Construction of a nontypeable Haemophilus influenzae-specific ectopic delivery vector

Complementation of chromosomal mutations in trans can introduce artifacts due to the number of episomal copies of the gene in question. One solution is to study the gene expressed at a single ectopic site in cis. We have designed and constructed a vector that allows homologous recombination into a gene encoding a frame-shifted IS1016-V6 protein in the Haemophilus influenzae Rd KW20 chromosome (HI1018). This site is the location of the > or = 35 kilobase capsule locus in encapsulated type b and d strains. This locus is not present in the nontypeable Rd KW20 strain, thus allowing ectopic expression of genes homologously recombined into HI1018 without polar effects.