Identification of amino acid residues important for the function of Agrobacterium tumefaciens Irr protein

Abstract

The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (Irr At ) were investigated. Several Irr At mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) ge ne was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of Irr At . Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of Irr At . H94 is essential for the iron responsiveness of Irr At . Furthermore, the Irr At mutant proteins showed differential abilities to complement the H 2 O 2 -hyper-resistant phenotype of an irr mutant. © 2012 Federation of European Microbiological Societies.