|Nisbet, David - Dave|
Submitted to: Methods in Enzymology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/20/2002
Publication Date: 12/20/2002
Citation: KWON, Y.M., KUBENA, L.F., NISBET, D.J., RICKE, S.C. FUNCTIONAL SCREENING OF BACTERIAL GENOME FOR VIRULENCE GENES BY TRANSPOSON FOOTPRINTING. METHODS IN ENZYMOLOGY. 2002. v. 358. p. 141-152. Interpretive Summary: Due to recent progress in genome sequencing, the entire DNA sequences of many disease causing bacteria have now been determined. However, the biological functions of many of the genes contained within these sequences are still unknown. In order to further our understanding of how some bacteria cause disease, we developed a simple genetic method to identify genes that play a role in the disease process. When used to identify disease causing genes in the foodborne bacterium Salmonella typhimurium, the method readily identified twelve previously unknown disease causing genes that enable the pathogen to survive and cause disease in animals. Because of its ease of use and wide applicability, this method will not only help researchers identify disease causing genes from numerous other foodborne pathogens but will enable them to develop effective control measures to prevent infection. Ultimately, this method will help American agriculture to produce safer, more affordable food for the American consumer.
Technical Abstract: A PCR-based approach was developed for the identification of bacterial virulence genes. Multiple transposon-flanking sequences in a pool of transposon mutants are simultaneously amplified and separated by gel electrophoresis, thus generating a footprinting pattern. As each transposon insertion generated a PCR product of unique length, the DNA bands on a footprint can serve as markers representing their corresponding transposon mutants. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, twelve Tn5 mutants with attenuated virulence were identified by the presence of PCR products in the footprints of the inoculum pool but not in bacteria recovered from infected mouse tissues. This approach termed "transposon footprinting" is an efficient method for functional screening of bacterial genomes without prior sequence information.