TABLE 3.
Bacterial strains and plasmids
| Genotype/description | Source/reference | |
|---|---|---|
| Strains | ||
| DH5α | F− Φ80lacZΔM15 Δ (lacZYA-argF) U169 recA1 endA1 hsdR17 (rK−, mK+) phoA supE44 λ− thi-1 gyrA96 relA1 | Invitrogen |
| XL-1 Blue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac (F′ proAB lacIqZΔM15 Tn10 (Tetr)) | Agilent Technologies |
| BL21(DE3)star | F− ompT hsdSB (rB−, mB−) gal dcm rne131 (DE3) | Invitrogen |
| BTH101 | F- cya-99, araD139, galE15, galK16, rpsL1 (strr), hsdR2, mcrA1, mcrB1 | Ref. 40 |
| FM433 | SpcraraD139 Δ (argF-lac)U169 ptsF25 deoC1 relA1 flbB5301 rpsE13 Δ (srl-recA)306::Tn10, conjugation donor | Ref. 41 |
| WL400 | CmrStrraraD139 Δ (argF-lac)U169, ptsF25 deoC1 relA1 flbB5301 rpsL150 ΔselD204::cat, conjugation recipient | W. Leinfelder, unpublished data |
| Plasmids | ||
| pHT | kanr pET24d derivative T7 expression vector with N-terminal His6 tag and TEV protease cleavage site | Ref. 31 |
| pHTVirB8bp | kanr T7 promoter vector for the expression of His6-tagged periplasmic domain of B. suis VirB8 | Ref. 31 |
| pHTTraEp | kanr T7 promoter vector for the expression of His6-tagged periplasmic domain of pKM101 TraE | This work |
| pET21aCagVp | ampr T7 promoter vector for the expression of His6-tagged periplasmic domain of H. pylori CagV | This work |
| pHTTraEp E97A | pHTTraEp modified to encode TraE with amino acid change E97A | This work |
| pHTTraEp Y214A | pHTTraEp modified to encode TraE with amino acid change Y214A | This work |
| pHTTraEp E97A/Q105A | pHTTraEp modified to encode TraE with amino acid change E97A/Q105A | This work |
| pUT18 | pUC19 derivative including the T18 fragment (amino acids 225–399 of B. pertussis CyaA) N-terminal to the multiple cloning site | Ref. 40 |
| pKNT25 | pSU40 derivative including the T25 fragment (amino acids 1–224 of B. pertussis CyaA) N-terminal to the multiple cloning site | Ref. 40 |
| pUT18C | pUC19 derivative including the T18 fragment (amino acids 225–399 of B. pertussis CyaA) C-terminal to the multiple cloning site | Ref. 40 |
| pKT25 | pSU40 derivative including the T25 fragment (amino acids 1–224 of B. pertussis CyaA) C-terminal to the multiple cloning site | Ref. 40 |
| pUT18CVirB10b | ampr, pUT18C harboring 1,176-bp XbaI/KpnI virB10 fragment from B. suis (encoding full-length VirB10) | Ref. 30 |
| pKT25VirB10b | kanr, pKT25 harboring 1,176-bp XbaI/KpnI virB10 fragment from B. suis (encoding full-length VirB10) | Ref. 30 |
| pUT18CVirB10a | ampr, pUT18C harboring 1,134-bp XbaI/KpnI virB10 fragment from A. tumefaciens (encoding full-length VirB10) | This work |
| pKT25VirB10a | kanr, pKT25 harboring 1,134-bp XbaI/KpnI virB10 fragment from A. tumefaciens (encoding full-length VirB10) | This work |
| pUT18CVirB8b | ampr, pUT18C harboring 720-bp XbaI/KpnI virB8 fragment from B. suis (encoding full-length VirB8) | Ref. 19 |
| pKT25VirB8b | kanr, pKT25 harboring 720-bp XbaI/KpnI virB8 fragment from B. suis (encoding full-length VirB8) | Ref. 19 |
| pUT18CVirB8a | ampr, pUT18C harboring 711-bp XbaI/KpnI virB8 fragment from A. tumefaciens (encoding full-length VirB8) | This work |
| pKT25VirB8a | kanr, pKT25 harboring 711-bp XbaI/KpnI virB8 fragment from A. tumefaciens (encoding full-length VirB8) | This work |
| pUT18CCagV | ampr, pUT18C harboring 765-bp XbaI/KpnI cagV fragment from H. pylori (encoding full-length CagV) | This work |
| pKT25CagV | kanr, pKT25 harboring 765-bp XbaI/KpnI cagV fragment from H. pylori (encoding full-length CagV) | This work |
| pUT18CTraE | ampr, pUT18C harboring 699-bp XbaI/KpnI traE fragment from pKM101 (encoding full-length TraE) | This work |
| pKT25TraE | kanr, pKT25 harboring 699-bp XbaI/KpnI traE fragment from pKM101 (encoding full-length TraE) | This work |
| pUT18CTraE E97A | pUT18CB8 modified to encode TraE with amino acid change E97A | This work |
| pUT18CTraE Q105A | pUT18CB8 modified to encode TraE with amino acid change Q105A | This work |
| pUT18CTraE K168A | pUT18CB8 modified to encode TraE with amino acid change K168A | This work |
| pUT18CTraE Y214A | pUT18CB8 modified to encode TraE with amino acid change Y214A | This work |
| pUT18CTraE E97A/Q105A | pUT18CB8 modified to encode TraE with amino acid change E97A/Q105A | This work |
| pUT18CTraE K168A/Y214A | pUT18CB8 modified to encode TraE with amino acid change K168A/Y214A | This work |
| pKT25TraE E97A | pKT25B8 modified to encode TraE with amino acid change E97A | This work |
| pKT25TraE Q105A | pKT25B8 modified to encode TraE with amino acid change Q105A | This work |
| pKT25TraE K168A | pKT25B8 modified to encode TraE with amino acid change K168A | This work |
| pKT25TraE Y214A | pKT25B8 modified to encode TraE with amino acid change Y214A | This work |
| pKT25TraE E97A/Q105A | pKT25B8 modified to encode TraE with amino acid change E97A/Q105A | This work |
| pKT25TraE K168A/Y214A | pKT25B8 modified to encode TraE with amino acid change K168A/Y214A | This work |
| pTrc200 | Strr, Spcr, pVS1 derivative, LacIq, trc promoter expression vector | Ref. 42 |
| pTrc200TraE | pTrc200, traC PCR fragment cloned downstream of the trc promoter | This work |
| pTrc200TraE E97A | pTrc200TraE modified to encode TraE with amino acid change E97A | This work |
| pTrc200TraE Q105A | pTrc200TraE modified to encode TraE with amino acid change Q105A | This work |
| pTrc200TraE K168A | pTrc200TraE modified to encode TraE with amino acid change K168A | This work |
| pTrc200TraE Y214A | pTrc200TraE modified to encode TraE with amino acid change Y214A | This work |
| pTrc200TraE E97A/Q105A | pTrc200TraE modified to encode TraE with amino acid change E97A/Q105A | This work |
| pTrc200TraE K168A/Y214A | pTrc200TraE modified to encode TraE with amino acid change K168A/Y214A | This work |
| pKM101 | Ampr, mucA, mucB, TraI-TraIII region for DNA processing, DNA transfer, and entry exclusion | Ref. 43 |
| pKM101traE | Ampr, Kmr, pKM101traE1228::Tn5 | Ref. 3 |