Characterization of Bacillus subtilis urease and the Klebsiella aerogenes UreEF protein

In vivo assembly of the urease metallocenter in most bacteria typically requires the actions of four accessory proteins: UreD, UreE, UreF, and UreG. The urease gene cluster of Bacillus subtilis possesses only the structural genes (ureABC), and this organism lacks known accessory genes in its genome. Nevertheless, the organism can produce functional Ni-containing urease. The activation properties of recombinant B. subtilis urease were examined in both Escherichia coli and B. subtilis hosts. Overexpression of B. subtilis ureABC alone unexpectedly confers urease activity, however the level is low even in the presence of excess Ni in the cultures. Although the B. subtilis urease shares high sequence similarity to those of many other bacteria, it does not interact with other heterologous accessory proteins to enhance the urease activity. It still remains unclear whether the organism has unidentified non-homologous accessory gene(s) or if it lacks accessory genes in its genome so that its urease activates spontaneously.; The UreF accessory protein is poorly characterized because it is insoluble when ureF is overexpressed in E. coli. To produce a soluble form of UreF for biochemical and structural studies, the K. aerogenes UreEF fusion protein was generated by a translational fusion of ureE and ureF. The fusion protein was purified and biochemically characterized for oligomerization and metal binding properties. The UreF portion of the UreEF is fully functional on the basis of its interactions with other urease components and its ability to activate urease. In contrast, the function of the UreE portion in UreEF was greatly compromised because the fusion prevented its dimerization and altered its metal binding properties as opposed to the wild type UreE. Serial deletion mutant studies on the UreEF protein provided the first evidence for the existence of distinct sub-domains of UreF. Finally, I propose a model for UreF action in urease activation by combining the results from my studies and previous investigations in the lab.