
| pBHK371 | Ermr, fabK C-12T of pZL157 | This work |
| pBHK372 | Ermr, fabK T-19A of pZL157 | This work |
| pBHK373 | Ermr, fabK T-19C of pZL157 | This work |
| pMC1871 | Tetr, promoterless lacZ reporter plasmid | 32 |
| pBHK322 | Ermr, lacZ from pMC1871 cloned into BamHI site of pTRKL2, p(lacZ) | This work |
| pBHK323 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, p(WT) | This work |
| pBHK394 | Ermr, pTRKL2 fabK (−275 to +35)::lacZ | This work |
| pBHK324 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, C-12 to G, p(S1) | This work |
| pBHK325 | Ermr, pTRKL2 P32::fabK (−136 to + 35)::lacZ, C-12 deletion, p(S2) | This work |
| pBHK326 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, A-9 to G, p(S3) | This work |
| pBHK327 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, T-19 to G, p(S4) | This work |
| pBHK334 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, A-9 to T | This work |
| pBHK335 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, A-9 to C | This work |
| pBHK336 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, C-12 to A | This work |
| pBHK337 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, C-12 to T | This work |
| pBHK338 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, T-19 to A | This work |
| pBHK339 | Ermr, pTRKL2 P32::fabK (−136 to +35)::lacZ, T-19 to C | This work |