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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 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.</long_description><tag>xref:EuropePMC:PMC12055562</tag><tag>xref:EuropePMC:PMC11142078</tag><repository>ENA</repository></additional><is_claimable>false</is_claimable><name></name><description>Inducible transposon mutagenesis for genome-scale forward genetics</description><dates><last_updated>2024-06-23</last_updated><first_public>2024-06-23</first_public></dates><accession>PRJNA1113708</accession><cross_references/></HashMap>