Bacillus subtilis aconitase is required for efficient late-sporulation gene expression

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Bacillus subtilis aconitase (CitB) is a bifunctional protein with enzymatic and RNA-binding activities, similar to eukaryotic iron regulatory protein 1 (IRP-1).
• Aconitase's dual role suggests complex regulatory functions beyond its canonical metabolic pathway involvement.

Purpose of the Study:

• To investigate the specific roles of Bacillus subtilis aconitase's enzymatic and RNA-binding activities.
• To elucidate the function of aconitase in bacterial sporulation, particularly its impact on gene expression.

Main Methods:

• Site-directed mutagenesis of the C-terminal region of the Bacillus subtilis citB gene to separate aconitase activities.
• Analysis of sporulation defects and gene expression (sigmaK-dependent genes, gerE mRNA, GerE protein) in the mutant strain.
• In vitro gel mobility shift assays using purified Bacillus subtilis aconitase and gerE mRNA.

Main Results:

• Mutagenized Bacillus subtilis exhibited high catalytic activity but a defect in late-stage sporulation.
• The sporulation defect was linked to delayed accumulation of gerE mRNA and GerE protein, crucial for spore coat assembly.
• Purified aconitase demonstrated direct binding to the 3' untranslated region of gerE mRNA.

Conclusions:

• The RNA-binding activity of Bacillus subtilis aconitase is critical for stabilizing gerE mRNA.
• This stabilization is essential for efficient GerE protein synthesis, ensuring proper timing of spore coat assembly during sporulation.
• Aconitase's RNA-binding function highlights a novel regulatory mechanism in bacterial sporulation.