TABLE 1.
List of bacterial strains, plasmids, and primers used in this studya
| Strain, plasmid, or primer | Relevant properties | Reference or source |
|---|---|---|
| Strains | ||
| Escherichia coli MG1655 | F-, λ-, ilvG-, rfb-50, rph-1 | 76 |
| ΔtrhAEC | F-, λ-, ilvG-, rfb-50, rph-1, ΔtrhAEC::Km | This work |
| ΔfadR | F-, λ-, ilvG-, rfb-50, rph-1, ΔfadR | 67 |
| ΔfadRΔfabR | P1 transduction of ΔfabR::Km (Keio collection) in ΔfadR. Km cassette removed using pCP20 plasmid. | This work |
| MG1655 (pRH005) | MG1655 containing empty (pRH005) (Km, Cm) | This work |
| ΔtrhAEC (pRH005) | ΔtrhAEC containing empty (pRH005) (Km, Cm) | This work |
| ΔtrhAEC (pRH005 trhAEC) | ΔtrhAEC containing (pRH005 trhAEC) (Km, Cm) | This work |
| MG1655 (pBAD33) | MG1655 containing empty (pBAD33) (Cm) | This work |
| ΔtrhAEC (pBAD33) | ΔtrhAEC containing empty (pBAD33) (Cm) | This work |
| ΔtrhAEC (pBAD33 trhAEC) | ΔtrhAEC containing (pBAD33 trhAEC) (Cm) | This work |
| MG1655 (pTrc99a) | MG1655 containing empty (pTrc99a) (Ap) | This work |
| ΔtrhAEC (pTrc99a) | ΔtrhAEC containing empty (pTrc99a) (Ap) | This work |
| ΔtrhAEC (pTrc99a trhAEC) | ΔtrhAEC containing (pTrc99a trhAEC), (Ap) | This work |
| Escherichia coli Top10 | General cloning strain | Invitrogen |
| Escherichia coli UB1005 | F-, λ-, gyrA37(NalR), relA1, spoT1, metB1, λR | 77 |
| UB1005 ΔtrhAEC | F-, λ-, gyrA37(NalR), relA1, spoT1, metB1, λR, ΔtrhA::Km | This work |
| Bacillus subtilis 168 | trpC2 | 78; BGSC |
| ΔtrhABS | trpC2, ΔyplQ::Erm | 78; BGSC |
| Plasmids | ||
| pUA66 | ori sc101, GFPmut2 (Km) | 40 |
| pUA-fabB | fabB promoter in pUA66 | 67 |
| pUA-fabA | fabA promoter in pUA66 | 67 |
| pUA-trhAEC | trhAEC promoter in pUA66 | 40 |
| pUA-trhAECmut | Site-directed PCR mutagenesis in the FabR binding site on the pUA- trhAEC plasmid | This work |
| pJL72 | TAP tag sequence | 68 |
| pUA-TAP | TAP tag sequence from pJL72 (BamHI/EcoRV) in pUA66 (BamHI/HincII) | This work |
| pUA-TrhAEC-TAP | trhAEC gene (promoter and ORF) cloned in phase with TAP sequence in pEB1588(XhoI/BamHI) | This work |
| pDONR221 | Gateway-based cloning vector (Km) | Invitrogen |
| pRH005 | Gateway-based destination vector expressing proteins fused with YFP at the C terminus; ori RK2oriV, (Km, Cm) | 51 |
| pRH005 trhAEC | pRH005 containing WT trhAEC, (Km, Cm) | This work |
| pTrc99a | Expression vector with inducible LacI promoter; ori ColE1/pMB1/pBR322, (Ap) | 79 |
| pTrc99a trhAEC | pTrc99a containing trhAEC (Cm) | This work |
| pBAD33 | Expression vector with arabinose-inducible promoter; ori pACYC184/p15A (Cm) | 80 |
| pBAD33 trhAEC | pBAD33 containing trhAEC (Cm) | This work |
| Primers | ||
| trhAEC Fwd | CACCATGGTTCAGAAGCCCCTC | |
| trhAEC Rev | TTACGCCTGCCCAATATA | |
| UP trhAEC XhoI | ttgctcgagGAAAATATGACCCTGACTGAACTG | |
| trhAEC BamHI Rev | cgggatccCGCCTGCCCAATATACAAATAGATC | |
| trhAECmut FabR site Fwd | CAGAATTTATTTTAGCTAGTGGGTGTTCACTGGAAC | |
| trhAECmut FabR site Rev | GTTCCAGTGAACACCCACTAGCTAAAATAAATTCTG | |
| GW trhAEC Fwd | GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGTTCAGAAGCCCCTCATT | |
| GW trhAEC Rev | GGGGACCACTTTGTACAAGAAAGCTGGGTCCGCCTGCCCAATATACAA | |
| GW pgk Fwd | GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTCTGTAATTAAGATGACC | |
| GW pgk Rev | GGGGACCACTTTGTACAAGAAAGCTGGGTCCTTCTTAGCGC GCTCTTC | |
| pRH005 ccdB mut Fwd | ATGTTCTAAAGCAGGTAAATGTCAGGC | |
| pRH005 ccdB mut Rev | GCCTGACATTTACCTGCTTTAGAACAT | |
| XbaI trhAEC Fwd | TAAGCATCTAGATCTAGGAGGTAAGTTATGGTT | |
| HindIII trhAEC Rev | TGCTTAAAGCTTTTACGCCTGCCCAATATA | |
a
Km, kanamycin; Cm, chloramphenicol; Ap, ampicillin; Erm, erythromycin; BGSC, Bacillus Genetic Stock Center. The restriction enzymes used are underlined as part of the primer name, and the location of these restriction sites within the primer is underlined.