Integrative in vivo analysis of the ethanolamine utilization bacterial microcompartment in Escherichia coli .

Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although approximately 20% of sequenced bacterial genomes contain operons encoding putative BMCs, few have been thoroughly characterized, nor any in the most studied Escherichia coli strains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon in E. coli K-12. The eut genotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of the E. coli Eut BMCs were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and the in vivo stoichiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome-scale modeling and 13 C-fluxomics. Our results reveal that contrary to previous suggestions, ethanolamine serves both as a nitrogen and a carbon source in E. coli K-12, while also contributing to significant metabolic overflow. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli .IMPORTANCEThe properties of bacterial microcompartments make them an ideal tool for building orthogonal network structures with minimal interactions with native metabolic and regulatory networks. However, this requires an understanding of how BMCs work natively. In this study, we combined genetic manipulation, multi-omics, modeling, and microscopy to address this issue for Eut BMCs. We show that the Eut BMC in Escherichia coli turns ethanolamine into usable carbon and nitrogen substrates to sustain growth. These results improve our understanding of compartmentalization in a widely used bacterial chassis.