HmuP Is a Coactivator of Irr-Dependent Expression of Heme Utilization Genes in Bradyrhizobium japonicum

Abstract

Utilization of heme as an iron source by Bradyrhizobium japonicum involves induction of the outer membrane heme receptor gene hmuR and other genes within the heme utilization locus. Here, we discovered the hmuP gene located upstream of hmuR and transcribed divergently from it along with hmuTUV. hmuP encodes a small protein that accumulated under iron limitation and is transcriptionally controlled by the global iron-responsive regulator Irr, as were all genes within the heme utilization locus. Cross-linking and immunoprecipitation experiments showed that Irr occupies the hmuR-hmuP promoter in vivo. An hmuP mutant did not grow on heme as an iron source, but retained the ability to use ferric chloride. Correspondingly, induction of hmuR mRNA under iron limitation was severely diminished in an hmuP strain, but other genes within the Irr regulon were unaffected. HmuP occupied the hmuR-hmuP promoter, and thus it plays a direct regulatory role in gene expression. HmuP was not required for Irr occupancy, nor was ectopic expression of hmuP from an Irr-independent promoter sufficient to induce the hmuR gene. Thus, both HmuP and Irr occupancy are necessary for hmuR induction. We suggest that HmuP is a coactivator of Irr-dependent expression of hmuR.

The Small Protein HemP Is a Transcriptional Activator for the Hemin Uptake Operon in Burkholderia multivorans ATCC 17616

“…3), the HemP protein most likely regulates at least the expression of other, currently unidentified genes (other than the hmu operon) for alternative hemin uptake systems. The presence of such predicted alternative systems is unlikely in the two alphaproteobacterial species and B. pseudomallei, because their derivatives lacking the identified heme-receptor-encoding genes demonstrated the loss of heme-utilizing ability (25)(26)(27). To our knowledge, the predicted multiple regulatory roles of HemP in ATCC 17616 represent a novel property that is not observed for the other HemP superfamily proteins.…”

“…The HmuP proteins from Bradyrhizobium japonicum and Sinorhizobium meliloti are transcriptional activators for the genes encoding outer membrane heme receptors in the heme uptake system (26,27). Although the overall amino acid sequence similarity of the two proteins with the ATCC 17616 HemP protein is approximately 30% ( Fig.…”

“…If this is also the case in vivo, then the HemP-heme complex might contribute to modulating its interaction with the hmuRupstream region so as to more effectively regulate transcription of the hmu operon. In spite of the ability of HemP to bind to its target DNA sequence, no canonical DNAbinding helix-turn-helix motifs were predicted in HemP, and this was also the case with other HemP superfamily proteins, including the HmuP proteins from the two alphaproteobacterial species (26,27). Three-dimensional structure modeling of the S. meliloti HmuP protein has predicted that it consists of three ␤-sheets and one small ␣-helix, probably with a dimer form, and that the KLILXK motif is located within the C-terminal ␤-sheet (26).…”

“…S1B) (26,27). Despite of the high level of conservation of these proteins among various proteobacteria, their experimental characterization has been limited to the HmuP proteins from S. meliloti and B. japonicum, and both proteins are required for heme uptake (26,27).…”

“…S1B) (26,27). Despite of the high level of conservation of these proteins among various proteobacteria, their experimental characterization has been limited to the HmuP proteins from S. meliloti and B. japonicum, and both proteins are required for heme uptake (26,27). Each HumP protein is a transcriptional activator for the gene encoding the outer membrane receptor protein for heme uptake but not for the hmuP-containing operon that encodes the remaining proteins for the heme uptake system (Fig.…”