TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant characteristic(s) | Source or reference |
|---|---|---|
| E. coli strains | ||
| DH5-α | supE44 hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | 10 |
| BL21(DE3) | hsdS gal (λcIts857 ind-1 Sam7 nin-5 lacUV5-T7 gene 1) | 30 |
| N. meningitidis strains | ||
| MC58 | Clinical isolate; sequenced strain | 32 |
| MC-Fko | fur mutant; derivative of MC58 in which the fur gene is replaced by a kanamycin cassette; fur Kmr | This study |
| MC-Fko-C | Complemented fur mutant; derivative of MC-Fko in which the fur gene under the control of its own promoter was inserted along with an erythromycin cassette in the noncoding region between the NMB1074 and NMB1075 genes; fur+ Kmr Eryr | This study |
| MC-furlacZ | Derivative of MC58 containing 317 bp consisting of the 5′ end of the fur gene and the entire upstream region fused to a promoterless lacZ gene chromosomally located between the NMB1074 and NMB1075-genes; Eryr | This study |
| MC-smpAlacZ | Derivative of MC58 containing 277 bp consisting of the 5′ end of the smpA gene and the entire upstream intergenic region fused to a promoterless lacZ gene chromosomally located between the NMB1074 and NMB1075 genes; Eryr | This study |
| Plasmids | ||
| pET15b | Expression plasmid for expression of recombinant proteins with the N-terminal His tag and thrombin site for removal of the tag | Invitrogen |
| pGem3Z | Cloning vector | Promega |
| pGemT | Cloning vector | Promega |
| pILL600 | Plasmid containing the kanamycin cassette from Campylobacter coli | 14 |
| pSL1190 | Cloning vector | Pharmacia |
| pCMVβ | Plasmid containing the lacZ gene of E. coli | Clontech |
| pET15furB | Derivative of pET15b containing a 435-bp NdeI-BamHI fragment of the fur coding region obtained by PCR on MC58 DNA using primers Fmb-F and Fmb-R | This study |
| pGemFkoB:Km | pGem3Z derivative containing a 587-bp EcoRI-BamHI region upstream of the fur gene obtained by PCR with primers FkoB-1 and FkoB-2, a 1.4-kb BamHI fragment of the kanamycin gene from plasmid pILL600, and a 465-bp BamHI-PstI region downstream of the fur gene obtained by PCR with primers FkoB-3 and FkoB-4; this plasmid was selected because it contains the kanamycin cassette oriented in the same direction as that of fur gene transcription | This study |
| pSLFur-C1 | pSL1190 derivative containing a 510-bp SpeI-XhoI PCR fragment of the NMB1074 locus with primers Fla-UP-L and Fla-UP-R, a 1.1-kb XhoI-PstI PCR fragment of the erm gene obtained with primers Eryt-DO and Eryt-UP, a 658-bp NsiI-BamHI PCR fragment of the fur promoter and coding region obtained with primers Fur-N and Fmb-R, and a 909-bp BamHI-XmaI PCR fragment of the NMB1075 locus obtained with primers Fla-DO-L and Fla-DO-R | This study |
| pSL-furlacZ | pSL1190 derivative containing a 510-bp SpeI-XhoI PCR fragment of the NMB1074 locus obtained with primers Fla-UP-L and Fla-UP-R, a 1.1-kb XhoI-PstI PCR fragment of the ermAM gene obtained with primers Eryt-DO and Eryt-UP, a 317-bp NsiI-SphI fragment of the fur promoter region amplified with primers Fur-N and Fur-P2, a 3.4-kb SmaI-BamHI fragment carrying the lacZ gene from plasmid pCMVβ (Clontech), and a 909-bp BamHI-XmaI PCR fragment of the NMB1075 locus obtained with primers Fla-DO-L and Fla-DO-R | This study |
| pSL-smpAlacZ | pSL-furlacZ derivative in which the fur promoter region was replaced with a 277-bp NsiI-SphI PCR fragment of the smpA promoter region with primers Upf-N2 and Upf-S | This study |
| pGemT-Fur | Derivative of pGemT containing the fur promoter region cloned as a 322-bp PCR product with primers Fur-P1 and Fur-P2 | This study |