Method of isolation of regulated determinants from bacterial pathogens

A method of isolating and identifying bacterial genes that relate to virulence determinants, the method comprising:

• (a) obtaining a pool of mutants of bacteria that over-express DNA of wild-type bacteria;
• (b) selecting a mutant bacteria from step (a) that displays the enhanced virulence phenotype during interaction with a host cell relative to the wild-type bacteria;
• (c) isolating the DNA that contains a sequence that is a factor in the enhanced virulence phenotype in the bacteria selected in step (b);
• (d) constructing random mutations in the DNA isolated in step (c);
• (e) transferring the DNA of step (d) into wild-type bacteria;
• (f) selecting the bacteria of step (e) that no longer display the enhanced virulence phenotype in order to identify an active site of the DNA necessary to confer the enhanced virulence phenotype to the wild-type bacteria;
• (g) constructing mutations in the active site of the DNA identified in step (f);
• (h) transferring the DNA of step (g) into wild-type bacteria;
• (i) comparing the resulting phenotype and DNA of the mutant bacteria from step (h) to the wild-type bacteria to identify the DNA responsible for the enhanced virulence phenotype.