Construction of Hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and...

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformable Neisseria mutants. By random insertion of a selectable marker into the conjugative Neisseria plasmid ptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned in Escherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of an E. coli replicon that does not support autonomous replication in Neisseria, e.g. ColE1, p15A, or ori(fd), fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformable Neisseria strain involves a three-step process: (i) insertion of the desired gene into a +Hermes vector; (ii) transformation of Hermes into a Neisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the final Neisseria recipient. Several applications for the genetic manipulation of pathogenic Neisseriae are described.