Heme as an iron source and as a target for manganese-mediated regulation of iron metabolism in Bradyrhizobium japonicum
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Iron is required for a wide variety of biological functions but can be a limiting nutrient in most bacterial niches. Many bacteria can utilize heme as an alternative source of nutritional iron. However heme degrading enzymes called heme oxygenases (HOs) have been identified in only a few bacterial pathogens of animals. Non-pathogenic rhizobia including Bradyrhizobium japonicum can utilize heme as an iron source, but no potential HO candidates have been identified in their annotated genomes. Here, bioinformatics analyses of the B. japonicum genome identified two paralogous genes renamed hmuQ ( bll7075 ) and hmuD ( bll7423 ) that encode proteins with weak similarity to the heme-degrading monooxygenase IsdG from Staphylococcus aureus. The hmuQ gene is clustered with known heme transport genes in the genome. Recombinant HmuQ bound heme with a K d value of 0.8 μM, and showed spectral properties consistent with a HO. In the presence of a reductant, HmuQ catalyzed the degradation of heme and the formation of biliverdin. The hmuQ and hmuD genes complemented a Corynebacterium ulcerans heme oxygenase mutant in trans for utilization of heme as the sole iron source for growth. Furthermore, homologs of hmuQ and hmuD were identified in many bacterial genera, and the recombinant homolog from Brucella melitensis bound heme and catalyzed its degradation. These findings show that hmuQ and hmuD encode heme oxygenases, and indicate that the IsdG family of heme-degrading monooxygenases is not restricted to Gram positive pathogenic bacteria. In addition to being a source of iron, heme also serves an important signaling function that contributes to the control of iron homeostasis in B. japonicum. The iron response regulator (Irr) protein in this bacterium senses iron through the status of heme biosynthesis to globally regulate iron-dependent gene expression. Heme binds directly to Irr to trigger its degradation. Here, we show that severe manganese limitation created by growth of a Mn 2+ transport mutant in manganese limited media resulted in a cellular iron deficiency. In wild type cells, Irr levels were attenuated under manganese limitation, resulting in reduced promoter occupancy of target genes, and altered iron-dependent gene expression. Irr levels were high regardless of manganese availability in a heme-deficient mutant, indicating that manganese normally affects heme-dependent degradation of Irr. Manganese altered the secondary structure of Irr in vitro, and inhibited binding of heme to the protein. We propose that manganese limitation destabilizes Irr under low iron conditions by lowering the threshold of heme that can trigger Irr degradation. Thus, manganese controls iron homeostasis in B. japonicum by interfering with the signaling activity of the heme molecule.