Construction of Plasmids and Mutants. We used the sequence of NMB0279 and NMB0280 from N. meningitidis strain MC58 (www.tigr.org) to design primers to clone and subsequently inactivate the imp gene in strain H44/76. Part of the gene upstream of imp, NMB0279, was cloned from H44/76 DNA by using primers A and B (Table 1). The 3' end of the imp gene was obtained by PCR with primers C and D. Both PCR products were cloned into pCR2.1-TOPO (Invitrogen), resulting in plasmids pCR2.1-NMB0279 and pCR2.1-3' Imp. An AccI–XbaI fragment of pCR2.1-NMB0279 was ligated into AccI–XbaI-restricted pCR2.1-3' Imp. The resulting plasmid was cut with AccI to allow insertion of a kanamycin-resistance cassette. This cassette was PCR-amplified from plasmid pACYC177 (New England Biolabs) by using primers E and F (Table 1), which introduced terminal AccI sites and a Neisserial DNA uptake sequence. The final construct, called pMB25, contained the kanamycin-resistance cassette in the same orientation as the transcription direction of the imp gene. Approximately 200 ng of a purified PCR product amplified from pMB25 with primers A and D was added to wild- type H44/76 bacteria growing in TSB plus 10 mM MgCl₂ for 6 h. Bacteria were plated on GC plates containing kanamycin. Transformants were screened by PCR using primer pairs AD, AF, and DE.

For complementation experiments, we cloned the imp gene from H44/76 genomic DNA by PCR by using the primer pair D and G (Table 1). The PCR product was cloned in pCR2.1-TOPO, and was cut and ligated into pEN11 by using NdeI and AatII restriction, resulting in plasmid pEN11-Imp. Plasmid pEN11, a Neisseria-replicative plasmid, is a derivative of pRV2100, which contains the H44/76 omp85 gene behind a tandem lac promoter-operator (tac-lacUV5) sequence (1). In pEN11, the ATG initiation codon of the omp85 gene is replaced by an NdeI site to facilitate exchange of genes. The imp mutant was transformed with pEN11-Imp by coincubation of bacteria with plasmid for 6 h on a plate (1). Transformants were selected on plates containing chloramphenicol and tested for the presence of pEN11-Imp and the chromosomal imp::kan allele by PCR.

For antibody production, the H44/76 imp gene without its signal sequence was cloned in pET11a (Novagen) by using primers H and I (Table 1). The resulting plasmid pET11a-Imp was introduced into E. coli strain BL21(DE3; Novagen) to allow expression of the truncated imp gene from the T7 promoter present in pET11a.

Plasmid pEN11-pagL was obtained by subcloning the pagL gene of Bordetella bronchiseptica from plasmid pPagL_(Bb) (J.G., L. Steeghs, J. ten Hove, A. de Jong, P. van der Ley, and J.T., unpublished work) into pEN11 by using NdeI–AatII restriction. This plasmid was used to transform H44/76 L8 immunotype. Correct transformants were identified by PCR and the imp gene was subsequently inactivated as described above.

1. Voulhoux, R., Bos, M. P., Geurtsen, J., Mols, M. & Tommassen, J. (2003) Science 299, 262-265.