Colicins are a type of bacteriocin produced by some bacterial species in response to stress. Colicins are cytotoxic to competing cells. They are classified into two groups: group A, also called “Tol-dependant colicins” and group B called “Ton dependant colicins”. To exert their cytotoxic effects, colicins first need to be translocated into the target cells via the periplasm; however, the exact mechanism by which colicins achieve this translocation remains unclear. For Group A colicins, translocation requires an interaction between the N-terminal domain of the colicin and a series of membrane-bound and periplasmic proteins known as the Tol system. The translocation also includes interactions with more complicated assemblies. Recent developments in confocal laser scanning microscopy and in-cell nuclear magnetic resonance (CLSM and NMR, respectively) have prompted more studies of protein–protein interactions in living cells. CLSM and in-cell NMR spectroscopy were used to study the interaction between an overexpressed colicin translocation domain and periplasmic machinery. Cloning the full-length of the translocation domain, and the TolA, and TolB box sub-regions was accomplished. Recombinant protein expression was optimised to meet the requirements for CLSM and in-cell NMR analysis. Several factors, including culture medium and high cell density, were considered to have produced a successful and high level of recombinant proteins. The antimicrobial activity of colicin E9 on different E. coli cells expressing either the translocation domain or the TolA or TolB box was examined, and it was found that overexpressing the translocation domain, or part of it, provided some protection against colicin. Moreover, CLSM results indicate that TolA plays a key role in occupying the periplasm and preventing translocation of external colicin E9 to its target, which was found based on the high level of protection against ColE9. The same cells along with a positive control, protein GB1, were also used to develop a method to detect proteins of interest using in-cell NMR. Several parameters were investigated, including a labelling technique, culture medium, and the concentration of the in-cell sample. Analysis of 60% slurry (v/v) showed better NMR spectra, indicating the presence of an unfolded protein or the unstructured part of the translocation domain or TolB box. Furthermore, the data suggest that the expressed proteins, especially the TolA box, are involved in many interactions that can lead to the formation of large complexes responsible for occupying the periplasm and thus prevent translocation and consequently, activity of externally applied colicin.
|Date of Award
|Ian Cooper (Supervisor), Lara Barnes (Supervisor) & Lucas Bowler (Supervisor)