The Effect of Fluorescent tagging of 1,2-Propanediol Utilization Microcompartment Shell Proteins on the Shell Formation and Spatial Organization Within the Bacterial Cell

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.