Resolving the molecular mechanisms for bacterial selenium nanoparticle production
Brimilcombe, Claire Louise
Thesis or dissertation
University of Exeter
Thauera selenatis is a bacterium that can respire using selenate as the sole electron acceptor and produce elemental selenium in the form of nanospheres. Analysis of the secreted protein profile from T. selenatis that was respiring using selenate, identified the selenium export factor protein, SefA. SefA was associated with the selenium nanospheres and is thought to stabilise the assembly of the nanospheres in T. selenatis. The N-terminus of SefA shows similarity to the N-terminal regions of other mineralin family proteins and is therefore speculated to be involved with SefA-selenium interactions. It has been suggested that other proteins may also play a role in the assembly of the selenium nanospheres and their exportation out of the T. selenatis cell, and they could interact with SefA. SefA has been expressed in Escherichia coli and two His-tagged recombinant forms of the protein (His-SefA-His and His-SefA) have been purified. A SefA construct was also produced, which had an exposed N-terminal region, by proteolytic cleavage of the His-tag from the N-terminus of His-SefA-His using thrombin. Interactions between the N-terminal of SefA and soluble proteins from T. selenatis grown in selenate were investigated and three potentially interacting proteins were identified using pull-down experiments. MALDI-MS analysis identified peptide sequences of fragments from each of the three proteins and BLASTp analysis found that the closest matches for these proteins were threonyl-tRNA synthetase, glucosamine-fructose-6 phosphate aminotransferase, and the DNA protection during starvation proteins. Whilst attempts to crystallise the His-SefA-His protein were unsuccessful, the His-SefA-His protein was determined to be a tetramer and stabilising conditions for the His-SefA-His protein were determined for use in future crystallisation trials. A transcriptome analysis of T. selenatis grown with or without selenite was undertaken and this identified some interesting differentially expressed genes, but also indicated that the culture was in fact a mixed culture of T. selenatis and Enterobacter cloacae.
MbyRes in Biological Sciences