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

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Copper is essential for life, but its transport and utilization in bacteria remain incompletely understood.
• The Gram-positive bacterium Bacillus subtilis possesses several copper-dependent proteins, yet mechanisms for copper recognition and trafficking are not fully elucidated.
• The ycnKJI operon in B. subtilis is known to encode proteins involved in copper homeostasis, including YcnI and YcnJ.

Purpose of the Study:

• To investigate the roles of YcnI and YcnJ in copper uptake and regulation in Bacillus subtilis.
• To characterize the copper-binding properties of the extracellular domain of YcnJ (YcnJ^CopC) and its interaction with YcnI.
• To elucidate the functional significance of these interactions for bacterial growth under copper-limited conditions.

Main Methods:

• Biochemical assays to determine the stoichiometry and affinity of Cu(II) binding by YcnJ^CopC.
• Protein exchange experiments between YcnJ^CopC and YcnI.
• Genetic studies involving gene knockouts and site-directed mutagenesis of YcnI and YcnJ.

Main Results:

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

Conclusions:

• Both YcnI and YcnJ possess functional Cu(II)-binding sites essential for efficient copper import in Bacillus subtilis under copper-limited conditions.
• A model is proposed where YcnI sequesters Cu(II) from YcnJ, regulating cytoplasmic copper entry and potentially storing copper extracellularly.
• This extracellular transfer of copper between membrane-bound proteins represents a potentially novel mechanism for bacterial copper management.