Table 1.
Strains, plasmids, and primers used in this study
| Strain name | Relevant genotype, description or sequence | Source |
|---|---|---|
| S. cerevisiae InvSc1 | MATa/MATα leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52 his3-Δ1/his3-Δ1 | Invitrogen |
| E. coli S17-1(λpir) | thi pro hsdR-hsdM+ΔrecA RP4–2∷TcMu-Km∷Tn7 | [55] |
| E. coli DH5α | lˉ f80dlacZDM15D(lacZYA-argF)U169 recA1 endA hsdR17(rKˉ mKˉ) supE44 thi-1 gyrA relA1 | Life Technologies |
| P aeruginosa PAO1 | Wild type | [54] |
| oprD∷IsphoA/hah | PAO1 with isphoA/hah insertion in opr∷Tcʳ | [54] |
| SMC214 | Imipenem intermediate P. aeruginosa clinical isolate; robust biofilm former | This study |
| SMC576 | Imipenem-sensitive P. aeruginosa clinical isolate; robust biofilm former | This study |
| SMC631 | Imipenem-sensitive clinical isolate; robust biofilm former | This study |
| SMC4972 | P. aeruginosa clinical isolate | This study |
| SMC4973 | P. aeruginosa clinical isolate | This study |
| SMC4974 | P. aeruginosa clinical isolate | This study |
| SMC4979 | P. aeruginosa clinical isolate | This study |
| SMC5806 | 631-F/oprD; imipenem-resistant derivative of SMC631; premature stop mutation in oprD gene | This study |
| SMC5810 | P. aeruginosa clinical isolate | This study |
| SMC5811 | P. aeruginosa clinical isolate | This study |
| SMC5812 | SMC576 pilW∷Mar19, Mariner insertion in pilW(769)a | This study |
| SMC5813 | SMC576 pilY1∷Mar8, Mariner insertion in pilY1 (3214) | This study |
| SMC5814 | SMC576 pilY1∷Mar13, Mariner insertion in pilY1 (1933) | This study |
| SMC5815 | SMC576 pilY1∷Mar18, Mariner insertion in pilY1 (3242) | This study |
| SMC5816 | SMC576 pilY1∷Mar20, Mariner insertion in pilY1(2156) | This study |
| SMC5817 | SMC576 pilY1∷Mar25, Mariner insertion in pilY1(1228) | This study |
| SMC5818 | SMC576 pslI∷Mar15, Mariner insertion in pslI | This study |
| SMC5819 | SMC576 algC∷Mar10, Mariner insertion in algC | This study |
| SMC5824 | SMC214 pilX∷Mar, Mariner insertion in pilX (84) | This study |
| SMC5825 | SMC214 pilW∷Mar, Mariner insertion in pilW(627) | This study |
| SMC5832 | P. aeruginosa ATCC 27853 | This study |
| SMC5833 | Escherichia coli ATCC 25922 | This study |
| Plasmid | Plasmid description | Reference |
| pMQ70 | Shuttle vector for cloning in yeast and for arabinose-inducible gene expression; Apr | [18] |
| pMQ72 | Shuttle vector for cloning in yeast and for arabinose-inducible gene expression; Gmr | [18] |
| pBT20 | Vector carrying mariner transposon; Apr; Gmr | [56] |
| poprD | oprD gene cloned in pMQ72; Gmr | This study |
| ppilY1 | His-tagged pilY1 gene cloned in pMQ70; Apr | This study |
| Primersb | Primer sequence (5′–3′) | |
| oprD comp 5′ | ttctccatacccgtttttttggggaaggagatatacatATGAAAGTGATGAAGTGGAG | |
| oprD comp 3′ | taatctgtatcaggctgaaaatcttctctcatccgccTCACAGGATCGACAGCGGATAG | |
| oprD seq a-f | ATGAAAGTGATGAAGTGGAGC | |
| oprD seq a-r | AGGGAGGCGCTGAGGTT | |
| oprD seq b-f | AACCTCAGCGCCTCCCT | |
| pilY1 comp 5′ | tctccatacccgtttttttgggctagcgaattcgaaggagatatacatATGAAATCGGTACTCCACCAG | |
| pilY1 comp 3′ | tcttctctcatccgccaaaacagccaagcttgcatgcctTCAgtggtgatggtggtggtgGTTCTTTCCGATGGGGC | |
| pilY1 seq Rev 2 | TGAACGGACAGGTACAGATCC | |
| pilY1 seq 3 | GGATCTGTACCTGTCCGTTC | |
| pilY1 seq 4 | GGCGAGTTTCTCAAGAAGACC | |
| pilY1 seq 5 | CTTCCAGGACATCCTCAACCG | |
| pilY1 seq 6 | AGCCCAGCGGTAACTACTCC | |
| pilY1 seq 7 | CAAGGTCAACCAGGACGATC | |
| P730 | GCAACTCTCTACTGTTTCTCC |
a
The number in parentheses indicates the nucleotide after which the transposon has inserted in the open reading frame
b
For primer sequences, lowercase letters indicate sequence identity to the cloning vector, uppercase letters indicate a Pseudomonas gene-specific sequence, boldface and lower case letters indicates a His tag sequence