TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmidRelevant 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
    MC58Clinical isolate; sequenced strain32
    MC-Fko fur mutant; derivative of MC58 in which the fur gene is replaced by a kanamycin cassette; fur KmrThis study
    MC-Fko-CComplemented 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 EryrThis study
    MC-furlacZDerivative 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; EryrThis study
    MC-smpAlacZDerivative 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; EryrThis study
Plasmids
    pET15bExpression plasmid for expression of recombinant proteins with the N-terminal His tag and thrombin site for removal of the tagInvitrogen
    pGem3ZCloning vectorPromega
    pGemTCloning vectorPromega
    pILL600Plasmid containing the kanamycin cassette from Campylobacter coli14
    pSL1190Cloning vectorPharmacia
    pCMVβPlasmid containing the lacZ gene of E. coliClontech
    pET15furBDerivative 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-RThis study
    pGemFkoB:KmpGem3Z 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 transcriptionThis study
    pSLFur-C1pSL1190 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-RThis study
    pSL-furlacZpSL1190 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-RThis study
    pSL-smpAlacZpSL-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-SThis study
    pGemT-FurDerivative of pGemT containing the fur promoter region cloned as a 322-bp PCR product with primers Fur-P1 and Fur-P2This study