The study of the activation of Bacillus subtilis urease in vivo

In the natural environment, calcium carbonate (CaCO3) precipitation often coincides with biological processes. It is well documented that microbes in soil can induce the precipitation of CaCO3 in both the laboratory and the natural setting through microbial induced calcium carbonate precipitation (MICP). MICP utilises microorganisms as a result of their active metabolism, to precipitate CaCO3. The most studied MICP mechanism is urea hydrolysis. This process is catalysed by urease, a metalloenzyme that hydrolyses urea producing ammonium. The accumulation of ammonium causes the increase in surrounding pH that leads to CaCO3 precipitation when sufficient calcium is present. Many soil microbes participate in MICP, including Bacillus subtilis; a model, Gram positive, spore-forming, soil bacterium that produces urease. However, little is known about MICP by B. subtilis. Increased understanding of the urease of non-pathogenic B. subtilis could enable the development of a bacterial system where urease expression is increased under mechanical pressure leading to the production of CaCO3 for soil improvement. This process has been explored in engineering terms, however, to fully harness MICP we must first increase our understanding of the genetics responsible for urease regulation and activation. Bacterial ureases are composed of three highly conserved structural subunits (encoded by ureABC) and four accessory proteins (encoded ureDEFG) essential for urease activation. Unlike other urease-producing bacteria, B. subtilis only contains urease structural genes (ureABC) and within its genome lacks any homologues to genes coding for accessory proteins. The research carried out in this thesis aims to understand the activation of urease in B. subtilis. The work in this thesis utilises molecular biology and bioinformatic analysis in order to characterise urease activation in B. subtilis 168; two main approaches were explored including altering growth conditions such as pH, nitrogen availability and metal ion concentrations; utilising a comparative proteomics approach to investigate the differentially-expressed proteins and assessing their role in the activation of urease in vivo. Urease assays highlighted specific genes of interest as being involved in urease activity, particularly peptide transporters, however the proteomic and bioinformatic analysis identified an increase in or similar level of urease structural unit expression. Any decrease in urease activity in specific knockouts was associated with poor urease activation, via enzyme assays and proteomic analysis. This confirmed the requirement for the combination of the enzyme assays and proteomic evaluations in understanding urease activity. Recombinant urease expression of structural proteins was then investigated utilising E. coli which identified the recombinant protein conformation was not optimum (trimer of trimers) as mostly the dimer of trimers was evident. This highlights the necessity for the recombinant expression method to be optimised in order to achieve a full understanding of urease activation in B. subtilis