Figure 1.
Summary of cloning steps in construction of Δopa plasmids.
A general scheme is depicted. Different steps were used for each opa gene, as described below and in the text. (i) The 5′ and 3′ ends of opaA, opaD or opaJ (black) were amplified by PCR (table 1) along with the adjacent locus-specific genes, depicted as nmb01 (blue) and nmb03 (green). Novel SalI and BamHI or BamHI and SacII restriction sites were introduced at the ends of the amplicons. Each PCR product was then cloned separately into pCR2.1-TOPO (not shown). The two ends of each opa were excised from the pCR2.1 plasmids and cloned sequentially into pBluescript, resulting in locus-specific plasmids pBS-ΔopaA-nmb, pBS-ΔopaD-nmb and pBS-ΔopaJ-nmb. These plasmids therefore contained a modified opa gene, Δopa, which contained a 185 bp deletion. (ii) A 1,192 bp BamHI fragment carrying ermC was cloned into pBS-ΔopaJ-nmb to produce pBS-ΔopaJ::ery-nmb. (iii) ΔopaJ::ery was amplified from pBS-ΔopaJ::ery-nmb using primers OpaFSalI and OpaRSacII (table 1), excluding opaJ locus-specific regions. The resulting amplicon was cloned into pCR2.1-TOPO to generate the generic plasmid pCR-Δopa::ery. (iv) ΔopaA and ΔopaD were amplified from their respective pBS-Δopa-nmb plasmids using primers OpaFSalI and OpaRSacII. Each modified opa was cloned into pCR2.1-TOPO, resulting in the generic plasmids pCR-ΔopaA and pCR-ΔopaD. (v) ΔopaA and ΔopaD were excised from the pCR2.1 plasmids by double digestion with SalI and SacII and cloned into pBluescript which had been similarly prepared, to create pBS-Δopa plasmids. (vi) A 1,194 bp SgrAI fragment carrying ermC was cloned into pCR-ΔopaD to produce pBS-ΔopaD::ery. (vii) A kanamycin (KanR) or tetracycline (TetR) resistance cassette was introduced into pBS-ΔopaD, or KanR was introduced into pBS-ΔopaA, resulting in generic Δopa plasmids containing selectable markers.
Table 1.
PCR primers used for amplification of opa genes from N. meningitidis and construction of Δopa plasmids.
Figure 2.
Homologous recombination in N. meningitidis.
The plasmid pBS-Δopa::ery-nmb contains sequences identical to the 5′ and 3′ ends of opa and adjacent locus-specific sequences flanking an erythromycin resistance cassette, EryR. Crossover events between chromosomal and plasmid DNA are illustrated using linearised plasmid. A double crossover event between homologous regions on the plasmid and chromosome allow EryR to be stably inserted into the chromosome at the opa locus. Generic plasmids containing Δopa without locus-specific regions are able to target multiple opa genes for homologous recombination.
Figure 3.
Summary of Opa-deficient mutant meningococci constructed from parent strain H44/76.
The plasmid used for each transformation and possible Opa expression of each new strain is indicated, as well as the gene disruptions that have been introduced. For each strain the underlined disruption is the one introduced by the most recent transformation. All possible opa combinations were created, including four single opa-deficient strains, six double opa-deficient strains, four triple opa-deficient strains, and an opa-negative strain.
Table 2.
Serum bactericidal antibody titres of pooled murine sera against 4 target strains, following immunisation with recombinant OpaA and OpaD, and Opa-positive and Opa-negative OMVs.
Table 3.
PCR primers used for screening opa genes following transformation of Neisseria meningitidis.