Genome-scale models of Escherichia coli K-12 MG1655 metabolism and regulation can predict growth phenotypes for wildtype and mutant strains in a variety of defined environments (1). With the development of high-throughput growth screens (such as Phenotype Microarrays, Biolog, Hayward, CA) the metabolic capabilities of organisms can be rapidly assessed (2). Such technology has been applied to study E. coli strains (2) and the results are used in this work. By evaluating each positive growth environment, in the context of a metabolic model, a subset of growth conditions can be found that can not be explained by the current metabolic model. These indicate that catabolic pathways or transporters are missing from the reconstructed metabolic network (3). A combined computational and experimental effort was used to uncover these missing metabolic components and the genes responsible for their activities. We use an optimization-based algorithm to identify potentially missing reactions (either enzymatic or transport) that are needed to enable the model to correctly predict growth, in agreement with experimental data. Knowing what enzymes or transporters to look for, potential candidate genes were identified through literature searches, sequence-based homology searches, context-based search methods, or through gene expression profiling. Subsequent experiments evaluating single gene knockout (4) phenotypes, gene expression measurements using quantitative RT-PCR, and enzyme assays (in one case), identified the genes responsible for missing metabolic enzymes and transporters. This combined computational and experimental effort led to updated functional assignments for eight open reading frames in E. coli with two new enzymatic activities and four transport functions (5). The study demonstrates the use of systems analysis to discover new metabolic and transport functions and their genetic basis by a combination of experimental and computational approaches.

REFERENCES: 1. Covert, M.W., Knight, E.M., Reed, J.L., Herrgard, M.J. & Palsson, B.O. (2004) Nature 429, 92-6. 2. Bochner, B.R., Gadzinski, P. & Panomitros, E. (2001) Genome Res 11, 1246-55. 3. Reed, J.L., Vo, T.D., Schilling, C.H. & Palsson, B.O. (2003) Genome Biol 4, R54. 4. Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L. & Mori, H. (2006) Molecular Systems Biology. 5. Reed, J.L, Patel, T.R., Chen, K.H., Joyce, A.R., Applebee, M.K., Herring, C.D., Bui, O.T., Knight, E.M., Fong, S.S., & Palsson, B.O. (2006) PNAS 103,17480-4.

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