Supersaturation mutagenesis reveals adaptive rewiring of essential genes among malaria parasites

Malaria parasites are highly divergent from model eukaryotes. Large-scale genome engineering methods effective in model organisms are frequently inapplicable, and systematic studies of gene function are few. We generated more than 175,000 transposon insertions in the Plasmodium knowlesi genome, averaging an insertion every 138 base pairs, and used this “supersaturation” mutagenesis to score essentiality for 98% of genes. The density of mutations allowed mapping of putative essential domains within genes, providing a completely new level of genome annotation for any Plasmodium species. Although gene essentiality was largely conserved across P. knowlesi , Plasmodium falciparum , and rodent malaria model Plasmodium berghei , a large number of shared genes are differentially essential, revealing species-specific adaptations. Our results indicated that Plasmodium essential gene evolution was conditionally linked to adaptive rewiring of metabolic networks for different hosts.