Storing copper in Streptomyces lividans: structural and biochemical properties of a copper storage protein

Copper (Cu) is essential to the growth and the morphological development of Streptomyces lividans. Understanding how Cu regulates the key development switches in this Gram-positive bacterium has been an area of extensive research. In particular, how Cu homeostasis and regulation are controlled and how metalation of enzymes important for morphological development is achieved have been previously investigated. To this end, this thesis reports the discovery of a cytosolic copper storage protein in S. lividans and offers new insight into intracellular Cu regulation, which is not under direct control of the Cu regulator protein CsoR (copper sensitive operon repressor). This copper storage protein belongs to a family of recently discovered cytosolic proteins known as Csp3. These members are exclusively found in the bacterial cytosol and comprise of a four-helix bundle that assemble into homotetramers and can bind between 70-80 Cu(I) ions through mainly Cys thiolate coordination. In Chapter 2 bioinformatic analyses reveals the phylogenetic distribution of Csp3 across Bacteria and Archaea and confirms the presence of Csp3 in S. lividans. Furthermore, the Csp3 in S. lividans is located in a gene environment that is sensitive to elevated Cu levels. Taxonomic distribution of these genes reveals a possible link to a novel transmembrane Cu export system that could facilitate removal of Cu from Csp3. X-ray structures of the apo and Cu(I) bound forms of the Csp3 from S. lividans have been determined and confirm a homotetramer assembly that can bind up to 80 Cu(I) ions (Chapter 3). The binding of Cu(I) ions in Csp3 is found to be cooperative with a Hill coefficient of 1.9 and Cu(I) can be transferred to Csp3 from a CopZ-like Cu(I) chaperone (Chapter 3). A Δcsp3 null-mutant in S. lividans reveals that Csp3 is operable at high Cu levels and this suggests it acts to provide an additional level of protection against Cu toxicity once the CsoR system becomes saturated (Chapter 3). The mechanism of Cu(I)-loading to Csp3 has also been investigated through X-ray crystallography, site-directed mutagenesis and stopped-flow reaction kinetics using aqueous Cu(I) and Cu(I) chelated by a donor. A clear role for a His residue (His107) leading to the formation of a tetranuclear [Cu4(μ2-S-Cys)4(Nδ1-His)] cluster is observed, followed by the loading of Cu(I) in a fluxional and dynamic manner (Chapters 4 and 5). Finally, over-expression studies of a putative transmembrane protein (SLI_RS17250) that is encoded by a neighbouring gene to the S. lividans Csp3 gene and could be part of a novel Cu export system, identified in Chapter 2, is described (Chapter 6).