A new vector for insertion of any DNA fragment into the chromosome of transformable neisseriae

A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence which is required for natural transformation; (4) a promoter capable of functioning in Neisseria; and (5) several unique restriction sites useful for cloning. pNLE1 integrates into the leuS region of the neisserial chromosome at high frequencies by transformation-mediated recombination. The usefulness of this vector has been demonstrated by cloning the tetracycline-resistance gene (tet) and subsequently inserting the tet gene into the meningococcal chromosome.