Vi-TIS: a transposon insertion sequencing framework for quantitative discrimination of viable and non viable mutants
Transposon-insertion sequencing (TIS) is a high-throughput approach that uses randomly generated transposon mutant libraries to assess bacterial gene essentiality and their contribution to fitness under defined conditions. The method integrates classical transposon mutagenesis with next-generation sequencing. However, conventional TIS workflows generally require at least one active growth step to enrich for replicating or fit mutants. The outcome of these experiments can be biased by the recovery of non-replicating cells or by the loss of slow-growing mutants. To overcome this limitation, we modified the traditional TIS workflow by incorporating propidium monoazide (PMA). PMA is membrane-impermeant and excluded from viable cells but can enter dead cells when the membrane has lost its integrity. PMA covalently crosslinks to DNA upon exposure to UV light. Such binding inhibits PCR amplification, an essential step in TIS library preparation. This property enables selective exclusion of DNA originating from dead cells and reduces the signal to noise ratio in TIS experiments. We refer to this modified approach as Viability-TIS (Vi-TIS). Applying Vi-TIS to our ultra-dense Escherichia coli K-12 BW25113 TIS library enhanced the accuracy of essential gene identification. We further exposed the library to subinhibitory concentrations of carbenicillin and found that Vi-TIS produced results consistent with those obtained using culture-based methodologies. In addition, Vi-TIS revealed previously unrecognized carbenicillin-susceptible mutants. Finally, we demonstrate that the mutations are linked to perturbations in peptidoglycan biosynthesis.