TABLE 1.
Bacterial strains and plasmids
| Description | Ref. | |
|---|---|---|
| Strains | ||
| E.coli DH5α | φ80d deoR lacZΔM15 endA1 recA1 hsdR17 (rK−mK−) supE44 thi-1 gyrA96 relA1 Δ (lacZYA-argF)U169 F− | |
| E. coli E69 | Prototroph, serotype O9a:K30:H12 | I. Orskov |
| E.coli W3110 | F− λ− IN (rrnD-rrnE)1 rph-1 | |
| CWG285 | E69 wzcK30::aacC1; wza22min::aadA (polar on etp and etk); GmR, SpR | 26 |
| CWG875 | E69 ΔgalE ugd::aaC1; GmR | This study |
| CWG876 | W3110 Δwzc Δetk ugd::kan; KmR | This study |
| Plasmids | ||
| pBAD18, pBAD24, pBAD33 | Arabinose-inducible expression vectors; KmR or ApR or CmR | 56 |
| pWQ173 | Allelic exchange suicide vector with pheS gene; CmR | 23 |
| pWQ500 | pHC79 derivative containing E. coli E69 chromosomal region surrounding rcsAK30; ApR | 57 |
| pWQ629 | pUC19 derivative containing E. coli E69 chromosomal region surrounding ugdK30 | 37, 58 |
| pWQ728 | pWQ173 derivative containing ugdK30 interrupted with gentamicin resistance cassette at SacI site; GmR, CmR | This study |
| pWQ729 | pBAD24 derivative expressing His6-UgdK-12; ApR | This study |
| pWQ730 | pBAD24 derivative expressing His6-UgdK30; ApR | This study |
| pWQ731 | pWQ729 derivative containing Y71F mutation; ApR | This study |
| pWQ732 | pWQ730 derivative containing Y71F mutation; ApR | This study |
| pWQ733 | pWQ729 derivative containing K323A mutation; ApR | This study |
| pWQ734 | pWQ729 derivative containing R324A mutation; ApR | This study |
| pWQ735 | pWQ729 derivative containing KR → AA mutations at residues 323–324; ApR | This study |
| pWQ736 | pBAD18 derivative expressing His6-WzcK-12 residues 447–704; ApR | This study |
| pWQ737 | pBAD18 derivative expressing His6-WzcK30 residues 447–704; ApR | This study |
| pWQ738 | pBAD33 derivative expressing His6-UgdK-12; CmR | This study |
| pWQ739 | pBAD33 derivative expressing His6-UgdK30; CmR | This study |