TABLE 1.
Bacterial strains, plasmids, and oligonucleotides used in this study
| Strain, plasmid, or oligonucleotide | Description or sequence | Source or reference |
|---|---|---|
| Strains | ||
| P. aeruginosa | ||
| FRD1 | Cystic fibrosis isolate; Alg+ | 40 |
| FRDΔalg8Gm | Δalg8::aacC1; Alg− | This study |
| FRDΔalg8 | Δalg8; Alg− | This study |
| PAO1 | Prototrophic wild-type strain; Alg− | 22 |
| PDO300 | ΔmucA22 variant of PAO1; Alg+ | 34 |
| PDO300Δalg8Gm | Δalg8::aacC1; Alg− | This study |
| PDO300Δalg8 | Δalg8; Alg− | This study |
| E. coli | ||
| TOP10 | E. coli cloning strain | Invitrogen |
| S17-1 | thi-1 proA hsdR17 (rK− mK+) recA1; tra gene of plasmid RP4 integrated in chromosome | 61 |
| Plasmids | ||
| pBBR1MCS-5 | Gmr; broad-host-range vector; Plac | 28 |
| pBBR1MCS-5:alg8 | HindIII-BamHI fragment comprising gene alg8 inserted into vector pBBR1MCS-5 | This study |
| pBBR1MCS-5:alg8His | HindIII-BamHI fragment encoding C terminally hexahistidine-tagged Alg8 inserted into vector pBBR1MCS-5 | This study |
| pBBR1MCS-5:alg8GFP | Translational Alg8-GFP fusion, inserted into vector pBBR1MCS-5 | This study |
| pBBR1MCS-5:alg8lacZ | Translational Alg8-LacZ fusion, inserted into vector pBBR1MCS-5 | This study |
| pBBR1MCS-5:alg8phoA | Translational Alg8-PhoA fusion, inserted into vector pBBR1MCS-5 | This study |
| pEX100T | Apr Cbr, gene replacement vector containing sacB gene for counterselection | 20 |
| pEX100TΔalg8Gm | Apr Cbr Gmr; vector pEX100T with SmaI-inserted alg8 deletion construct | This study |
| pPS856 | Apr Gmr; source of 1,100-bp BamHI fragment comprising aacC1 gene flanked by FRT signal sequences | 20 |
| pPFLP2 | Apr Cbr; broad-host-range vector encoding Flp recombinase | 20 |
| pPHO7 | Apr; phoA without signal sequence | 19 |
| pZsGreen | GFP translational fusion vector | BD Clontech |
| pJE608 | LacZ lacking the first 8 amino acids with promoter Ptac in pMMB67EH | 9 |
| Oligonucleotides | ||
| alg81N-Ec5 | CGCAGGATATCGGAAACTTACAAACGTGGCCTC | |
| alg81N-Ba | TAGAGGATCCGGTTCATCTTCTCCCACAGAG | |
| alg82C-Ba | TGATGGATCCGTTCACCATGCTGGTGCTGTTC | |
| alg82C-Ec5 | CCAGGATATCTCATACGATGGTCAGCAGCAC | |
| alg8up | AAGAACCTCTTTATCGCCCTCGGAC | |
| alg8down | TCACGGATCCCCAGGTAGGAGGTGATCAGGTAG | |
| alg8N(HiSDNd) | CCGGGAAGCTTGAGGAGCACAGCCATATGGAACTGATGATGGAAACTTACAAA | |
| alg8C(Ba) | CCCGGATCCTCATACGATGGTCAGCAGCACG | |
| alg8C(HisBa) | CTTGGATCCTCAATGGTGATGGTGATGGTGTACGATGGTCAGCAGCACGGCGAC | |
| alg8C(Δstop) | CCCGGATCCCATACGATGGTCAGCAGCACG |