Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.769804
Title: The effect of fluorescent tagging of 1,2-propanediol utilization microcompartment shell proteins on the shell formation and spatial organization within the bacterial cell
Author: Packwood, Sarah
ISNI:       0000 0004 7659 4466
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2017
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Abstract:
Bacterial microcompartments are isohedral structures composed entirely of protein and contain a 2-4 nm thick proteinaceous shell. This shell encases enzymes which act in a sequential manner to carry out a specific metabolic reaction. There are seven essential shell proteins encoded on the propanediol utilization microcompartment (Pdu) operon, Pdu -A -B -B' -J -K -N and - U, each having a critical role in the formation of the compartment. Other genes in the Pdu operon interact to form enzymes in the metabolic pathway with some having unknown functions such as PduV which has been shown to form filament-like structures. These filaments only appear in the presence of microcompartments and often co-localise with them and it is thought that PduV plays a role in the spatial distribution of microcompartments. This study has explored the interaction between PduV and the microcompartment shell. The shell protein involved in this interaction is thought to be PduK, and to characterise the mechanism of this interaction PduK truncations were generated and novel microcompartments containing these truncations were engineered. The ability of these microcompartments to form was examined, as well as the ineraction between PduV and these truncated forms of PduK. Filament formation of the small GTPase, PduV, was studied by site directed mutagenesis of the GTP binding site, which was then examined by microscopic and biochemical approaches. In addition, to further characterise how microcompartments form each individual shell protein was tagged with mCherry. Microscopy techniques such as live cell imaging and transfer electron microscopy were used in order to determine if these novel compartments were able to form correctly, if PduV filament formation was affected in any way and whether this subsequently had an effect on the distribution of microcompartments within the bacterial cell. The work presented has given insight into the formation of the microcompartment shell, higlighting the sensitivity of individual shell proteins to minor modifications such as fluorescent tagging and the effect this can have on the formation of the shell. It has also revealed possible previously undiscovered regulatory mechanisms of shell proteins on PduV filament formation and length, with the modification of shell proteins affecting PduV filaments. Finally, it has given greater evidence to previous suggestions that PduV may be a GTPase.
Supervisor: Warren, Martin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.769804  DOI: Not available
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