TABLE 1.
Bacterial strains and plasmids
| Bacterial strain or plasmid | Characteristics | Reference and/or source |
|---|---|---|
| Strains | ||
| E. coli DH5αe | Cloning host strain: F− φ80lacZΔM15 Δ(lacZYA-argF) U169 deoR recA1 endA1 hsdR17 (rK− mK+) gal phoA supE44 λ−thi-1 gyrA96 relA1 | 25; Invitrogen |
| C. jejuni strains | ||
| NCTC 11168 (11168-GS) | Wild-type genome sequenced strain | 41; NCTC |
| 480 (NCTC 12744) | Clinical isolate used as host strain for maintaining plasmids/reporter assays | 32; NCTC |
| KAR2 | NCTC 11168 cj0178::Kanr | This study |
| KAR3 | NCTC 11168 p19::Kanr | This study |
| JDR5 | NCTC 11168 ΔchuA::cat | 47 |
| JDR21 | NCTC 11168 Δcj0178::cat | This study |
| CEM3 | NCTC 11168 Δcj0174c::Kanr | This study |
| CEM5 | NCTC 11168 ΔcfrA::Kanr | This study |
| CEM8 | Complementation of cj0178::Kanr with a wild-type copy of cj0178, plus cat, into cj0752 | This study |
| Plasmids | ||
| pUC19 | Cloning/suicide vector; Ampr | New England Biolabs |
| pMW10 | E. coli/C. jejuni shuttle LacZ reporter vector; Kmr | 60 |
| p23E5 | Reporter construct; C. jejuni NCTC 11168 metK promoter region inserted into BamHI site of pMW10 | 59, 60 |
| pJDR13 | Reporter construct; C. jejuni NCTC 11168 chuA promoter region inserted into BamHI site of pMW10 | 47 |
| pAV35 | pBluescript vector containing a promotered Cmr (cat) cassette | 59 |
| pJMK30 | pUC19 containing C. coli (aph-3) Kmr gene flanked by multiple cloning regions | 59 |
| pcam193b8a | pUC19 containing C. jejuni NCTC 11168 genomic fragment (bases 174132 to 175858) at SmaI site | 41; from tiled genome library |
| pKAR2 | pcam193b8::Kanr; insertion of kan at PsiI site (base position 1128 of Cj0178) | This study |
| pGEMCWH01b | cj0752 flanks with internal multiple cloning region in pGEM T-vector backbone | 15 |
| pCEM3 | pUC19 containing C. jejuni NCTC 11168 cj0174c genomic fragment (bases 171562 to 169946) at KpnI and PstI sites | This study |
| pCEM5 | pUC19 containing C. jejuni NCTC 11168 cfrA genomic fragment (bases 705450 to 707540) at KpnI and PstI sites | This study |
| pCEM9 | pCEM3, cj0174c::Kanr; deletion of 1,477 bp of cj0174c ORF (bases 171520 to 170042) and insertion of Kanr (in forward orientation with respect to deleted gene) into BglII site created at point of deletion | This study |
| pCEM11 | pCEM5, ΔcfrA::Kanr; deletion of 1,966 bp of cfrA ORF (bases 705484 to 707451) and insertion of Kanr (in forward orientation with respect to deleted gene) into BglII site created at point of deletion | This study |
| pCEM14 | Reporter construct; C. jejuni NCTC 11168 cj0176c-cj0177 promoter region (bases 172915 to 172695) inserted into BamHI site of pMW10 (cj0176c orientation) | This study |
| pCEM15 | Reporter construct; C. jejuni NCTC 11168 cj0176c-cj0177 promoter region (bases 172695 to 172915) inserted into BamHI site of pMW10 (cj0177 orientation) | This study |
| pCEM16 | Reporter construct; C. jejuni NCTC 11168 cj0178 promoter region (bases 173477 to 173763) inserted into BamHI site of pMW10 | This study |
| pCEM17 | Reporter construct; C. jejuni NCTC 11168 exbB1 promoter region (bases 175801 to 176042) inserted into BamHI site of pMW10 | This study |
| pCEM18 | pUC19 containing C. jejuni NCTC 11168 genomic fragment (bases 172686 to 176113; promoter region, cj0177, and cj0178) at KpnI and PstI sites | This study |
| pCEM19 | pCEM18, Δcj0177; deletion of 764 bp of cj0177 ORF (bases 172943 to 173706) | This study |
| pCEM20 | pCEM19 plus cat; insertion of cat at PstI site downstream of cj0178 (in forward orientation with respect to wild type-copy of gene) | This study |
a
pcam193b8 was kindly provided by J. Parkhill, PSU, Sanger Centre, Cambridge, United Kingdom.
b
A gift from C. Penn, University of Birmingham, Birmingham, United Kingdom.