TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Relevant genotype and characteristic(s)a | Source or reference |
|---|---|---|
| E. coli | ||
| DH5α | Routine cloning vector | Stratagene |
| BL21(DE3) | gal ompT, lysogen of DE3 carrying T7 polymerase gene | 29 |
| S17-1 (IR747) | Carries RP4 tra genes and λ pir gene | 25 |
| IR1638 | IR747 harboring pWMZ1637 | This study |
| P. aeruginosa | ||
| IA614 | PAO1 derivative, siderophore deficient | 1 |
| IR1648 | IA614 derivative, chromosomal knockout of pigA, Genr | This study |
| IR4309 | IR1648, harbors pMMB66HE | This study |
| IR4310 | IR1648, harbors pMMB66HE | This study |
| IR4311 | IR1648, harbors pMCR2050(pigA) | This study |
| N. meningitidis | ||
| IR1072 | WT laboratory strain, serogroup C | 40 |
| 2855 | IR1072 derivative, chromosomal knockout of hemO | 40 |
| 4093 | 2855 derivative, chromosomal insertion of pigA | This study |
| Plasmids | ||
| pCR 2.1 Topo II | PCR cloning vector, Ampr Kanr | Invitrogen |
| pBluescript II SK(+) | ColE1 cloning phagemid, Ampr | Stratagene |
| pRK2013 | Kmr, ColE1 with Tra+ Mob+ | 6 |
| pET21a | T7 expression vector, Ampr | Novagene |
| pGMΩ | 0.9-kb gentamicin cassette cloned into pHP45Ω, Ampr Genr | 23 |
| pGP704 | pir-dependent suicide vector, Ampr | 14 |
| pCVD442 | pir-dependent suicide vector, Sucs, Ampr | 5 |
| pMMB66HE | Broad host range, tacP expression vector, Ampr | 8 |
| pWMZ1591a | 3-kb erm-lacIOP cassette cloned 5′ of hemO | 24 |
| pWMZ1605 | PCR product encoding PigA on pCR 2.1 Topo II | This study |
| pWMZ1606 | BamHI fragment containing pigA in pBluescript II SK(+) | This study |
| pWMZ1613 | HindIII-SacI fragment from pWMZ1606 in pUC18 | This study |
| pWMZ1621 | Gentamicin cassette cloned into KpnI site of pWMZ1613 | This study |
| pWMZ1629 | EcoRI fragment of pWMZ1621 cloned into pGP704 | This study |
| pWMZ1637 | XbaI-SmaI fragment from pWMZ1621 cloned into pCVD442 | This study |
| pWMZ1614 | 0.8-kb EcoRI-HindIII PCR product of hmbR in pCR 2.1 Topo II | This study |
| pWMZ1616 | 4-kb EcoRI-KpnI from pWMZ1591 and hmbR from pWMZ1614 in pBlueScript II SK(+) | This study |
| pWMZ1619 | pCR 2.1 Topo II minus EcoRI restriction site | This study |
| pWMZ1635 | 5-kb KpnI-HindIII fragment of pWMZ1616 into pWMZ1619 | This study |
| pWMZ1639a | EcoRI pigA fragment of pWMZ1605 in pWMZ1635 | This study |
| pWMZ1655 | PCR product encoding PigA-His tagged in pCR 2.1 Topo II | This study |
| pWMZ1656 | NdeI-XhoI fragment from pWMZ1655 cloned into pET21a | This study |
| pWMZ1665 | PCR product encoding native PigA in PCR 2.1 Topo II | This study |
| pWMZ1666 | NdeI-XhoI fragment from pWMZ1666 cloned into pET21a | This study |
| pMCR2050 | EcoRI fragment carrying pigA cloned into pMMB66HE | This study |
a
WT, wild type; Amp, ampicillin; Kan, kanamycin; Gen, gentamicin; Suc, sucrose.