Dataset Information

A single shell protein plays a major role in choline transport across the shell of the choline utilization microcompartment of Escherichia coli 536.

ABSTRACT: Bacterial microcompartments (MCPs) are widespread protein-based organelles that play important roles in the global carbon cycle and in the physiology of diverse bacteria, including a number of pathogens. MCPs consist of metabolic enzymes encapsulated within a protein shell. The main roles of MCPs are to concentrate enzymes together with their substrates (to increase reaction rates) and to sequester harmful metabolic intermediates. Prior studies indicate that MCPs have a selectively permeable protein shell, but the mechanisms that allow selective transport across the shell are not fully understood. Here we examine transport across the shell of the choline utilization (Cut) MCP of Escherichia coli 536, which has not been studied before. The shell of the Cut MCP is unusual in consisting of one pentameric and four hexameric bacterial microcompartment (BMC) domain proteins. It lacks trimeric shell proteins, which are thought to be required for the transport of larger substrates and enzymatic cofactors. In addition, its four hexameric BMC domain proteins are very similar in amino acid sequence. This raises questions about how the Cut MCP mediates the selective transport of the substrate, products and cofactors of choline metabolism. In this report, site-directed mutagenesis is used to modify the central pores (the main transport channels) of all four Cut BMC hexamers to assess their transport roles. Our findings indicate that a single shell protein, CmcB, plays the major role in choline transport across the shell of the Cut MCP and that the electrostatic properties of the CmcB pore also impact choline transport. The implications of these findings with regard to the higher-order structure of MCPs are discussed.

SUBMITTER: Ochoa JM

PROVIDER: S-EPMC10710832 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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A single shell protein plays a major role in choline transport across the shell of the choline utilization microcompartment of <i>Escherichia coli</i> 536.

Ochoa Jessica M JM Dershwitz Philip P Schappert Mary M Sinha Sharmistha S Herring Taylor I TI Yeates Todd O TO Bobik Thomas A TA

Microbiology (Reading, England) 20231101 11

Bacterial microcompartments (MCPs) are widespread protein-based organelles that play important roles in the global carbon cycle and in the physiology of diverse bacteria, including a number of pathogens. MCPs consist of metabolic enzymes encapsulated within a protein shell. The main roles of MCPs are to concentrate enzymes together with their substrates (to increase reaction rates) and to sequester harmful metabolic intermediates. Prior studies indicate that MCPs have a selectively permeable pro ...[more]

PMID: 37971493

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Project description: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 13C-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.

Project description:Bacterial microcompartments (BMCs) are self-assembled protein structures often utilized by bacteria as a modular metabolic unit, enabling the catalysis and utilization of less common carbon and nitrogen sources within a self-contained compartment. The ethanolamine (EA) utilization (eut) BMC has been widely demonstrated in enteropathogens, such as Salmonella enterica, and current research is exploring its activity in the commensal species that populate the human gut. Escherichia coli Nissle 1917 (EcN) is a strong colonizer and probiotic in gut microbial communities and has been used extensively for microbiome engineering. In this study, the utilization of ethanolamine as a sole carbon source and the formation of the eut BMC in EcN were demonstrated through growth assays and visualization with transmission electron microscopy. Subsequently, flux balance analysis was used to further investigate the metabolic activity of this pathway. It was found that not only is the utilization of the eut BMC for the degradation of EA as a carbon source in EcN comparable with that of Salmonella enterica but also that ammonium is released into solution as a byproduct in EcN but not in S. enterica. Control of EA-dependent growth was demonstrated using different concentrations of the operon inducer, vitamin B12. We show that vitamin B12-dependent EA utilization as the sole carbon source enables growth in EcN, and demonstrate the concurrent formation of the BMC shell and inducible control of the eut operon.ImportanceThe human gut is a complex environment of different bacterial species, nutrient sources, and changing conditions that are essential for human health. An imbalance can allow for the emergence of opportunistic pathogens. Bacterial microcompartments (BMCs) are utilized by bacteria to metabolize less common nutrients, conferring a growth advantage. Although widely studied in enteropathogens, there is limited research on BMC activity in commensal species. We demonstrate the formation of the eut BMC and utilization of ethanolamine as a carbon source in the human gut commensal Escherichia coli Nissle 1917 (EcN). Additionally, we found increased ammonium production when EcN utilized ethanolamine but did not see the same in Salmonella enterica, highlighting potential differences in how these species affect the wider microbial community.

Dataset Information

A single shell protein plays a major role in choline transport across the shell of the choline utilization microcompartment of Escherichia coli 536.

Publications

A single shell protein plays a major role in choline transport across the shell of the choline utilization microcompartment of <i>Escherichia coli</i> 536.

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