SIMULTANEOUS IDENTIFICATION OF BACTERIAL VIRULENCE GENES BY NEGATIVE SELECTION

An insertional mutagenesis system that uses transposons carrying uniqu e DNA sequence tags was developed for the isolation of bacterial virul ence genes. The tags from a mixed population of bacterial mutants repr esenting the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella t yphimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered f rom infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally disti nct from, the inv/spa family of S. typhimurium.