TABLE 1.
Strains and plasmids used in the current study
| Strain or plasmid | Relevant characteristic(s) | Reference or source |
|---|---|---|
| E. coli strains | ||
| RS218 | O18:K1:H7, isolated from cerebrospinal fluid of neonate with E. coli meningitis | 12 |
| EC100D | F−mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara leu)7697 galU galK λ−rpsL nupG pir+ (DHFR) | Epicentre Biotechnologies |
| DH5α | F′ φ80dlacZ ΔM15Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK− mK+)phoA supE44 λ−thi-1 | Lab stock |
| Δcnf1 mutant | RS218 (O18:K1:H7) cnf1 deletion mutant | 12 |
| Δfdx mutant | RS218 (O18:K1:H7) fdx deletion mutant | This study |
| CΔfdx mutant | RS218 (O18:K1:H7) fdx deletion mutant complemented with fdx under control of arabinose promoter; complementation achieved by Tn7 site-specific insertion into second benign site in chromosome | This study |
| Plasmids | ||
| pBC-KS | Cloning vector with chloramphenicol resistance gene | Stratagene |
| pBAD-Myc/His | Arabinose promoter expression vector, ampicillin resistance | Invitrogen |
| pCX340 | PBR322 derivative, cloning vector used to fuse CNF1 to the mature form of TEM-1 β-lactamase, tetracycline resistance | 3 |
| pCX311 | Negative control, fusion of MBP to TEM-1, tetracycline resistance | 3 |
| pCXN | CNF1 coding region cloned into KpnI site of pCX340, tetracycline resistance | This study |
| pKD3 | Containing chloramphenicol resistance gene, R6kγ replication origin | 6 |
| pKD47 | Derivative of pKD46 (5); the only modification is that blaM in pKD46 is replaced by the spectinomycin resistance gene | This study |
| pGRG36 | Tn7 insertion vector, ampicillin resistance, temperature sensitive | 21 |
| pFBI | Contains β-lactamase coding region (signal peptide genetically deleted); spectinomycin resistance; R6kγ replication origin | This study |
| pFBI-CNF1 | CNF1 translationally fused with β-lactamase in pFBI | This study |
| pNFB | DNA fragment downstream of cnf1 in RS218 genome was PCR amplified and cloned in pFBI-CNF1 | This study |
| pNBC | Chloramphenicol resistance gene was inserted right after β-lactamase in pNFB | This study |
| pSR | Tn5 vector; spectinomycin resistance; R6kγ replication origin | This study |
| pGRGM | Multiple cloning site of pGRG36 ligated into PvuII site of pBC-KS; chloramphenicol resistance | This study |
| pGAP | AraC and arabinose promoter (pBAD) cloned into AvrII and XhoI site of pGRGM | This study |
| pGAP-fdx | Coding region of fdx cloned into NdeI and NotI site of pGAP | This study |
| pG-fdx | DNA fragment containing fdx obtained from pGAP-fdx by digestion with AvrII and PacI; ligated into same sites of pGRG36 | This study |