Chemical genomics screening reveals novel functions for unannotated genes in Vibrio cholerae C6706

Vibrio cholerae, the causative agent of cholera, remains a major global health burden despite ongoing initiatives for vaccination and improved sanitation. Much of the V. cholerae lifecycle occurs in aquatic environments, requiring gene functions that enable survival under multiple stresses associated with this niche and during the transition to the human host. Although numerous V. cholerae genome sequences are available, functional annotation across its pan-genome remains incomplete, limiting our understanding of its biology. In this study, we employed a chemical genomics approach to profile fitness across 104 diverse stress conditions and identify growth phenotypes for unannotated genes using 3,026 single-gene deletion mutants of V. cholerae C6706, a widely used research strain. In total, we identified significant growth phenotypes in 1,658 mutants, of which 285 correspond to currently unannotated genes. Together, these data provide a comprehensive resource for exploring gene function in V. cholerae, supported by detailed quality metrics and open-access tools to facilitate community-driven discovery. AUTHOR SUMMARYAlthough many bacterial pathogens have been extensively sequenced, we still know relatively little about what many of their genes actually do. In many cases, gene functions are solely predicted by computer algorithms but have not been confirmed in the lab, and we can assume that some of those predictions may be incorrect. Chemical genomics--a method that tests thousands of single-gene mutants under different stresses--can help reveal what these genes are responsible for. Using this approach, we screened a library of Vibrio cholerae C6706 mutants under 104 different conditions. We found that more than half of the mutants showed measurable effects on growth, including many genes that were previously uncharacterized. Here, we describe our experimental pipeline, present key quality checks, and share how other researchers can use this dataset to explore gene function in V. cholerae.