DC Field | Value | Language |
---|---|---|
dc.contributor.author | Choe, Donghui | ko |
dc.contributor.author | Kim, Uigi | ko |
dc.contributor.author | Hwang, Soonkyu | ko |
dc.contributor.author | Seo, Sang Woo | ko |
dc.contributor.author | Kim, Donghyuk | ko |
dc.contributor.author | Cho, Suhyung | ko |
dc.contributor.author | Palsson, Bernhard | ko |
dc.contributor.author | Cho, Byung-Kwan | ko |
dc.date.accessioned | 2023-03-16T01:02:57Z | - |
dc.date.available | 2023-03-16T01:02:57Z | - |
dc.date.created | 2023-01-02 | - |
dc.date.issued | 2023-02 | - |
dc.identifier.citation | MSYSTEMS, v.8, no.1 | - |
dc.identifier.issn | 2379-5077 | - |
dc.identifier.uri | http://hdl.handle.net/10203/305628 | - |
dc.description.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. | - |
dc.language | English | - |
dc.publisher | AMER SOC MICROBIOLOGY | - |
dc.title | Revealing Causes for False-Positive and False-Negative Calling of Gene Essentiality in Escherichia coli Using Transposon Insertion Sequencing | - |
dc.type | Article | - |
dc.identifier.wosid | 000897780800001 | - |
dc.identifier.scopusid | 2-s2.0-85149142544 | - |
dc.type.rims | ART | - |
dc.citation.volume | 8 | - |
dc.citation.issue | 1 | - |
dc.citation.publicationname | MSYSTEMS | - |
dc.identifier.doi | 10.1128/msystems.00896-22 | - |
dc.contributor.localauthor | Cho, Byung-Kwan | - |
dc.contributor.nonIdAuthor | Choe, Donghui | - |
dc.contributor.nonIdAuthor | Kim, Uigi | - |
dc.contributor.nonIdAuthor | Seo, Sang Woo | - |
dc.contributor.nonIdAuthor | Kim, Donghyuk | - |
dc.contributor.nonIdAuthor | Palsson, Bernhard | - |
dc.description.isOpenAccess | N | - |
dc.type.journalArticle | Article | - |
dc.subject.keywordAuthor | gene essentiality | - |
dc.subject.keywordAuthor | subgenic-level essentiality | - |
dc.subject.keywordAuthor | Tn-Seq | - |
dc.subject.keywordAuthor | DNA-binding proteins | - |
dc.subject.keywordAuthor | nucleoid-associated proteins | - |
dc.subject.keywordPlus | SCALE | - |
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