TABLE 1.
Strains and plasmids used in this study
| Strain or plasmid | Relevant characteristic(s) | Source or reference |
|---|---|---|
| Strains | ||
| DH5α | recA endA1 gyrA96 hsdR17 thi-1 supE44 relA1 φ80dlacΖΔM15 | Lab collection |
| S17-1 | thi pro hsdR recA chr::RP4-2 | 29 |
| GM2163 | F− ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 rpsL136 (Strr) dam13::Tn9 (Cmr) xylA5 mtl-1 thi-1 mcrB1 hsdR2 | New England Biolabs |
| PAO1 | Wild type P. aeruginosa strain ATCC 15692 | Lab collection |
| PAO1ΔchpA | PAO1 with Tcr insertion in chpA | This study |
| Plasmids | ||
| pBluescript II KS | E. coli cloning vector with Ap selection | Stratagene |
| pOK12 | E. coli cloning vector with Km selection | 31 |
| pUC21 | E. coli cloning vector with Ap selection | 31 |
| pUCP19 | E. coli-P. aeruginosa cloning vector | 27 |
| pUCPSK | P. aeruginosa-E. coli shuttle vector | 32 |
| pMMB206 | Pseudomonas controlled expression level vector with Cm selection | 19 |
| pMOB3 | Counterselectable sacB/sacR genes from Bacillus subtilis | 26 |
| pSM-TET | Source of Tetr cartridge | 18 |
| MTP26 | Cosmid containing P. aeruginosa PAO1 chromosomal DNA from bp 448166-472109 | 10 |
| pAL1 | pBluescript II KS with modified multiple cloning site | This study |
| pAL5 | 3,168-bp SacII/EcoRV fragment of chpA from MTP26 in pAL1 | This study |
| pAL6 | 458-bp H912i PCR product in pUC21 | This study |
| pAL7 | 3,168-bp SacII/EcoRV fragment of chpA containing H1088 in pAL1 | This study |
| pAL8 | 1,297-bp S1231i/H1410i PCR product in pUC21 | This study |
| pAL9 | 611-bp H1570i PCR product in pUC21 | This study |
| pAL10 | 872-bp BamHI/KpnI fragment of chpA containing H1717 in pUC21 | This study |
| pAL11 | 1,396-bp EcoRV/KpnI fragment of chpA from MTP26 in pAL5 | This study |
| pAL13 | 1,561-bp EcoRV/AatII fragment of chpA from MTP26 containing H54 in pOK12 | This study |
| pAL14 | ChpA H54Q in pAL13 | This study |
| pAL16 | ChpA H912Q in pAL6 | This study |
| pAL17 | ChpA H1088Q in pAL7 | This study |
| pAL18 | ChpA S1231A in pAL8 | This study |
| pAL19 | ChpA H1410Q in pAL8 | This study |
| pAL20 | ChpA H1570Q in pAL9 | This study |
| pAL37 | 2,839-bp BamHI/ClaI fragment of chpA from MTP26 containing D2406N in pUC21 | This study |
| pAL40 | 4,291-bp SacI/ClaI chpA fragment in pUCPSK | This study |
| pAL41 | Full-length chpA under Plac control in pUCPSK | This study |
| pAL42 | ChpA D2406N in pAL37 | This study |
| pAL49 | Tcr cassette from pSM-TET (EcoRI/MluI) inserted into MfeI/AscI pAL11 | This study |
| pAL52 | 5-kb NotI fragment from pMOB3 ligated into pAL49 | This study |
| pAL57 | 7,972-bp NotI chpA fragment from MTP26 in pAL1 | This study |
| pAL64 | pAL41 with an internal double FLAG tag inserted between the native NotI/NruI sites of chpA | This study |
| pAL77 | 3,862-bp ClaI/SacI chpA fragment from pAL41 in pUCP19 | This study |
| pAL78 | Full-length chpA against Plac in pUCP19 | This study |
| pAL79 | 8-kb ClaI/SacI fragment from MTP26 ligated into pMMB206 with PtaclacUV | This study |
| pAL82 | 3,503-bp StuI fragment of chpA cut from pAL57 and ligated into pAL14 | This study |
| pAL83 | pAL79 with ChpA H54Q (mutation in HPt1 domain) | This study |
| pAL86 | pAL79 with ChpA H912Q (mutation in HPt2 domain) | This study |
| pAL87 | pAL79 with ChpA H1088Q (mutation in HPt3 domain) | This study |
| pAL88 | pAL79 with ChpA S1231A (mutation in SPt domain) | This study |
| pAL89 | pAL79 with ChpA H1410Q (mutation in HPt4 domain) | This study |
| pAL90 | pAL79 with ChpA H1570Q (mutation in HPt5 domain) | This study |
| pAL92 | pAL79 with ChpA D2406N (mutation in CheY domain) | This study |
| pAL79FLAG | pAL79 with a double FLAG tag inserted into the NotI/KpnI sites of chpA | This study |
| pAL86FLAG | pAL86 with a double FLAG tag inserted into the NotI/KpnI sites of chpA | This study |
| pAL87FLAG | pAL87 with a double FLAG tag inserted into the NotI/KpnI sites of chpA | This study |
| pAL92FLAG | pAL92 with a double FLAG tag inserted into the NotI/KpnI sites of chpA | This study |