In vitro mutagenesis of Brucella species.

Three major techniques have been employed for broad-range in vitro mutagenesis of Brucella species. Shotgun approaches capable of generating large libraries of randomly inserted transposon mutants include Tn5, mariner (Himar1), and mini-Tn5 signature-tagged mutagenesis. Allelic exchange has also been extensively employed for targeted gene deletion. In general, plasmid and transposon delivery into Brucella has relied upon electroporation; however, conjugation has also been successfully employed. Both approaches have been used to identify critical virulence determinants necessary for disease and intracellular survival of the organism. Perhaps more importantly these approaches have provided an opportunity to develop attenuated vaccine candidates of improved safety and efficacy. Future experiments are designed to identify individual functions that govern the interaction between host and agent and control intracellular trafficking and survival. Toward this goal, this chapter describes current approaches used to mutagenize Brucella spp.