The Bradyrhizobium japonicum Irr protein is a transcriptional repressor with high-affinity DNA-binding activity

The Irr protein is a global regulator of iron homeostasis in Bradyrhizobium japonicum, and a subset of genes within the Irr regulon are negatively controlled under iron limitation. However, repressor function, highaffinity DNA binding in vitro, or promoter occupancy in vivo of Irr for a negatively regulated gene has not been demonstrated. Here, we show that the blr7895 and bll6680 genes are negatively regulated by Irr as determined by derepression of transcript levels in iron-limited cells of an irr mutant strain. Electrophoretic gel mobility shift analysis showed that a component in extracts of wild-type cells grown under iron limitation bound the iron control elements (ICE) within the promoters of blr7895 and b116680 identified previously (G. Rudolph, G. Semini, F. Hauser, A. Lindemann, M. Friberg, H. Hennecke, and H. M. Fischer, J. Bacteriol. 188:733-744, 2006). Binding was not observed with extracts of cells from the parent strain grown under high iron conditions or with those from an irr mutant. Furthermore, gel mobility supershift experiments identified Irr as a component of the binding complex. Purified recombinant Irr bound to ICE DNA with high affinity in the presence of divalent metal, with [K.sub.d] values of 7 to 19 nM, consistent with a physiological role for Irr as a transcriptional regulator. In addition, in vitro transcription initiated from the blr7895 promoter was inhibited by Irr. Whole-cell cross-linking and immunoprecipitation experiments showed that Irr occupies the promoters of blr7895 and b116680 in vivo in an iron-dependent manner. The findings demonstrate that Irr is a transcriptional repressor that binds DNA with high affinity.