Cytosolic Copper Binding by a Bacterial Storage Protein and Interplay with Copper Efflux

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Escherichia coli possesses a copper (Cu) transporting ATPase (CopA) to detoxify the cytosol.
• The absence of CopA (ΔcopA strain) inhibits E. coli growth above 0.5 mM Cu, unlike wild-type (WT) E. coli, which tolerates up to 2.5 mM Cu.
• Bacterial cytosolic copper storage proteins (Csp3) are known to bind copper ions.

Purpose of the Study:

• To investigate the influence of Bacillus subtilis copper storage protein (BsCsp3) on copper handling in E. coli.
• To assess the protective effect of BsCsp3 against copper toxicity in both WT and ΔcopA E. coli strains.

Main Methods:

• Expression of BsCsp3 in WT and ΔcopA E. coli strains.
• Growth assays of E. coli strains in the presence of varying copper concentrations.
• Purification and copper-binding analysis of BsCsp3 from E. coli.

Main Results:

• BsCsp3 significantly enhances copper tolerance in both WT and ΔcopA E. coli, allowing growth at higher copper concentrations (~1.5 mM and ~3.5 mM, respectively).
• Higher BsCsp3 expression levels are required for copper resistance in the ΔcopA strain compared to the WT strain.
• Purified BsCsp3 binds significant amounts of Cu(I) (up to ~4 equivalents per monomer), indicating its role in copper sequestration.

Conclusions:

• BsCsp3 effectively protects E. coli from copper toxicity by sequestering cuprous ions in the cytosol.
• Csp3 can acquire and retain Cu(I) even in the absence of the primary copper efflux system (CopA) in a heterologous host.
• This study highlights the potential of Csp3 as a tool for managing copper homeostasis in bacteria.