The yeast Saccharomyces cerevisiae contains a plasma membrane reductase activity associated with the gene product of the FRE1 locus. This reductase is required for Fe(III) uptake by this yeast; transcription from FRE1 is repressed by iron (Dancis, A., Klausner, R. D., Hinnebusch, A. G., and Barriocanal, J. G.(1990) Mol. Cell. Biol. 10, 2294-2301). We show here that Cu(II) is equally efficient at repressing FRE1 transcription and is an excellent substrate for the Fre1p reductase. This reductase activity is required for 50-70% of the uptake of 64Cu by wild type cells. Under conditions of low Fre1-dependent activity, cells retain 30-70% of Cu(II) reductase activity but only 8-25% of Fe(III) reductase activity. While Fre1p-dependent activity is 100% inhibitable by Pt(II), this residual Cu(II) reduction is insensitive to this inhibitor. The data suggest the presence of a Fre1p-independent reductase activity in the yeast plasma membrane which is relatively specific for Cu(II) and which supports copper uptake in the absence of FRE1 expression. The gene product of MAC1, which is required for regulation of FRE1 transcription, is also required for expression of Cu(II) reduction activity. This is due in part to its role in the regulation of FRE1; however, it is required for expression of the putative Cu(II) reductase, as well. Similarly, a gain-of-function mutation, MAC1up1, which causes elevated and unregulated transcription from FRE1 and elevated Fe(III) reduction and 59Fe uptake exhibits a similar phenotype with respect to Cu(II) reduction and 64Cu uptake. Ascorbate, which reduces periplasmic Cu(II) to Cu(I), suppresses the dependence of 64Cu uptake on plasma membrane reductase activity as is the case for ascorbate-supported 59Fe uptake. The close parallels between Cu(II) and Fe(III) reduction, and 64Cu and 59Fe uptake, strongly suggest that Cu(II) uptake by yeast involves a Cu(I) intermediate. This results in the reductive mobilization of the copper from periplasmic chelating agents, making the free ion available for translocation across the plasma membrane. The yeast Saccharomyces cerevisiae contains a plasma membrane reductase activity associated with the gene product of the FRE1 locus. This reductase is required for Fe(III) uptake by this yeast; transcription from FRE1 is repressed by iron (Dancis, A., Klausner, R. D., Hinnebusch, A. G., and Barriocanal, J. G.(1990) Mol. Cell. Biol. 10, 2294-2301). We show here that Cu(II) is equally efficient at repressing FRE1 transcription and is an excellent substrate for the Fre1p reductase. This reductase activity is required for 50-70% of the uptake of 64Cu by wild type cells. Under conditions of low Fre1-dependent activity, cells retain 30-70% of Cu(II) reductase activity but only 8-25% of Fe(III) reductase activity. While Fre1p-dependent activity is 100% inhibitable by Pt(II), this residual Cu(II) reduction is insensitive to this inhibitor. The data suggest the presence of a Fre1p-independent reductase activity in the yeast plasma membrane which is relatively specific for Cu(II) and which supports copper uptake in the absence of FRE1 expression. The gene product of MAC1, which is required for regulation of FRE1 transcription, is also required for expression of Cu(II) reduction activity. This is due in part to its role in the regulation of FRE1; however, it is required for expression of the putative Cu(II) reductase, as well. Similarly, a gain-of-function mutation, MAC1up1, which causes elevated and unregulated transcription from FRE1 and elevated Fe(III) reduction and 59Fe uptake exhibits a similar phenotype with respect to Cu(II) reduction and 64Cu uptake. Ascorbate, which reduces periplasmic Cu(II) to Cu(I), suppresses the dependence of 64Cu uptake on plasma membrane reductase activity as is the case for ascorbate-supported 59Fe uptake. The close parallels between Cu(II) and Fe(III) reduction, and 64Cu and 59Fe uptake, strongly suggest that Cu(II) uptake by yeast involves a Cu(I) intermediate. This results in the reductive mobilization of the copper from periplasmic chelating agents, making the free ion available for translocation across the plasma membrane.
The Fet3 protein (Fet3p) is a multinuclear copper oxidase essential for high-affinity iron uptake in yeast. Fet3p contains one type 1, one type 2, and a strongly antiferromagnetically coupled binuclear Cu(II)−Cu(II) type 3 copper. The type 2 and type 3 sites constitute a structurally distinct trinuclear cluster at which dioxygen is reduced to water. In Fet3p, as in ceruloplasmin, Fe(II) is oxidized to Fe(III) at the type 1 copper; this is the ferroxidase reaction that is fundamental to the physiologic function of these two enzymes. Using site-directed mutagenesis, we have generated type 1-depleted (T1D), type 2-depleted (T2D), and T1D/T2D mutants. None were active in the essential ferroxidase reaction catalyzed by Fet3p. However, the spectroscopic signatures of the remaining Cu(II) sites in any one of the three mutants were indistinguishable from those exhibited by the wild type. Although the native protein and the T1D mutant were isolated in the completely oxidized Cu(II) form, the T2D and T1D/T2D mutants were found to be completely reduced. This result is consistent with the essential role of the type 2 copper in dioxygen turnover, and with the suggestions that cuprous ion is the valence state of intracellular copper. Although stable to dioxygen, the Cu(I) sites in both proteins were readily oxidized by hydrogen peroxide. The double mutant was extensively analyzed by X-ray absorption spectroscopy. Edge and near-edge features clearly distinguished the oxidized from the reduced form of the binuclear cluster. EXAFS was strongly consistent with the expected coordination of each type 3 copper by three histidine imidazoles. Also, copper scattering was observed in the oxidized cluster along with scattering from a ligand corresponding to a bridging oxygen. The data derived from the reduced cluster indicated that the bridge was absent in this redox state. In the reduced form of the double mutant, an N/O ligand was apparent that was not seen in the reduced form of the T1D protein. This ligand in T1D/T2D could be either the remaining type 2 copper imidazole ligand (from His416) or a water molecule that could be stabilized at the type 3 cluster by H-bonding to this side chain. If present in the native protein, this H2O could provide acid catalysis of dioxygen reduction at the reduced trinuclear center.
Abstract A saturable and temperature-dependent copper uptake pathway has been identified in Chlamydomonas reinhardtii. The uptake system has a high affinity for copper ions (Km approximately 0.2 [mu]M) and is more active in cells that are adapted to copper deficiency than to cells grown in a medium containing physiological (submicromolar to micromolar) copper ion concentrations. The maximum velocity of copper uptake by copper-deficient cells (169 pmol h-1 106 cells-1 or 62 ng min-1 mg-1 chlorophyll) is up to 20-fold greater than that of fully copper-supplemented cells, and the Km (approximately 2 x 102 nM) is unaffected. Thus, the same uptake system appears to operate in both copper-replete and copper-deficient cells, but its expression or activity must be induced under copper-deficient conditions. A cupric reductase activity is also increased in copper-deficient compared with copper-sufficient cells. The physiological characteristics of the regulation of this cupric reductase are compatible with its involvement in the uptake path-way. Despite the operation of the uptake pathway under both copper-replete and copper-deficient conditions, C. reinhardtii cells maintained in fully copper-supplemented cells do not accumulate copper in excess of their metabolic need. These results provide evidence for a homeostatic mechanism for copper metabolism in C. reinhardtii.
Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomyces cerevisiae. Fet3p is functionally homologous to ceruloplasmin (Cp) in that both are ferroxidases. However, by sequence homology Fet3p is more similar to fungal laccase, and both contain a type 1 Cu site that lacks the axial methionine ligand present in the functional type 1 sites of Cp. To determine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circular dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-depleted mutants. The spectroscopic features of the type 1 Cu site of Fet3p are nearly identical to those of fungal laccase, indicating a very similar three-coordinate geometry. We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics. From poised potential redox titrations, the E degrees of the type 1 Cu site is 427 mV, which is low for a three-coordinate type 1 Cu site. The kinetics of reduction of the type 1 Cu sites of four different multicopper oxidases with two different substrates were compared. The type 1 site of a plant laccase (Rhus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky organic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.029 and 0.013 s(-)(1), respectively). On the other hand, the type 1 site of a fungal laccase (Coprinus cinereus) is reduced very rapidly by both substrates (k(obs) > 23 s(-)(1)). In contrast, both Fet3p and Cp are rapidly reduced by Fe(II) (k(obs) > 23 s(-)(1)), but only very slowly by 1,4-hydroquinone (10- and 100-fold more slowly than plant laccase, respectively). Semiclassical theory is used to analyze the origin of these differences in reactivity in terms of type 1 Cu site accessibility to specific substrates.
The plasma-membrane of Saccharomyces cerevisiae contains high affinity permeases for Cu(I) and Fe(II). A low affinity Fe(II) permease has also been identified, designated Fet4p. A corresponding low affinity copper permease has not been characterized, although yeast cells that lack high affinity copper uptake do accumulate this metal ion. We demonstrate in the present study that Fet4p can function as a low affinity copper permease. Copper is a non-competitive inhibitor of (55)Fe uptake through Fet4p (K(i)=22 microM). Fet4p-dependent (67)Cu uptake was kinetically characterized, with K(m) and V(max) values of 35 microM and 8 pmol of copper/min per 10(6) cells respectively. A fet4-containing strain exhibited no saturable, low affinity copper uptake indicating that this uptake was attributable to Fet4p. Mutant forms of Fet4p that exhibited decreased efficiency in (55/59)Fe uptake were similarly compromised in (67)Cu uptake, indicating that similar amino acid residues in Fet4p contribute to both uptake processes. The copper taken into the cell by Fet4p was metabolized similarly to the copper taken into the cell by the high affinity permease, Ctr1p. This was shown by the Fet4p-dependence of copper activation of Fet3p, the copper oxidase that supports high affinity iron uptake in yeast. Also, copper-transported by Fet4p down-regulated the copper sensitive transcription factor, Mac1p. Whether supplied by Ctr1p or by Fet4p, an intracellular copper concentration of approx. 10 microM caused a 50% reduction in the transcriptional activity of Mac1p. The data suggest that the initial trafficking of newly arrived copper in the yeast cell is independent of the copper uptake pathway involved, and that this copper may be targeted first to a presumably small 'holding' pool prior to its partitioning within the cell.
ABSTRACT The recent identification of the iron response regulator (Irr) in Bradyrhizobium japonicum raised the question of whether the global regulator Fur is present in that organism. A fur gene homolog was isolated by the functional complementation of an Escherichia coli fur mutant. The B. japonicum Fur bound to a Fur box DNA element in vitro, and a fur mutant grown in iron-replete medium was derepressed for iron uptake activity. Thus, B. japonicum expresses at least two regulators of iron metabolism.
The plasma-membrane of Saccharomycescerevisiae contains high affinity permeases for Cu(I) and Fe(II). A low affinity Fe(II) permease has also been identified, designated Fet4p. A corresponding low affinity copper permease has not been characterized, although yeast cells that lack high affinity copper uptake do accumulate this metal ion. We demonstrate in the present study that Fet4p can function as a low affinity copper permease. Copper is a non-competitive inhibitor of 55Fe uptake through Fet4p (Ki = 22µM). Fet4p-dependent 67Cu uptake was kinetically characterized, with Km and Vmax values of 35µM and 8pmol of copper/min per 106 cells respectively. A fet4-containing strain exhibited no saturable, low affinity copper uptake indicating that this uptake was attributable to Fet4p. Mutant forms of Fet4p that exhibited decreased efficiency in 55/59Fe uptake were similarly compromised in 67Cu uptake, indicating that similar amino acid residues in Fet4p contribute to both uptake processes. The copper taken into the cell by Fet4p was metabolized similarly to the copper taken into the cell by the high affinity permease, Ctr1p. This was shown by the Fet4p-dependence of copper activation of Fet3p, the copper oxidase that supports high affinity iron uptake in yeast. Also, copper-transported by Fet4p down-regulated the copper sensitive transcription factor, Mac1p. Whether supplied by Ctr1p or by Fet4p, an intracellular copper concentration of approx. 10µM caused a 50% reduction in the transcriptional activity of Mac1p. The data suggest that the initial trafficking of newly arrived copper in the yeast cell is independent of the copper uptake pathway involved, and that this copper may be targeted first to a presumably small 'holding'pool prior to its partitioning within the cell.
