Dimeric Brucella abortus Irr protein controls its own expression and binds haem

Area of Science:

• Microbiology
• Bacterial Pathogenesis
• Molecular Biology

Background:

• Heme synthesis is crucial for Brucella abortus intracellular replication and virulence.
• Iron availability significantly impacts bacterial pathogenesis.
• Iron response regulator (Irr) proteins coordinate heme biosynthesis and iron homeostasis in related bacteria.

Purpose of the Study:

• To identify and characterize regulatory proteins involved in the heme biosynthetic pathway of Brucella abortus.
• To investigate the role of an iron response regulator (Irr) orthologue in Brucella abortus.
• To understand the regulation of heme synthesis in response to iron availability.

Main Methods:

• Bioinformatic search for irr orthologue gene in Brucella abortus.
• Gene cloning, sequencing, and disruption to create a null mutant.
• Phenotypic analysis of the mutant under iron deprivation.
• Complementation studies using a plasmid with the wild-type irr gene.
• Biochemical characterization of purified recombinant Brucella abortus Irr.
• In vivo expression analysis and transcriptional regulation studies using lacZ fusion.

Main Results:

• A Brucella abortus irr gene orthologue was identified and cloned.
• A null mutant accumulated protoporphyrin IX under iron deprivation, indicating impaired heme synthesis.
• The null mutant phenotype was rescued by complementation with the wild-type irr gene.
• Purified recombinant Brucella abortus Irr formed a stable dimer and bound heme.
• Irr protein was detected in vivo only under iron-limited conditions.
• Brucella abortus Irr downregulated its own transcription, and iron did not directly regulate irr transcription.

Conclusions:

• Brucella abortus Irr is a homodimeric protein that accumulates in iron-limited cells.
• Brucella abortus Irr regulates its own transcription.
• Irr downregulates the biosynthesis of heme precursors in Brucella abortus, linking iron availability to heme synthesis.