title: Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.
abstract: Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.
authors.affiliation: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom.
authors.forename: Gemma C
authors.initials: GC
authors.lastname: Langridge
authors.forename: Minh-Duy
authors.initials: MD
authors.lastname: Phan
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Turner
authors.forename: Timothy T
authors.initials: TT
authors.lastname: Perkins
authors.forename: Leopold
authors.initials: L
authors.lastname: Parts
authors.forename: Jana
authors.initials: J
authors.lastname: Haase
authors.forename: Ian
authors.initials: I
authors.lastname: Charles
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Peters
authors.forename: Gordon
authors.initials: G
authors.lastname: Dougan
authors.forename: John
authors.initials: J
authors.lastname: Wain
authors.forename: Julian
authors.initials: J
authors.lastname: Parkhill
authors.forename: A Keith
authors.initials: AK
authors.lastname: Turner
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001646:Bile / Q000502:physiology; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial / Q000235:genetics*; D019295:Computational Biology / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D020043:Genes, Essential; D016254:Mutagenesis, Insertional; D009154:Mutation; D012485:Salmonella typhi / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements