Table 1.
Bacterial strains, plasmids, and primers used in this study
| Bacterial strain, plasmid, or primer | Relevant genotype or phenotypea or primer sequence | Source or reference |
|---|---|---|
| E. coli strains | ||
| BW25113 | E. coli Κ-12 Wt [Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) λ− rph-1 Δ(rhaD-rhaB)568 hsdR514] | CGSCb |
| TMY32 | BW25113 fabH::camR(pET-PA5174); Kanr Camr | 56 |
| TMT47 | BW25113 fabH::camR(pET-PA3286); Kanr Camr | This study |
| P. aeruginosa strains | ||
| NB52019 | P. aeruginosa PAO1 prototroph K767 | K. Poole |
| TMT01 | NB52019 ΔPA3333 (fabH2) | 56 |
| TMT02 | NB52019 ΔPA0999 (pqsD) | 56 |
| TMT12 | NB52019 ΔPA3286 | 56 |
| TMT15 | NB52019 ΔPA0998 (pqsC) | 56 |
| TMT16 | NB52019 ΔPA0998 ΔPA0999 ΔPA3333 ΔPA3286 | 56 |
| TMT39 | NB52019 ΔfabY (PA5174)::aacC1; Gmr | 56 |
| TMT44 | TMT12 attB::PA3286+; Gmr | This study |
| Plasmids | ||
| pMini-CTX1 | Chromosomal integration vector; FRT oriT int+ori (pMB1) tetR-FRT attP+ MCS; Tetr | 26 |
| pRC9 | pMini-CTX-1 with araC-PBAD promoter cassette | C. Dean |
| pTMT131 | pRC9 with the PA3286 gene and 483 bp upstream | This study |
| pFLP2 | E. coli-P. aeruginosa shuttle vector with Flp recombinase; Carbr | 25 |
| pET24b(+) | IPTG-inducible T7 promoter for protein expression; Kanr | Novagen |
| pET-PA3286 | pET24b(+) with PA3286 and C-terminal His tag; Kanr | This study |
| pKD3 | oriRλ camR; Camr | 13 |
| pTMT123 | pEX18ApGW-PA5174::aacC1 fabHEc; Gmr | 56 |
| pTMT124 | pEX18ApGW-PA5174::aacC1 fabH′ fragment; Gmr | 56 |
| Primers | ||
| CTX-PA3286 EcoRVc | TGCGACGCTGGCGATCTTCCTTGTGAATAC | This study |
| CTX-PA3286 XhoIc | CGGGCCCCCCCTCGAAACCAGCTCGCGGAAG | This study |
| PA3286 for NdeId | GTAGCTCATATGCATAAAGCCGTCATC | This study |
| PA3286 rev HindIIId | GTAGCTAAGCTTGTGTTTACGCAGGATC | This study |
| fabH Ec KO P1 | CGCCACATTGCCGCGCCAAACGAAACCGTTTCAACCATGGGCCGCCTACCTGTGACGGAA | 56 |
| fabH Ec KO P2 | CCGCCCCAGATTTCACGTATTGATCGGCTACGCTTAATGCATGAACTTCATTTAAATGGCGCG | 56 |
a
Carbr, carbenicillin resistant; Camr, chloramphenicol resistant; Kanr, kanamycin resistant; Gmr, gentamicin resistant; Tetr, tetracycline resistant; FRT, Flp recombinase target; MCS, multicloning site.
b
Strain CGSC7636 at the Coli Genetic Stock Center (CGSC).
c
Cloned using the In-fusion system (Clontech).
d
Cloned into vectors by DNA digestion and T4 DNA-catalyzed ligation.