A Biochemical And Genetic Investigation Of Redox-Active Transition Metal Ion Homeostasis
Iron and copper are essential trace nutrients required for cell growth and proliferation. In excess, these metals pose a serious threat to the cell, either by interacting with oxygen species, generating free radicals, or by replacing other metal cofactors within the metal binding sites of enzymes. For these reasons, it is of vital importance that bacteria be able to tightly control the intracellular concentrations of both iron and copper. In Bacillus subtilis, a Gram positive model soil bacterium, this control is exerted via a number of uptake, efflux, and storage systems. Iron limitation is answered by means of a newly discovered sRNA mediated iron-sparing system. Using global analytical techniques, I identified the regulon of the iron-sparing response; mediated by the Fur regulated small RNA A (FsrA) and the Fur regulated basic proteins (FbpABC). In times of iron starvation, the mRNAs of iron binding targets are post-transcriptionally affected by their interaction with the iron-sparing response elements. Through this regulatory process, FsrA and its chaperones prioritize iron usage within the cell during iron starvation, ensuring that newly acquired iron is earmarked for essential functions. I also characterized the direct repression of a single target of the iron-sparing response, the iron-utilizing lactate oxidoreductase operon, lutABC. The regulation of this operon was shown to be dependent on the expression of FsrA as well as the translation of the RNA chaperone-like peptide, FbpB. The iron responsive regulation of the lut operon plays an important role in the cell's ability to grow and form biofilms on lactate as a carbon source in iron limiting environments. Finally, I characterized how the cell senses and responds to copper accumulation via the copper sensitive operon repressor, CsoR, and not the previously identified copper regulator YhdQ. I determined the high affinity binding of CsoR to a site overlapping the copper efflux operon, copZA, repressing its expression when copper is bound. The work presented here furthers our understanding of how B. subtilis regulates and responds to changing levels of iron and copper.
iron; copper; Fur; CsoR; FsrA; sparing response; regulation
Helmann, John D
Shapleigh, James P; Crane, Brian
Ph.D. of Biochemistry
Doctor of Philosophy
dissertation or thesis