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Appl Environ Microbiol. 2012 May;78(9):3098-107. doi: 10.1128/AEM.06865-11. Epub 2012 Feb 24.

Genome scanning for conditionally essential genes in Salmonella enterica Serotype Typhimurium.

Author information

1
Cell and Molecular Biology Program, University of Arkansas, Fayetteville, Arkansas, USA.

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.

PMID:
22367088
PMCID:
PMC3346488
DOI:
10.1128/AEM.06865-11
[Indexed for MEDLINE]
Free PMC Article

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