DEVELOPMENT AND APPLICATION OF A NEGATIVE SELECTION SYSTEM FOR STUDYING THE GENETIC BASIS OF VIRULENCE IN ISOLATES OF ENTEROBACTERIACEAE

Abstract

We have designed a general negative selection strategy based on inducible toxins. Combined with an appropriate positive selection marker, multiple variants of this strategy are efficient for making chromosomal alterations in both lab strains of E. coli and clinical isolates of uropathogenic and enterohemorrhagic E. coli, and Salmonella enterica. Importantly, no strain- or species-specific optimization is needed. Furthermore, our negative selection cassette demonstrates up to 60-fold greater selection stringency than all other reported negative selection systems and up to a 1000-fold greater than the most widely used negative selection system in E. coli. Taking advantage of the high stringency of our system, we have worked towards establishing mass allelic exchange between different strains of E. coli. By combining sexual genetics and linkage analysis, we hope to decipher causal genotype-phenotype associations in bacteria. We have also enabled an experimental method to identify regions of the bacterial genome that have variable stability.