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Kim, Donghyuk
Systems Biology and Machine Learning Lab
Research Interests
  • Systems Biology, Computational Biology, Bacterial Pathogens, Host-Pathogen Interaction, Anti-Microbial Resistance

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Revealing Causes for False-Positive and False-Negative Calling of Gene Essentiality in Escherichia coli Using Transposon Insertion Sequencing

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Title
Revealing Causes for False-Positive and False-Negative Calling of Gene Essentiality in Escherichia coli Using Transposon Insertion Sequencing
Author
Choe, DonghuiKim, UigiHwang, SoonkyuSeo, Sang WooKim, DonghyukCho, SuhyungPalsson, BernhardCho, Byung-Kwan
Issue Date
2023-02
Publisher
AMER SOC MICROBIOLOGY
Citation
MSYSTEMS, v.8, no.1, pp.e00896-22
Abstract
Transposon mutagenesis is an efficient way to explore gene essentiality of a bacterial genome. However, there was a discrepancy between the essential gene set determined by transposon mutagenesis and that determined using single-gene knockout strains. The massive sequencing of transposon insertion mutant libraries (Tn-Seq) represents a commonly used method to determine essential genes in bacteria. Using a hypersaturated transposon mutant library consisting of 400,096 unique Tn insertions, 523 genes were classified as essential in Escherichia coli K-12 MG1655. This provided a useful genome-wide gene essentiality landscape for rapidly identifying 233 of 301 essential genes previously validated by a knockout study. However, there was a discrepancy in essential gene sets determined by conventional gene deletion methods and Tn-Seq, although different Tn-Seq studies reported different extents of discrepancy. We have elucidated two causes of this discrepancy. First, 68 essential genes not detected by Tn-Seq contain nonessential subgenic domains that are tolerant to transposon insertion, which leads to the false assignment of an essential gene as a nonessential or dispensable gene. These genes exhibited a high level of transposon insertion in their subgenic nonessential domains. In contrast, 290 genes were additionally categorized as essential by Tn-Seq, although their knockout mutants were available. The comparative analysis of Tn-Seq and high-resolution footprinting of nucleoid-associated proteins (NAPs) revealed that a protein-DNA interaction hinders transposon insertion. We identified 213 false-positive genes caused by NAP-genome interactions. These two limitations have to be considered when addressing essential bacterial genes using Tn-Seq. Furthermore, a comparative analysis of high-resolution Tn-Seq with other data sets is required for a more accurate determination of essential genes in bacteria.IMPORTANCE Transposon mutagenesis is an efficient way to explore gene essentiality of a bacterial genome. However, there was a discrepancy between the essential gene set determined by transposon mutagenesis and that determined using single-gene knockout strains. In this study, we generated a hypersaturated Escherichia coli transposon mutant library comprising approximately 400,000 different mutants. Determination of transposon insertion sites using next-generation sequencing provided a high-resolution essentiality landscape of the E. coli genome. We identified false negatives of essential gene discovery due to the permissive insertion of transposons in the C-terminal region. Comparisons between the transposon insertion landscape with binding profiles of DNA-binding proteins revealed interference of nucleoid-associated proteins to transposon insertion, generating false positives of essential gene discovery. Consideration of these findings is required to avoid the misinterpretation of transposon mutagenesis results.
URI
https://scholarworks.unist.ac.kr/handle/201301/60688
URL
https://journals.asm.org/doi/10.1128/msystems.00896-22
DOI
10.1128/msystems.00896-22
ISSN
2379-5077
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