1. Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, Canada
2. Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M4K 1X8, Canada
3. Affinium Pharmaceuticals, 100 University Avenue, Toronto, Ontario M5J 1V6, Canada
4. Department of Biochemistry and
5. Department of Medical Genetics and Microbiology, University of Toronto, Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada

Correspondence to: Andrew Emili^1,5 Correspondence and requests for materials should be addressed to A.E. (Email: andrew.emili@utoronto.ca) or J.G. (Email: jack.greenblatte@utoronto.ca).

Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism¹, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames ( 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data². This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.

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