Mark R. O'Brian

A whole genome view of prokaryotic haem biosynthesis.

Haem is the prosthetic group of proteins involved in many processes, and is also a regulatory molecule that mediates cellular responses to oxygen, iron and haem itself. Haem is synthesized in a multistep biosynthetic pathway with well-defined intermediates. Accordingly, the enzymes of the pathway are, to a large extent, conserved among prokaryotes and eukaryotes. Although individual haem biosynthesis genes and enzymes have been studied in many prokaryotic systems, only in few organisms has the entire pathway been examined. The availability of whole genome sequences allows an initial snapshot of the entire pathway at the genetic level in many diverse organisms. Unlike model systems, the genome represents a starting point for the detailed characterization of some unusual prokaryotes, and therefore examining haem synthesis genes can give important insight into core metabolism on which to lay the foundation for future experiments.

The bacterial irr protein is required for coordination of heme biosynthesis with iron availability.

Heme is a ubiquitous macromolecule that serves as the active group of proteins involved in many cellular processes. The multienzyme pathway for heme formation culminates with the insertion of iron into a protoporphyrin ring. The cytotoxicity of porphyrins suggests the need for coordination of its biosynthesis with iron availability. We isolated a mutant strain of the bacteriumBradyrhizobium japonicum that, under iron limitation, accumulated protoporphyrin and showed aberrantly high expression ofhemB, an iron-regulated gene encoding the heme synthesis enzyme δ-aminolevulinic acid dehydratase. The strain carries a loss of function mutation in irr, a newly described gene that encodes a putative member of the GntR family of bacterial transcriptional regulators. Irr accumulated only under iron limitation, and turned over rapidly upon an increase in iron availability. A separate role for Irr in controlling the cellular iron level was inferred based on a deficiency in high affinity iron transport activity in the irr strain, and suggests that regulation of the heme pathway is coordinated with iron homeostasis. A high level of protoporphyrin accumulation is not a normal consequence of nutritional iron deprivation, thus a mechanism for iron-dependent control of heme biosynthesis may be present in other organisms.

Interaction between the Bacterial Iron Response Regulator and Ferrochelatase Mediates Genetic Control of Heme Biosynthesis.

The heme biosynthetic pathway culminates with the insertion of iron into protoporphyrin catalyzed by ferrochelatase. The Bradyrhizobium japonicum iron response regulator (Irr) protein represses the pathway at an early step under iron limitation to prevent protoporphyrin synthesis from exceeding iron availability. Here, we show that Irr interacts directly with ferrochelatase and responds to iron via the status of heme and protoporphyrin localized at the site of heme synthesis. In the presence of iron, ferrochelatase inactivates Irr, followed by heme-dependent Irr degradation to derepress the pathway. Under iron limitation, protoporphyrin relieves the inhibition of Irr by ferrochelatase, probably by promoting protein dissociation, allowing genetic repression. Thus, metabolic control of the heme pathway involves a regulatory function of a biosynthetic enzyme to affect gene expression. Furthermore, heme can serve as a signaling molecule without accumulating freely in cells.

Bradyrhizobium japonicum senses iron through the status of haem to regulate iron homeostasis and metabolism

The Irr protein from the bacterium Bradyrhizobium japonicum is expressed under iron limitation to mediate iron control of haem biosynthesis. The regulatory input to Irr is the status of haem and its precursors iron and protoporphyrin at the site of haem synthesis. Here, we show that Irr controls the expression of iron transport genes and many other iron‐regulated genes not directly involved in haem synthesis. Irr is both a positive and negative effector of gene expression, and in at least some cases the control is direct. Loss of normal iron responsiveness of those genes in an irr mutant, as well as a lower total cellular iron content, suggests that Irr is required for the correct perception of the cellular iron status. Degradation of Irr in iron replete cells requires haem. Accordingly, control of Irr‐regulated genes by iron was aberrant in a haem‐defective strain, and iron replete mutant cells behave as if they are iron‐limited. In addition, the haem mutant had an abnormally high cellular iron content. The findings indicate that B. japonicum senses iron via the status of haem biosynthesis in an Irr‐dependent manner to regulate iron homeostasis and metabolism.

Fur-independent regulation of iron metabolism by Irr in Bradyrhizobium japonicum

Bradyrhizobium japonicum expresses both Fur and Irr, proteins that mediate iron-dependent regulation of gene expression. Control of irr mRNA accumulation by iron was aberrant in a fur mutant strain, and Fur repressed an irr::lacZ promoter fusion in the presence of iron. Furthermore, metal-dependent binding of Fur to an irr gene promoter was demonstrated in a region with no significant similarity to the Fur-binding consensus DNA element. These data suggest that the modest control of irr transcription by iron is mediated by Fur. However, Irr protein levels were regulated normally by iron in the fur strain, indicating that Fur is not required for post-transcriptional control of the irr gene. Accordingly, regulation of hemB, a haem biosynthesis gene regulated by Irr, was controlled normally by iron in a fur strain. In addition, the hemA gene was shown to be controlled by Fur, but not by Irr. It was concluded that Fur cannot be the only protein by which B. japonicum cells sense and respond to iron, and that Irr may be involved in Fur-independent signal transduction. Furthermore, iron-dependent regulation of haem biosynthesis involves both Irr and Fur.