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. 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. Heme carries out a wide range of biological functions in prokaryotes and eukaryotes. Heme has long been known to be essential for respiration, oxygen metabolism, and electron transfer as the prosthetic group of hemoglobins, hydroxylases, catalases, peroxidases, and cytochromes. More recently, roles for heme as a biosensor of diatomic gases (1Gilles-Gonzalez M.A. Ditta G.S. Helinski D.R. Nature. 1991; 350: 170-172Crossref PubMed Scopus (417) Google Scholar, 2Lowenstein C.J. Snyder S.H. Cell. 1992; 70: 705-707Abstract Full Text PDF PubMed Scopus (739) Google Scholar, 3Verma A. Hirsch D.J. Glatt C.E. Ronnett G.V. Snyder S.H. Science. 1993; 259: 381-384Crossref PubMed Scopus (1377) Google Scholar) and as a modulator of protein activity (4Mendez R. Moreno A. de Haro C. J. Biol. Chem. 1992; 267: 11500-11507Abstract Full Text PDF PubMed Google Scholar, 5Lathrop J.T. Timko M.P. Science. 1993; 259: 522-525Crossref PubMed Scopus (246) Google Scholar, 6Zhang L. Guarente L. Genes Dev. 1994; 8: 2110-2119Crossref PubMed Scopus (88) Google Scholar) have been described. The biosynthesis of protoheme involves seven sequential enzymatic steps from the first universal heme precursor ALA 1The abbreviations used are: ALAδ-aminolevulinic acidMOPS4-morpholinepropanesulfonic acidPAGEpolyacrylamide gel electrophoresisbpbase pair(s)kbkilobase pairs. ; other cellular hemes arise from modifications of protoheme. Ferrous iron is inserted into protoporphyrin IX in the final step of heme biosynthesis catalyzed by ferrochelatase. Synthesis of heme presents several regulatory problems because it must be controlled in accordance with cellular function and coordinated with cellular heme levels (5Lathrop J.T. Timko M.P. Science. 1993; 259: 522-525Crossref PubMed Scopus (246) Google Scholar, 7Hamilton J.W. Bement W.J. Sinclair P.R. Sinclair J.F. Alcedo J.A. Wetterhahn K.E. Arch. Biochem. Biophys. 1991; 289: 387-392Crossref PubMed Scopus (105) Google Scholar), hemoprotein apoprotein formation (4Mendez R. Moreno A. de Haro C. J. Biol. Chem. 1992; 267: 11500-11507Abstract Full Text PDF PubMed Google Scholar, 6Zhang L. Guarente L. Genes Dev. 1994; 8: 2110-2119Crossref PubMed Scopus (88) Google Scholar, 8Schilke B.A. Donahue T.J. J. Bacteriol. 1995; 177: 1929-1937Crossref PubMed Google Scholar), and other factors associated with cellular differentiation (4Mendez R. Moreno A. de Haro C. J. Biol. Chem. 1992; 267: 11500-11507Abstract Full Text PDF PubMed Google Scholar, 9Page K.M. Guerinot M.L. J. Bacteriol. 1995; 177: 3979-3984Crossref PubMed Google Scholar, 10Chauhan S. O'Brian M.R. J. Bacteriol. 1997; 179: 3706-3710Crossref PubMed Google Scholar). δ-aminolevulinic acid 4-morpholinepropanesulfonic acid polyacrylamide gel electrophoresis base pair(s) kilobase pairs. The cytotoxicity of porphyrins has been readily demonstrated in animals (11Kappas A. Sassa S. Galbraith R.A. Nordman Y. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Stanbury J.B. Wyngaarden J.B. Frederickson D.S. The Metabolic and Molecular Bases of Inherited Disease. 3. McGraw-Hill, New York1995: 2103-2159Google Scholar), plants (12Mock H.-P. Grimm B. Plant Physiol. 1997; 113: 1101-1112Crossref PubMed Scopus (84) Google Scholar), and bacteria (13Nakahigashi K. Nishimura K. Miyamoto K. Inokuchi H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10520-10524Crossref PubMed Scopus (61) Google Scholar), which is caused by their ability to catalyze light-dependent formation of reactive oxygen species. The pathological consequences of abnormal porphyrin accumulation in humans ranges from light sensitivity to neurological disorders (11Kappas A. Sassa S. Galbraith R.A. Nordman Y. