TABLE 1.
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 |
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.