Identification of a Soybean Protein That Interacts with GAGA Element Dinucleotide Repeat DNA

Dinucleotide repeat DNA with the pattern (GA)n/(TC)n, so-called GAGA elements, control gene expression in animals, and are recognized by a specific regulatory protein. Here, a yeast one-hybrid screen was used to isolate soybean (Glycine max) cDNA encoding a GAGA-binding protein (GBP) that binds to (GA)n/(CT)nDNA. Soybean GBP was dissimilar from the GAGA factor ofDrosophila melanogaster. Recombinant GBP protein did not bind to dinucleotide repeat sequences other than (GA)n/(CT)n. GBP bound to the promoter of the heme and chlorophyll synthesis gene Gsa1, which contains a GAGA element. Removal of that GAGA element abrogated binding of GBP to the promoter. Furthermore, insertion of the GAGA element to a nonspecific DNA conferred GBP-binding activity on that DNA. Thus, the GAGA element of the Gsa1 promoter is both necessary and sufficient for GBP binding. Gbp mRNA was expressed in leaves and was induced in symbiotic root nodules elicited by the bacterium Bradyrhizobium japonicum. In addition,Gbp transcripts were much higher in leaves of dark-treated etiolated plantlets than in those exposed to light for 24 h. Homologs of GBP were found in other dicots and in the monocot rice (Oryza sativa), as well. We suggest that interaction between GAGA elements and GBP-like proteins is a regulatory feature in plants.

Fur Is Involved in Manganese-Dependent Regulation of mntA (sitA) Expression in Sinorhizobium meliloti

ABSTRACT Fur is a transcriptional regulator involved in iron-dependent control of gene expression in many bacteria. In this work we analyzed the phenotype of a fur mutant in Sinorhizobium meliloti, an α-proteobacterium that fixes N2 in association with host plants. We demonstrated that some functions involved in high-affinity iron transport, siderophore production, and iron-regulated outer membrane protein expression respond to iron in a Fur-independent manner. However, manganese-dependent expression of the MntABCD manganese transport system was lost in a fur strain as discerned by constitutive expression of a mntA::gfp fusion reporter gene in the mutant. Thus, Fur directly or indirectly regulates a manganese-dependent function. The data indicate a novel function for a bacterial Fur protein in mediating manganese-dependent regulation of gene expression.

Oxidative stress promotes degradation of the Irr protein to regulate haem biosynthesis in Bradyrhizobium japonicum

The haem proteins catalase and peroxidase are stress response proteins that detoxify reactive oxygen species. In the bacterium Bradyrhizobium japonicum, expression of the gene encoding the haem biosynthesis enzyme δ‐aminolevulinic acid dehydratase (ALAD) is normally repressed by the Irr protein in iron‐limited cells. Irr degrades in the presence of iron, which requires haem binding to the protein. Here, we found that ALAD levels were elevated in iron‐limited cells of a catalase‐deficient mutant, which corresponded with aberrantly low levels of Irr. Irr was undetectable in wild‐type cells within 90 min after exposure to exogenous H2O2, but not in a haem‐deficient mutant strain. In addition, Irr did not degrade in response to iron in the absence of O2. The findings indicate that reactive oxygen species promote Irr turnover mediated by haem, and are involved in iron‐dependent degradation. We demonstrated Irr oxidation in vitro, which required haem, O2 and a reductant. A truncated Irr mutant unable to bind ferrous haem does not degrade in vivo, and was not oxidized in vitro. We suggest that Irr oxidation is a signal for its degradation, and that cells sense and respond to oxidative stress through Irr to regulate haem biosynthesis.

The hmuQ and hmuD Genes from Bradyrhizobium japonicum Encode Heme-Degrading Enzymes

Utilization of heme by bacteria as a nutritional iron source involves the transport of exogenous heme, followed by cleavage of the heme macrocycle to release iron. Bradyrhizobium japonicum can use heme as an iron source, but no heme-degrading oxygenase has been described. 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 microM and showed spectral properties consistent with a heme oxygenase. 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. The 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.

Biochemistry, regulation and genomics of haem biosynthesis in prokaryotes.

Haems are involved in many cellular processes in prokaryotes and eukaryotes. The biosynthetic pathway leading to haem formation is, with few exceptions, well-conserved, and is controlled in accordance with cellular function. Here, we review the biosynthesis of haem and its regulation in prokaryotes. In addition, we focus on a modification of haem for cytochrome c biogenesis, a complex process that entails both transport between cellular compartments and a specific thioether linkage between the haem moiety and the apoprotein. Finally, a whole genome analysis from 63 prokaryotes indicates intriguing exceptions to the universality of the haem biosynthetic pathway and helps define new frontiers for future study.