title: Genome scanning for conditionally essential genes in Salmonella enterica Serotype Typhimurium.
abstract: As more whole-genome sequences become available, there is an increasing demand for high-throughput methods that link genes to phenotypes, facilitating discovery of new gene functions. In this study, we describe a new version of the Tn-seq method involving a modified EZ:Tn5 transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This Tn-seq method was applied to a genome-saturating Salmonella enterica serotype Typhimurium mutant library recovered from selection under 3 different in vitro growth conditions (diluted Luria-Bertani [LB] medium, LB medium plus bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes in S. Typhimurium that are conditionally essential under at least one of the above selective conditions. Competition assays using 4 deletion mutants (pyrD, glnL, recD, and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other approximately 70 bacterial species for which EZ:Tn5 mutagenesis has been established.
authors.affiliation: Cell and Molecular Biology Program, University of Arkansas, Fayetteville, Arkansas, USA.
authors.forename: Anita
authors.initials: A
authors.lastname: Khatiwara
authors.forename: Tieshan
authors.initials: T
authors.lastname: Jiang
authors.forename: Sam-Sun
authors.initials: SS
authors.lastname: Sung
authors.forename: Turki
authors.initials: T
authors.lastname: Dawoud
authors.forename: Jeong Nam
authors.initials: JN
authors.lastname: Kim
authors.forename: Dhruva
authors.initials: D
authors.lastname: Bhattacharya
authors.forename: Hee-Bal
authors.initials: HB
authors.lastname: Kim
authors.forename: Steven C
authors.initials: SC
authors.lastname: Ricke
authors.forename: Young Min
authors.initials: YM
authors.lastname: Kwon
mesh_terms: D004251:DNA Transposable Elements; D017353:Gene Deletion; D005798:Genes, Bacterial; D020043:Genes, Essential; D005820:Genetic Testing / Q000379:methods; D005827:Genetics, Microbial / Q000379:methods*; D016680:Genome, Bacterial; D016254:Mutagenesis, Insertional / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements