Control of ferric iron acquisition and reduction by Irr in the bacterium Bradyrhizobium japonicum
Small, Sandra Kay
MetadataShow full item record
Iron utilization by bacteria in aerobic environments involves uptake as a ferric chelate from the environment by ferric siderophore receptors, followed by reduction to the ferrous form. Ferric siderophore receptors are components of high-affinity iron-chelate transport systems in gram-negative bacteria. Here, we identified the genes encoding the outer membrane receptors for ferrichrome, pyoverdine PL-9, and rhodotorulic acid in Bradyrhizobium japonicum . These genes, as well as two additional putative ferric siderophore receptor genes, are positively controlled by the regulatory protein Irr, as observed by the low level of mRNA transcripts in an irr mutant in iron-limited cells. Potential Irr binding sites with iron control element (ICE)-like motifs were found upstream and distal to the transcription start sites of the five receptor genes. However, purified recombinant Irr bound only some of those elements. Nevertheless, dissection of the ferrichrome gene promoter region showed that a component in extracts of wild type cells grown in iron limited media bound only in the ICE motif region of the promoter. This binding was not observed with extracts of cells from the parent strain grown under high iron, or from an irr mutant strain. Furthermore, gel mobility supershift experiments identified Irr as the binding protein in cell extracts. In vivo crosslinking and immunoprecipitation experiments demonstrated that Irr occupies the promoters of the five ferric iron transport genes in vivo . Following internalization of the siderophore, the iron is presumed to be reduced, though ferric iron reduction is poorly understood in most bacterial species. We identified Bradyrhizobium japonicum frcB ( bll3557 ) as a gene adjacent to, and co-regulated with the ferric-pyoverdine receptor gene. FrcB is a membrane-bound, di-heme protein, as is found for eukaryotic ferric reductases. Heme was essential for FrcB stability, as were conserved histidine residues in the protein that likely coordinate the heme moieties. Expression of the frcB gene in E. coli conferred ferric reductase activity on those cells. In addition, purified recombinant FrcB was oxidized by iron, which is an aspect of ferric reductase activity. Furthermore, B. japonicum cells showed elevated ferric reductase activity in iron-limited cells that was diminished in an frcB mutant. Steady state levels of frcB mRNA were strongly induced under iron limitation, but transcript levels were low and unresponsive to iron in an irr mutant. In addition, Irr occupied the frcB promoter in vivo in iron-limited cells as determined by cross-linking and immunoprecipitation experiments, showing that Irr is a direct positive regulator of the frcB gene. FrcB belongs to a family of proteins found in many Proteobacteria and some cyanobacteria. This suggests that membrane-bound, heme-containing ferric reductase proteins are not confined to eukaryotes, but rather they may be common in bacteria. Moreover, we conclude that Irr coordinates ferric iron transport and assimilation with iron availability B. japonicum .