CONSTRUCTION OF HERMES SHUTTLE VECTORS - A VERSATILE SYSTEM USEFUL FOR GENETIC COMPLEMENTATION OF TRANSFORMABLE AND NON-TRANSFORMABLE NEISSERIA MUTANTS

A versatile shuttle system has been developed for genetic complementat ion with cloned genes of transformable and non-transformable Neisseria mutants. By random insertion of a selectable marker into the conjugat ive Neisseria plasmid ptetM25.2, a site within this plasmid was identi fied 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 t he construction of the Hermes vectors. Hermes vectors are composed of an E. coli replicon that does not support autonomous replication in Ne isseria, e.g. ColE1, p15A, or ori(fd), fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the int egration region of ptetM25.2. Complementation of a non-transformable N eisseria strain involves a three-step process: (i) insertion of the de sired gene into a Hermes vector; (ii) transformation of Hermes into a Neisseria strain containing pretM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugativ e transfer of the hybrid ptetM25.2 into the final Neisseria ia recipie nt. Several applications for the genetic manipulation of pathogenic Ne isseriae are described.