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Stanbury J.B. Wyngaarden J.B. Frederickson D.S. The Metabolic and Molecular Bases of Inherited Disease. 3. McGraw-Hill, New York1995: 2103-2159Google Scholar). Iron must be acquired exogenously and may be a limiting nutrient, thus a prima facie argument can be made for the coordination of the heme pathway with the cellular iron level to prevent protoporphyrin synthesis from exceeding iron availability. This problem has been partially addressed in mammalian erythroid cells, where the iron-regulatory proteins (reviewed in Refs. 14Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1132) Google Scholar and 15Rouault T.A. Klausner R.D. Trends Biochem. Sci. 1996; 21: 174-177Abstract Full Text PDF PubMed Scopus (232) Google Scholar) inhibit translation of mRNA encoding the heme synthesis enzyme ALA synthase under low iron conditions (16Melefors O. Goossen B. Johansson H.E. Stripecke R. Gray N.K. Hentze M.W. J. Biol. Chem. 1993; 268: 5974-5978Abstract Full Text PDF PubMed Google Scholar, 17Bhasker C.R. Burgiel G. Neupert B. Emery-Goodman A. Kuhn L.C. May B.K. J. Biol. Chem. 1993; 268: 12699-12705Abstract Full Text PDF PubMed Google Scholar). However, it has not been established that this mechanism is sufficient to regulate the heme pathway as a whole or that it prevents excess protoporphyrin synthesis under iron limitation. Similarly, little is known about regulation of the heme pathway by iron in prokaryotes, and this is addressed in the present study. Bradyrhizobium japonicum is a good system for studying heme biosynthesis because its ability to live either as a free-living cell or as a nitrogen-fixing endosymbiont of soybean necessitates the expression and regulation of diverse systems for respiratory metabolism (18Sangwan I. O'Brian M.R. Science. 1991; 251: 1220-1222Crossref PubMed Scopus (30) Google Scholar, 19O'Brian M.R. J. Bacteriol. 1996; 178: 2471-2478Crossref PubMed Google Scholar). Expression of B. japonicum hemA and hemB, the genes encoding ALA synthase and ALA dehydratase, respectively, are positively affected by iron (20Page K.M. Connolly E.L. Guerinot M.L. J. Bacteriol. 1994; 176: 1535-1538Crossref PubMed Google Scholar,21Chauhan S. Titus D.E. O'Brian M.R. J. Bacteriol. 1997; 179: 5516-5520Crossref PubMed Google Scholar). hemB is particularly responsive, with mRNA and protein levels varying over 80-fold as a function of the iron concentration in which the cells are grown (21Chauhan S. Titus D.E. O'Brian M.R. J. Bacteriol. 1997; 179: 5516-5520Crossref PubMed Google Scholar). In the present study, we show that loss of function of a new gene called irruncouples heme biosynthesis from iron availability, thereby demonstrating that the pathway as a whole is regulated by iron, and providing insight into the mechanism of control. Furthermore, this regulation is integrated with iron homeostasis. B. japonicum LO and I110 were the parent strains used in the present work. Both strains were analyzed because strain I110 is the most commonly used strain, but strain LO can be mutagenized with Tn5 with higher efficiency. Strain LODTM5 is a mutant derivative of strain LO that contains a transposon Tn5 within the irr gene. Strain I110ek4 is a hemH mutant constructed as described previously (22Frustaci J.M. O'Brian M.R. J. Bacteriol. 