Copper acquisition in Bacillus subtilis involves Cu(II) exchange between YcnI and YcnJ

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Copper (Cu) is essential for life, but its transport and utilization in bacteria remain incompletely understood.
• The Gram-positive bacterium Bacillus subtilis possesses a ycnKJI operon encoding proteins involved in Cu homeostasis, including YcnI and YcnJ.
• Mechanisms of Cu recognition and intracellular trafficking to metalloproteins require further elucidation.

Purpose of the Study:

• To investigate the roles of YcnI and YcnJ in copper uptake and regulation in Bacillus subtilis.
• To characterize the interaction between YcnJ and YcnI in copper binding and transfer.
• To elucidate the functional significance of these interactions for bacterial growth under copper limitation.

Main Methods:

• Biochemical assays to determine Cu(II) binding stoichiometry and affinity of YcnJ extracellular domain (YcnJ^CopC).
• Investigation of Cu(II) exchange between YcnJ^CopC and YcnI.
• Genetic studies involving gene knockouts and site-directed mutagenesis to assess the impact on bacterial growth.

Main Results:

• The YcnJ^CopC domain binds Cu(II) with high affinity in a 1:1 ratio, utilizing a histidine brace motif.
• YcnJ^CopC and YcnI were shown to readily exchange Cu(II).
• Disruption of YcnI or YcnJ, or mutation of critical Cu-binding residues, resulted in impaired bacterial growth under copper-limited conditions.

Conclusions:

• Both YcnI and YcnJ contribute to efficient copper import in Bacillus subtilis, particularly under conditions of copper scarcity.
• A model is proposed where YcnI sequesters Cu(II) from YcnJ, regulating cytoplasmic entry and potentially storing copper extracellularly.
• This extracellular transfer mechanism between membrane-bound proteins suggests a novel paradigm for bacterial copper management.