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Updated: Jan 9, 2026
13:31
Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis
Published on: October 31, 2014
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Bacteriophage genome-wide transposon mutagenesis
Alex Chan1, Wearn-Xin Yee1, Deepto Mozumdar1
1Department of Microbiology & Immunology, University of California, San Francisco.
Biorxiv : the Preprint Server for Biology
|December 8, 2025
Summary
We developed a phage transposon sequencing (TnSeq) platform to identify essential genes in bacteriophages. This scalable technology efficiently maps gene functions for large lytic phages like ΦKZ.
Area of Science:
- Bacteriophage functional genomics
- Molecular biology
- Genetics
Background:
- Bacteriophage genomes are large and gene-rich, with many genes of unknown function.
- Traditional genetic methods for phages are laborious and not scalable.
- Unbiased functional genomics approaches are crucial for understanding phage biology.
Purpose of the Study:
- To develop a scalable transposon sequencing (TnSeq) platform for bacteriophage functional genomics.
- To identify essential and non-essential genes in the jumbo phage ΦKZ.
- To enable conditional genetic screens and functional analysis of phage genes.
Main Methods:
- Developed a phage TnSeq platform using a mariner transposase and an anti-CRISPR selectable marker.
- Employed CRISPR-Cas13a-based enrichment of transposed phages.
- Utilized pooled sequencing to identify fitness-conferring and dispensable genes in phage ΦKZ.
Main Results:
- Identified approximately 110 fitness-conferring genes in phage ΦKZ, including essential genes for nucleus formation, protein trafficking, and virion assembly.
- Generated knockouts for hundreds of non-essential genes.
- Discovered conditionally essential genes required under specific conditions (clinical isolates, environmental temperature, or nuclease presence).
Conclusions:
- Phage TnSeq is a facile and scalable technology for defining essential phage genes.
- This platform enables comprehensive functional genomics and conditional genetic screens in bacteriophages.
- Provides a broadly applicable resource for studying large lytic phages.
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