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A structural and biochemical comparison of wild-type and recombinant propanediol utilising bacterial microcompartments

Mayer, Matthias (2016) A structural and biochemical comparison of wild-type and recombinant propanediol utilising bacterial microcompartments. Doctor of Philosophy (PhD) thesis, University of Kent. (KAR id:54756)

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Abstract

Bacterial microcompartments (BMCs) likely represent the largest protein complex found in bacterial cells (estimated 18,000 subunits). BMCs are associated with specific metabolic processes such as carbon fixation in the case of carboxysomes or carbon utilisation in the case of the 1,2-propanediol utilisation metabolosome. With all BMCs there is still much to learn about the structure and function of these supramolecular assemblies.

In this project the structure and function of the Pdu BMC has been studied through a combination of recombinant DNA technology, proteomics, fluorescence microscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM). Initially, wild type Citrobacter freundii (C. freundii) Pdu BMCs were compared to recombinant BMCs that had been produced in Escherichia coli (E. coli). These were found to have very similar profiles in terms of BMC shell composition, size (120-130 nm on average) and shape. However, recombinant empty BMCs (eBMCs), generated by the coexpression of the genes for just the shell proteins, were found to be distinctly different in that they incorporated more of the first shell protein of the operon, PduA, and were also significantly smaller (55-80 nm on average) than the complete BMCs. This work provides the first recorded application of AFM for the study of BMCs and revealed that the eBMCs were stiffer and less flexible than complete BMCs and it is unclear if differences in shell protein composition or the lack of the internal scaffold is causing the reported biophysical differences.

One key property of the BMCs is their ability to absorb substrate and cofactor molecules to allow the internalised metabolic pathway to operate. Adenosylcobalamin (Ado-B12) is required as a coenzyme for the diol dehydratase conversion of 1,2-propanediol (1,2-PD), but the uptake of cobalamin into the BMC was not shown. By feeding cells exogenous cobalamin, it was possible to demonstrate that cobalamin is specifically partitioned from the cytoplasm into the BMC. Moreover the synthesis of fluorescently labelled versions of cobalamin allowed this partitioning to be followed ex-vivo with purified BMCs.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Warren, Martin J.
Uncontrolled keywords: 1,2-propanediol utilisation, bacterial organelles, cobalamin, atomic force microscopy, transmission electron microscopy
Subjects: Q Science
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: [UNSPECIFIED] University of Kent / BBSRC
Depositing User: Users 1 not found.
Date Deposited: 31 Mar 2016 09:26 UTC
Last Modified: 20 May 2021 13:32 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/54756 (The current URI for this page, for reference purposes)
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