TABLE 1.
Bacterial strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant characteristicsa | Source or reference |
|---|---|---|
| Strains | ||
| Escherichia coli DH5α | hsdR recA lacZYA φ80 lacZΔM15 | GIBCO-BRL |
| P. aeruginosa | ||
| PAK | Wild type | Steve Lory |
| LecA strain | PAK LecA::Gmr | This study |
| LecB strain | PAK LecB::Gmr | This study |
| PAKpilG::lacZ | PAK harboring pDN19lacΩ with pilGHIJK promoter fused to lac | This study |
| PAKlecB::lacZ strain | LecB strain harboring pDN19lacΩ with pilGHIJK promoter fused to lacZ | This study |
| lecB+ strain | lecB complemented | This study |
| PAK-NP | PAK pilA::Tcr | 25 |
| Plasmids | ||
| pUC19 | Cloning vector, Ampr | New England Biolabs |
| pUC7G | Plasmid with a gentamicin cassette | |
| pUClecA::Gm | pUC19 containing a 2.0-kb PAK lecA gene with gentamicin cassette | This study |
| pUClecB::Gm | pUC19 containing a 2.0-kb PAK lecB gene with gentamicin cassette | This study |
| pMMB67EH | Broad-host-range cloning vector, tac promoter, Carbr | 7 |
| pDN19lacΩ | Promoterless lacZ oriV oriT Tcr Strr Ω | 28 |
| Primers | ||
| LecAFor | 5′-cccaaaggatccGCCTGCCGTCGCAGATCAACC-3′ | BamHI site inserted |
| LecARev | 5′-cccaaaaagcttGCGGGCGGCAGACCGTCC-3′ | HindIII site inserted |
| LecBFor | 5′-cccaaaggatccGCTGCACACATGGTGGCGC-3′ | BamHI site inserted |
| LecBRev | 5′-cccaaaaagcttGGAACGCATCGCCGGCCAGG-3′ | HindIII site inserted |
| LecA1 | 5′-GGGCAGGTAACGTCGATTATCTGCAGTCCGGGCGATGTCATTACCAT-3′ | Two bases incorporated to create PstI site |
| LecA2 | 5′-ATGGTAATGACATCGCCCGGACTGCAGATAATCGACGTTACCTGCCC-3′ | Two bases incorporated to create PstI site |
| LecB1 | 5′-CGAGACGGCCGCGACCTGCAGCGGGCAAAGCACCA-3′ | One base incorporated to create PstI site |
| LecB2 | 5′-TGGTGCTTTGCCCGCTGCAGGTCGCGGCCGTCTCG-3′ | One base incorporated to create PstI site |
a
Gmr, gentamicin resistance; Ampr, ampicillin resistance; Carbr, carbenicillin resistance; Tcr, tetracycline resistance; Strr, streptomycin resistance. In primer sequences, lowercase denotes nucleotides added to facilitate restriction digestion at the marked sites in bold. Nucleotides added to create a restriction site are underlined.