Dual transposon sequencing profiles the genetic interaction landscape in bacteria.

Gene redundancy complicates systematic characterization of gene function as single-gene deletions may not produce discernible phenotypes. We report dual transposon sequencing (dual Tn-seq), a platform for assaying the fitness of a comprehensive double mutant pool in parallel. Dual Tn-seq couples random barcode transposon site sequencing with the Cre-lox system, enabling deep sampling of 73% of the 1.3 million possible double gene deletions in Streptococcus pneumoniae. The genetic interactions identified span a wide range of biochemical processes, revealing new factors in presumably well-studied pathways, exemplified by a cytidine triphosphate synthase PyrJ. Moreover, this approach should permit further investigation of growth condition–specific genetic interactions. Because dual Tn-seq does not require the construction of a large array of single mutants, it should be readily adaptable to various microorganisms. Editor's summary: Understanding gene function is often complicated by gene redundancy, in which disrupting a single gene yields no clear phenotype. Zik et al. developed dual-transposon sequencing, a high-throughput method to systematically uncover genetic interactions. This approach uses the Cre-lox system to fuse two DNA barcodes identifying random transposon insertions within a single cell. Sequencing these recombined barcodes enabled parallel fitness assessment of millions of double mutants. Applied to Streptococcus pneumoniae, this revealed functions for previously uncharacterized genes across diverse biological pathways. This method offers a powerful tool to explore the vast genetic "dark matter" in microorganisms. —Di Jiang INTRODUCTION: Next-generation DNA sequencing (NGS) has considerably increased the amount of sequencing data in public databases. However, millions of these sequences still remain as genetic "dark matter." About one-third of bacterial genes have no known functions, partly due to challenges such as gene redundancy. We report dual transposon sequencing (dual Tn-seq), a high-throughput platform designed to systematically mine genetic interactions and help identify gene functions. Understanding these unknown genes is especially important for developing new strategies to combat the growing threat of antimicrobial resistance. RATIONALE: Transposon sequencing (Tn-seq) is a widely used, unbiased approach for studying gene function. This technique measures changes in the abundance of randomly inserted, gene-inactivating transposons within a saturated mutant pool, helping to identify genes necessary for survival under different conditions. One approach is to perform Tn-seq in a single-gene knockout background to uncover genetic interactions, in which combinations of mutations lead to unexpected results such as synthetic lethality. Genes that interact are often functionally related. However, traditional Tn-seq is not designed to systematically identify genetic interaction networks because it cannot detect random double transposon mutants that are far apart in the genome, which limits its scope. RESULTS: To address this limitation, we developed dual Tn-seq, which enables high-throughput fitness assessment of a large pool of double transposon mutants. Dual Tn-seq combines randomly barcoded (RB) Tn-seq with the cre-lox system. Cre recombinase brings the two distant barcodes close together, allowing for the capture of both insertions within a single sequencing read. We applied this system to the human pathogen Streptococcus pneumoniae by building two barcoded transposon libraries and combining them to generate more than a billion double mutants. After inducing Cre and sequencing, we used a probabilistic model to compare observed double mutant frequencies with the expected levels calculated from the frequencies of the individual mutants. This screen reported 244 high-confidence genetic interactions, ranging from synthetic lethality to partial growth impairment. These interactions involved diverse biochemical pathways, allowing us to define functions of hypothetical genes, such as the alternative CTP synthase PyrJ and a regulator of peptidoglycan synthesis. CONCLUSION: Dual Tn-seq provides a scalable and cost-effective approach to uncover genetic interactions in bacteria. It only requires a few components: an inducible promoter, the ability to generate transposon mutants, and a functioning cre cassette. As sequencing costs continue to decline, this approach will become more accessible in a wide range of organisms. Moreover, the scope of dual Tn-seq can be broadened by growing the double mutant libraries under different conditions, such as with antibiotic treatments or various nutritional and growth conditions. Dual Tn-seq opens new avenues for exploring microbiology and accelerating the discovery of drug targets. Dual Tn-seq probes genetic interactions in bacteria.: In a pool of double transposon mutants, two transposons can be identified as originating from a single cell by sequencing a dual-barcode identifier after inducing site-specific recombination with Cre recombinase. The abundance of a double mutant can then be quantified and compared with the theoretical, calculated frequency to identify mutants that deviate from expectations, thus revealing genetic interactions. [ABSTRACT FROM AUTHOR]