
==== Front
bioRxiv
BIORXIV
bioRxiv
Cold Spring Harbor Laboratory

10.1101/2024.05.21.595064
preprint
1
Article
Inducible transposon mutagenesis for genome-scale forward genetics
Basta David W. http://orcid.org/0000-0003-4176-6566

Campbell Ian W. http://orcid.org/0000-0003-3019-2560

Sullivan Emily J. http://orcid.org/0000-0001-9558-3883

Hotinger Julia A. http://orcid.org/0000-0003-2427-5482

Hullahalli Karthik http://orcid.org/0000-0003-3064-2090

Waldor Matthew K. http://orcid.org/0000-0003-1843-7000

21 5 2024
2024.05.21.595064http://biorxiv.org/lookup/doi/10.1101/2024.05.21.595064
nihpp-2024.05.21.595064.pdf
Abstract

Transposon insertion sequencing (Tn-seq) is a powerful method for genome-scale functional genetics in bacteria. However, its effectiveness is often limited by a lack of mutant diversity, caused by either inefficient transposon delivery or stochastic loss of mutants due to population bottlenecks. Here, we introduce “InducTn-seq”, which leverages inducible mutagenesis for temporal control of transposition. InducTn-seq generates millions of transposon mutants from a single colony, enabling the sensitive detection of subtle fitness defects and transforming binary classifications of gene essentiality into a quantitative fitness measurement across both essential and non-essential genes. Using a mouse model of infectious colitis, we show that InducTn-seq bypasses a highly restrictive host bottleneck to generate a diverse transposon mutant population from the few cells that initiate infection, revealing the role of oxygen-related metabolic plasticity in pathogenesis. Overall, InducTn-seq overcomes the limitations of traditional Tn-seq, unlocking new possibilities for genome-scale forward genetic screens in bacteria.
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pmc
