K-131. E. coli-Synthetic Genetic Arrays (eSGA): Uncovering Functional Relationships on a Genome-Wide Scale

G. Butland¹, M. Babu², H. Lo², B. Gold¹, C. Christopolous², W. Yang¹, R. Prathapam¹, O. Pogoutse², J. Li², J. Wasniewski², P. Venn², F. Bohdana², A. Safavi-Naini², N. Sourour², S. Caron², L. Laigle², S. Phanse², A. Deshpande², S. Joe², H. Ding², M. Maris², G. Moreno-Hagelseib³, J. Greenblatt², A. Emili²;
¹Lawrence Berkeley Natl. Lab., Berkeley, CA, ²Univ. of Toronto, Toronto, ON, CANADA, ³Wilfrid Laurier Univ., Waterloo, ON, CANADA.

Interactions define the molecular organization of the cell. Physical interactions, such as protein-protein interactions, reveal how individual polypeptide chains come together to form protein complexes which perform many of the biochemical processes in a cell. Other types of interactions, such as functional interactions, propose organizational relationships between gene products and protein complexes within the cell. Epistatic (genetic) interactions, one of the most commonly encountered forms of functional interaction, exist between genes which encode proteins involved in parallel pathways or overlapping processes. High-throughput experimental systems to examine epitasis on a genome-wide scale have been devised for yeast, worm, and mammalian cells, but have not been previously reported for Prokaryotes. Here, we report the development of a quantitative screening procedure for systematically monitoring genetic interactions among double gene deletion mutants in E. coli on a global manner based on the natural genetic system of conjugation. The assay detects both aggravating genetic interactions, resulting in the retarded growth or synthetic lethality of double gene mutants, which provides information regarding functional redundancy among pathways, and alleviating interactions, which buffer phenotypes, allowing for the classification of bacterial gene products into their respective functional modules. Initial studies are focused on two different but analogous systems for iron sulfur cluster biosynthesis, encoded by the isc and suf operons. Mutations in each member of these systems have been combined in a pair-wise manner with one of ~4000 E. coli single gene deletion mutants and the resulting double mutants assayed for fitness. These global functional interaction screens have confirmed previously reported synthetic lethality phenotypes observed for various combinations of isc and suf operon mutations. We have also uncovered multiple novel interactions, including the apparent synthetic lethality of mutations in grxD, a monothiol glutaredoxin, with members of the isc iron-sulfur cluster biosynthesis system.