Large-scale identification of essential Salmonella genes by trapping lethal insertions.

Abstract

A novel screening approach based on insertion-duplication mutagenesis (IDM) was established to efficiently screen for essential genes of Salmonella enterica serovar Typhimurium under laboratory conditions. Small, randomly generated genomic fragments were cloned into a conditionally replicating vector, and the resulting library of single Salmonella clones was grown under permissive conditions. Upon switching to non-permissive temperature, discrimination between lethal and non-lethal insertions following homologous recombination allowed the trapping of genes with essential functions. Further characterization of a total of 498 fragments resulting in such lethal knockout revealed 145 known essential genes and 112 functionally characterized or hypothetical genes not yet shown to encode essential genes, among them three Salmonella-specific genes. The essentiality was demonstrated for a prioritised set of 15 putative indispensable genes by creating conditional lethal phenotypes. The results of this large-scale screening indicate that in rich media, the class of Salmonella genes indispensable for growth is composed of approximately 490 genes.