Quantitative Genome-Wide Genetic Interaction Screens Reveal Global Epistatic Relationships of Protein Complexes in Escherichia coli

Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among gamma-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems. Author Summary Genome-wide genetic interaction (GI) screens have been performed in yeast, but no analogous large-scale studies have yet been reported for bacteria. Here, we have used E. coli synthetic genetic array (eSGA) technology developed by our group to quantitatively map GIs to reveal epistatic dependencies and functional cross-talk among similar to 600,000 digenic mutant combinations. By combining this epistasis information with functional modules derived by our group's earlier efforts from proteomic and genomic context (GC)-based methods, we identify several unexpected pathway-level dependencies, functional links between protein complexes, and biological roles of uncharacterized bacterial gene products. As part of the study, two of our pathway predictions from GI screens were validated experimentally, where we confirmed the role of these new components in iron-sulphur biogenesis and ribosome integrity. We also extrapolated the epistatic connectivity diagram of E. coli to 233 distantly related gamma-proteobacterial species lacking GI information, and identified co-conserved genes and functional modules important for bacterial pathogenesis. Overall, this study describes the first genome-scale map of GIs in gram-negative bacterium, and through integrative analysis with previously derived protein-protein and GC-based interaction networks presents a number of novel insights into the architecture of bacterial pathways that could not have been discerned through either network alone.