1992; 174: 4223-4229Crossref PubMed Google Scholar). The B. japonicum strains were routinely grown at 29 °C in GSY medium (23Frustaci J.M. Sangwan I. O'Brian M.R. J. Bacteriol. 1991; 173: 1145-1150Crossref PubMed Google Scholar). The medium was supplemented with kanamycin and streptomycin at 100 and 50 μg/ml for solid and liquid cultures, respectively, for strain LODTM5. Strain I110ek4 was grown with kanamycin and 15 μm hemin to fulfill its heme auxotrophy. Escherichia coli strains DH5α or XL1-Blue were used for propagation of plasmids and were grown in LB medium containing 100 μg/ml ampicillin. Strain LO was mutagenized with Tn5 as described previously (22Frustaci J.M. O'Brian M.R. J. Bacteriol. 1992; 174: 4223-4229Crossref PubMed Google Scholar). Colonies were screened for those that fluoresced under ultraviolet light on plates containing low iron medium. One protoporphyrin-accumulating strain was cultured, and theEcoRI restriction fragment containing the Tn5 and flanking genomic DNA was isolated and reintroduced into the strain LO genome by homologous recombination to construct LODTM5. The position of Tn5 in the genome of strain LODTM5 was confirmed by Southern blot analysis. Precautions were taken to avoid exposure of strain LODTM5 to light in order to prevent the formation of light-dependent reactive oxygen species catalyzed by protoporphyrin. The fluorescent compound produced by the mutant strains was purified and identified as protoporphyrin as described elsewhere (22Frustaci J.M. O'Brian M.R. J. Bacteriol. 1992; 174: 4223-4229Crossref PubMed Google Scholar, 24Smith K.M. Porphyrins and Metalloporphyrins. Elsevier Scientific, Amsterdam1975Google Scholar). Genomic DNA flanking the Tn5 was used as a hybridization probe to isolate a 13-kb HindIII fragment from the wild type genome, and a 3.4-kb EcoRI fragment subclone contained the region mutated in strain LODTM5. The EcoRI fragment and subclones ligated into the broad host range plasmid pLAFR3 were tested for complementation. Although strain LODTM5 grew on plates, introduction of pLAFR3 alone into the mutant yielded pinpoint colonies that could not be cultured in liquid medium. Although unexplained, it is possible that the mutant could not bear the burden of replication of the 22-kb plasmid. Thus, we were able to use growth as a criterion for complementation as well as colony fluorescence. The nucleotide sequence of both strands of a SmaI-SacI fragment corresponding the mutated locus of strain LODTM5 was determined, which contained a 492-bp open reading frame. The exact location of Tn5 in the mutant was determined by sequencing of a portion of the mutated fragment in the region of the transposon. The medium used for culturing cells under iron limitation was a modified GSY medium in which 0.5 g/liter yeast extract (Difco) is used instead of 1 g/liter, and no exogenous iron source was added. The actual iron concentration of the medium was 0.3 μm as determined with a Perkin-Elmer model 1100B atomic absorption spectrometer. Cells grew well in this medium to A540 of 0.6, but mutant strain LODTM5 grew more slowly than the parent strain at higher cell densities. Cells were grown to A540 of 0.4–0.6 for all experiments except for protoporphyrin accumulation experiments, in which cells were grown to A540 of 0.8. For RNA and protein analysis of hemB, hemH, andirr, the cells were grown in medium with no added iron as the iron-limited condition or 6 μm FeCl3 as the iron replete condition. For the metal-dependent protoporphyrin accumulation experiments, FeCl3 was added to the final concentration indicated, and other metals were added as the chloride salt to a final concentration of 11 μm. Total RNA was prepared from cultured cells grown to mid-log phase as described previously (10Chauhan S. O'Brian M.R. J. Bacteriol. 