TABLE 2.
List of strains, plasmid and primers
| Strain, plasmid, or primer | Relevant genotype, phenotype, and/or sequence | Reference or source |
|---|---|---|
| Escherichia coli | ||
| DH5α | ϕ80lacZΔM15 recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 deoR Δ(lacZYA-argF)U169 | Invitrogen (Bethesda Research Laboratories) |
| MTP216 | Tcr, PAO1 fragment 4068269–4093431 (pMO013722) | 44 |
| Pseudomonas aeruginosa | ||
| PA2192 | Alg+ mucA180 | 36 |
| PAO1 | Alg− prototypic strain, nonmucoid | 45 |
| PDO300 | Alg+ mucA22, constitutively mucoid (PAOmucA22) | 5 |
| PKM800 | Alg− sap22 mucA22 alg107-1 | 29 |
| PKM816 | Alg− sap26 mucA22 alg107-2 | This study |
| PKM817 | Alg− sap27 mucA22 alg107-3 | 46 |
| PKM818 | Alg− sap31 mucA22 alg107-4 | This study |
| PKM819 | Alg− sap42 mucA22 algO55-1 | This study |
| PKM820 | Alg− sap32 mucA22 algO336-1 | This study |
| PKM821 | Alg− sap16 mucA22 algO500-1 | This study |
| PKM822 | Alg− sap17 mucA22 algO500-2 | This study |
| PKM823 | Alg− sap23 mucA22 algO500-3 | This study |
| PKM824 | Alg− sap24 mucA22 algO500-4 | This study |
| PKM825 | Alg− sap25 mucA22 algO500-5 | This study |
| PKM826 | Alg− sap46 mucA22 algO516-1 | This study |
| PKM827 | Alg− sap47 mucA22 algO516-2 | This study |
| PKM828 | Alg− sap30 mucA22 mucP159-1 | This study |
| PKM829 | Alg− sap36 mucA22 mucP304-1 | This study |
| PKM830 | Alg− sap20 mucA22 mucP392-1 | This study |
| PKM831 | Alg− sap21 mucA22 mucP392-2 | This study |
| PKM832 | Alg− ΔalgO | PAOΔalgO (46) |
| PKM833 | Alg− mucA22 ΔalgO | PDOΔalgO (46) |
| PKM834 | Alg− ΔalgW | PAOΔalgW (46) |
| PKM835 | Alg− mucA22 ΔalgW | PDOΔalgW (46) |
| PKM836 | Alg− ΔmucE | PAOΔmucE; this study |
| PKM837 | Alg+ mucA22 ΔmucE | PDOΔmucE; this study |
| Plasmids | ||
| pCD10 | Apr Kmr; pCRII-TOPO with 300-bp fragment containing mucE | This study |
| pCD11 | Apr Kmr; pCRII-TOPO with 1,400-bp fragment containing mucD | This study |
| pCD12 | Apr Kmr; pCRII-TOPO with 1,400-bp fragment containing algW | This study |
| pCD13 | Apr Kmr; pCRII-TOPO with 2,000-bp fragment containing algO | This study |
| pCD20 | Apr/Cbr; pMF54 with 300-bp fragment containing mucE | pMucE; this study |
| pCD21 | Apr/Cbr; pMF54 with 1,400-bp fragment containing mucD | pMucD; this study |
| pCD22 | Apr/Cbr; pMF54 with 1,400-bp fragment containing algW | pAlgW; this study |
| pCD23 | Apr/Cbr; pMF54 with 2,000-bp fragment containing algO | pAlgO; this study |
| pCRII-TOPO | Apr Kmr; colE1 lacZa | Invitrogen |
| pJG293 | Tcr Hgr Kmr pAlgT/U oriT (RK2-Tra+)::Tn501-250 | 29 |
| pLAFR3 | Tcr; cosmid vector derived from pLAFR1; IncP1 λ cos+ rlx | 47 |
| pLVF48 | Apr Kmr; pCRII-TOPO with 1,400-bp fragment containing mucP | This study |
| pLVF52 | Apr/Cbr; pMF54 with 1,400-bp fragment containing mucP | pMucP; this study |
| pLVF60 | pMO013722 (mucP::Tn5-200) | This study |
| pME6030 | Tcr oriV pVS1 oriV P15A oriT | 48 |
| pMF54 | Apr/Cbr ori ColE1-SF oriT (RK2) Ptrc, lacIq | 49 |
| pMO013722 | Tcr PAO1 fragment 4068269–4093431 | 44 |
| pRK2013 | Kmr colE1 RK2− Tra+ | 50 |
| pRK600 | Cmr colE1 RK2− Tra+ | 51 |
| Primers | ||
| cos-1 | 5′-CGCCCTCTGGTAAGGTTG-3′ | 44 |
| CC_algO_F | 5′-CTCGACACGACCTTCGATACCTTGC-3′ | This study |
| CC_algO_R | 5′-TTTATGACGCTCCCGCTGAACGCTAGC-3′ | This study |
| CC_MucD_F | 5′-TCTAGAAAGAAGGAGATATACATGCATACCCTAAAACG-3′ | This study |
| CC_MucD_R | 5′-AAGCTTGCCGGCTTATTCGGCCAGCTT-3′ | This study |
| CC_mucE_F | 5′-TTTTCTAGAAAGAAGGAGATATACATGGGTTTCCGGCCAGTTAGCC-3′ | This study |
| CC_mucE_R | 5′-AAGCTTTCGCGTTCAAAACACCCAG-3′ | This study |
| LF_mucE_UF (P1) | 5′-GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTGAAGCGCTCGTAGAGATATT-3′ | This study |
| LF_mucE_UR (P) | 5′-CTATTTCAGATTCGTCGCGTCTAGTTAGCTAGCATGGCTACGACTCCTTGATAGG-3′ | This study |
| LF_mucE_DF (P2) | 5′-CCTATCAAGGAGTCGTAGCCATGCTAGCTAACTAGACGCGACGAATCTGAAATAG-3′ | This study |
| LF_mucE_DR (P2) | 5′-CCAGGCAAATTCTGTTTTATCAGACCGCTTCTGCGTTCTGATCGCGGCTTGCAACGAGTA-3′ | This study |
| LF_MucP_F | 5′-CCATGGTTTAAAGAAGGAGATATACCATGAGTGCGCTTTACATGATCG-3′ | This study |
| LF_MucP_R | 5′-AAGCTTCTACAGACGACTCAGATCGTTGACC-3′ | This study |
| KAN-2 FP-1 | 5′-ACCTACAACAAAGCTCTCATGAACC-3′ | EpiCenter |
| KAN-2 RP-1 | 5′-GCAATGTAACATCAGAGATTTTGAG-3′ | EpiCenter |