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.