TABLE 1.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Relevant information | Source or reference |
|---|---|---|
| Strains | ||
| E. coli | ||
| DH5α | hsdR recA lacZYA φ80 lacZΔM15 | GIBCO-BRL |
| XL1 blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZ ΔM15 Tn10 (Tetr)] | Stratagene |
| BL21(pLysS) | F−ompT hsdSB(rB− mB−) gal dcm(DE3), T7 lysozyme gene on pLysS | Novagen Inc., Madison, Wis. |
| S. cerevisiae Y190 | MATagal4 gal80 his3 trpl-901 ade2-101 ura3-52 leu2-3 | M. S. Swanson |
| P. aeruginosa | ||
| PAK | Wild-type clinical isolate | 6 |
| PAK-Q | PAK fleQ::Gmr | 1 |
| PAK-N | PAK fleN::Gmr | 6 |
| PAK-R | PAK fleR::Gmr | 23 |
| PAO/T7(ADD1976) | PAO1 Tcr Cbs mini-D180, T7 polymerase gene in chromosome | A. Darzin |
| Plasmids | ||
| pGBT9 | Shuttle plasmid containing the GAL4 DNA binding domain | Clontech |
| pGBTQ | fleQ cloned into EcoRI-BamHI sites of pGBT9 | This study |
| pGBTN | fleN cloned into EcoRI-BamHI sites of pGBT9 | This study |
| pGBKT7-M | flgM cloned into NdeI-BamHI sites of pGADT7 | Frisk et al., submitted |
| pGBKT7-A | fliA cloned into NdeI-BamHI sites of pGADT7 | Frisk et al., submitted |
| pGAD424 | Shuttle plasmid containing the GAL4 activation domain | Clontech |
| pGADQ | fleQ cloned into EcoRI-BamHI sites of pGAD424 | This study |
| pGADN | fleN cloned into EcoRI-BamHI sites of pGAD424 | This study |
| pGADN1 | pGADNΔFleN amino acids 107–280 | This study |
| pGADN2 | pGADNΔFleN amino acids 191–280 | This study |
| pGADN3 | pGADNΔFleN amino acids 251–280 | This study |
| pGADN4 | pGADNΔFleN amino acids 261–280 | This study |
| pGADN5 | pGADNΔFleN amino acids 271–280 | This study |
| pGADN-C | pGADNΔFleN amino acids 1–106 | This study |
| pGADN24K→Q | pGADN mutated at FleN24K→Q | This study |
| pET15bVP | oriV cloned as a PstI fragment in bla of pET15b | 6 |
| pET-fleN | fleN inserted as a PCR product into the NdeI/BamHI sites of pET15bVP | 6 |
| pET-fleNΔN | fleNΔFleN amino acids 1–106 inserted as a PCR product into the NdeI/BamHI sites of pET15bVP | This study |
| pET-fleNΔC | fleNΔFleN amino acids 107–280 inserted as a PCR product into the NdeI/BamHI sites of pET15bVP | This study |
| pET-fleN24K→Q | fleN24K→Q inserted as a PCR product into the NdeI/BamHI sites of pET15bVP | This study |
| pDN19lacΩ | Promoterless lacZ oriV oriT Tetr Strr Ω fragment | 6 |
| pMSZ5 | pDN19lacΩ containing the pilA promoter region | 6 |
| placΩA | pDN19lacΩ containing the flhA promoter region | Arora, unpublished |
| pIH1119 | Expression vector to generate MBP fusion proteins | New England BioLabs |
| pIH-fleQ | fleQ cloned into EcoRI-BamHI sites of pIHI119 to express MBP-FleQ | B. Ritchings |
| pIH-fleN | fleN cloned as a PCR product into EcoRI-BamHI sites of pIHI119 to express MBP-FleN | This study |
| pMMB67EH | Broad host-range cloning vector, Ampr | S. Lory |
| pMMBQ | fleQ cloned into EcoRI-BamHI sites of pMMB67EH to express FleQ | This study |
| pMMBN | fleN cloned into EcoRI-BamHI sites of pMMB67EH to express FleN | This study |
| pMMBN4 | EcoRI-BamHI insert from pGADN4 cloned into pMMB67EH to express FleNΔ261–280 | This study |
| pMMBN5 | EcoRI-BamHI insert from pGADN5 cloned into pMMB67EH to express FleNΔ271–280 | This study |
| pMMBN24K→Q | EcoRI-BamHI insert from pGADN24K→Q cloned into pMMB67EH to express FleN24K→Q | This study |
| Primersa | ||
| fleQ5PRI | 5′ cccgaattcATGTGGCGC 3′, EcoRI site incorporated | |
| Q3Pbam | 5′ cccaaaggatccTCAATCATCCGACAG 3′, BamHI site incorporated | |
| fln5peco | 5′ cccaaagaattcATGAAGCAGATGGGGTAG 3′, EcoRI site incorporated | |
| flnBam | 5′ CCTTGCTATACgggaTCCAGAGGCCGCTG 3′, BamHI site incorporated | |
| flnN1bam | 5′ cccaaaggatcctcaCTGCATCGGCGAGAGGTG 3′, BamHI site incorporated | |
| flnN2bam | 5′ cccaaaggatcctcaCCTTCCTGCGGGCTGTGG 3′, BamHI site incorporated | |
| flnN3bam | 5′ cccaaaggatcctcaGACCTTCTGCGCGACCGCC 3′, BamHI site incorporated | |
| flnN4bam | 5′ cccaaaggatcctcaGCGCGGGTTGGCCGGC 3′, BamHI site incorporated | |
| flnN5bam | 5′ cccaaaggatcctcaCAGTCGTTCGACGAAG 3′, BamHI site incorporated | |
| flnCeco | 5′ cccaaagaattcCTCGCCGATGCAGCATGC 3′, EcoRI site incorporated | |
| flnNde | 5′ GACAACACAAcatATGAAGCAGATGGG 3′, NdeI site incorporated | |
| flnCNde | 5′ cccaaaaaacatatgTCTCGCCGATGCAGCATG 3′, NdeI site incorporated | |
| mutN1 | 5′ GGGCGGCGTCCGGCcAGACCAATGTGTCGG 3′ | |
| mutNcomp2 | 5′ CCGACACATTGGTCTgGCCGGACGCCGCCC 3′ | |
| GADf | 5′ CACTGTCACCTGGTTGGAC 3′ |
a
Lowercase denotes nucleotides added or modified to facilitate restriction digestion or mutagenesis at the sites marked in bold.