TABLE 1.
Bacterial strains and plasmids used in this study
| Strain, plasmid, or primer | Genotype, phenotype, or sequencea | Source or reference |
|---|---|---|
| P. aeruginosa | ||
| PAO1 | Algwt prototroph | P. Phibbs |
| PAO1VE2ΔalgW | Alg+ PAO1 mucE+oe (himar1 Gmr::PGM::mucE) ΔalgW | 20 |
| P. fluorescens Pf-5 | Algwt prototroph | ATCC |
| E. coli DH5α | F− ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK− mK+) phoA supE44 λ−thi-1 gyrA96 relA1 | Laboratory strain |
| B. pseudomallei Bp50 | Δ(amrRAB-oprA) derivative of wild-type strain 1026b | 5 |
| Plasmids | ||
| pBAD/Thio-TOPO | araC-PBADoripUC Apr (4,454 bp) | Invitrogen |
| pHERD20T | pUCP20T Plac replaced with 1.3-kb AflII-EcoRI fragment of araC-PBAD cassette (5,087 bp) | This study |
| pHERD30T | pUCP30T Plac replaced with 1.3-kb AflII-EcoRI fragment of araC-PBAD cassette (5,216 bp) | This study |
| pHERD26T | pUCP26 Plac replaced with 2.4-kb AdhI-EcoRI fragment of araC-PBAD cassette and oriT (6,166 bp) | This study |
| pHERD28T | pUCP28T Plac replaced with 1.3-kb AflII-EcoRI fragment of the araC-PBAD cassette (4,993 bp) | This study |
| pHERD30T-mucE | mucE in pHERD30T EcoRI/HindIII | This study |
| pHERD20T-algU | algU in pHERD20T EcoRI/HindIII | This study |
| pHERD20T-oprF | oprF (PA1777) in pHERD20T EcoRI/HindIII | This study |
| pHERD20T-oprF-WVF | oprF allele encoding OprF ending with the WVF motif cloned in pHERD20T EcoRI/HindIII | This study |
| Primers | ||
| pBAD-F | AGTATACCTTAAGGAATCCCCAAATTATGACAACTTGACGGCTACATCAT | This study |
| pBAD-R | AGGATCCCCGGGTACCGAGCTCGAATTCTTATCAGATCCCATGGGTATGTATA | This study |
| pHERD-SF | ATCGCAACTCTCTACTGTTTCT | This study |
| pHERD-SR | TGCAAGGCGATTAAGTTGGGT | This study |
| algU-F | AGAATTCGATGCTAACCCAGGAACAGGA | This study |
| algU-R | CAAGCTTTCAGGCTTCTCGCAACAAAGGCTGCA | This study |
| algW-F | AGAATTCGATGCCCAAGGCCCTGCGTTTCCT | This study |
| algW-R | TGCCAAGCTTTCACTCGCCGCCGTCCTGTTT | This study |
| mucE-F | AGAATTCGATGGGTTTCCGGCCAGTTA | This study |
| mucE-R | GAAGCTTCAAAACACCCAGCGCAACTCGTC | This study |
| oprF-F | AGAATTCGATGAAACTGAAGAACACCTTA | This study |
| oprF-R | CAAGCTTTTACTTGGCTTCAGCTTCTACTTCGGCT | This study |
| oprF-WVF-R | AAGCTTAAAACACCCAGCGCTTGGCTTCAGCTTCTACTTCGGCT | This study |
| lacZ-RT-For2 | GTCGTGACTGGGAAAACC | This study |
| lacZ-RT-Rev2 | GCCTCTTCGCTATTACGC | This study |
| Bp23S_F | GTAGACCCGAAACCAGGTGA | This study |
| Bp23S_R | CACCCCTATCCACAGCTCAT | This study |
a
Algwt, wild-type nonmucoid phenotype; Alg−, nonmucoid phenotype; Alg+, mucoid phenotype. Primers used for cloning carried built-in restriction sites (underlined), with F denoting forward and R denoting reverse primers, respectively.