S. uvarum Tn7 transposon insertional mutagenesis sequencing

To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often translated to the increasing number of newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented comparative functional analysis by high-throughput experimental testing of gene dispensability in Saccharomyces uvarum, a sister species of S. cerevisiae. We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in S. uvarum to identify species dependent essential genes, with the goal of detecting genes with divergent function. Comprehensive gene dispensability comparisons with S. cerevisiae revealed that approximately 12% of conserved orthologs are predicted to display diverged dispensability, which were enriched for gene ontology categories that include DNA replication, protein binding and structural constituent of the ribosome. Surprisingly, despite their differences in essentiality, these genes are capable of cross-species complementation, demonstrating that other trans-acting factors that are background dependent contribute to differential gene essentiality. This data set provides direct experimental evidence of gene function across species, which can inform comparative genomic analyses, improve gene annotation and be applied across a diverse set of microorganisms to further our understanding of gene function evolution.