1997; 179: 3706-3710Crossref PubMed Google Scholar). Steady state levels of irr, hemB, or hemH mRNA were analyzed by an RNase protection assay as described previously (10Chauhan S. O'Brian M.R. J. Bacteriol. 1997; 179: 3706-3710Crossref PubMed Google Scholar,21Chauhan S. Titus D.E. O'Brian M.R. J. Bacteriol. 1997; 179: 5516-5520Crossref PubMed Google Scholar) using antisense RNA probes to the respective genes. The effect of iron on the rate of irr mRNA synthesis was determined by transcriptional runoff analysis essentially as described previously (10Chauhan S. O'Brian M.R. J. Bacteriol. 1997; 179: 3706-3710Crossref PubMed Google Scholar). The presence of ALA dehydratase or Irr in whole cells or cell extracts was detected by immunoblot analysis of 10% or 15% SDS-PAGE gels using antibodies raised against the respective protein. Anti-ALA dehydratase antibodies were prepared as described previously (25Chauhan S. O'Brian M.R. J. Biol. Chem. 1995; 270: 19823-19827Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). A modified irrgene was constructed by a polymerase chain reaction such that anNdeI restriction site was introduced at the second methionine codon of the open reading frame and cloned into pET3c for overexpression in E. coli Bl21(DE3)(pLysS). The 5′ primer used to modify the sequence was 5′-AACCATATGCTCCAGTC-3′. The M13 reverse primer complementary to pUC19 vector in which the B. japonicum DNA was cloned was used as the 3′ polymerase chain reaction primer. Antibodies were raised against the Irr protein derivative purified from inclusion bodies using a protocol described previously (26Frustaci J.M. Sangwan I. O'Brian M.R. J. Biol. Chem. 1995; 270: 7387-7393Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). For Irr turnover experiments, 1 liter of cells was grown to mid-log phase in low iron medium and then separated into two 500-ml cultures. To one culture, 6 μm FeCl3was added at time 0, and for the control an equivalent volume of buffer with no iron was added. Incubation with shaking was continued, and 25-ml samples were removed at various times after iron addition for immunoblot analysis. Autoradiograms were quantified using a Bio-Rad model GS-700 imaging densitometer in the transmittance mode and the Molecular Analyst software package, version 1.4.1. Ferrochelatase activity was measured as the formation of mesoheme by cell extracts from ferrous iron and mesoporphyrin as described previously (22Frustaci J.M. O'Brian M.R. J. Bacteriol. 1992; 174: 4223-4229Crossref PubMed Google Scholar). Protoporphyrinogen oxidase activity was measured in membrane preparations as the oxidation of protoporphyrinogen to protoporphyrin as described previously (22Frustaci J.M. O'Brian M.R. J. Bacteriol. 1992; 174: 4223-4229Crossref PubMed Google Scholar). A unit of activity is defined as the nanomoles of product formed/h/mg of protein. Cells were grown to mid-log phase in low or high iron media, centrifuged, washed twice, and resuspended in uptake buffer to an A540 of 0.4. Uptake buffer contained 0.2 m MOPS, 20 mm citrate, and 2% (w/v) glucose, pH 6.8. 30 ml of cells were placed into a 125-ml flask and preincubated at 30 °C with shaking. At time 0,59FeCl3 was added to a final concentration of 0.05 μm (1.8 μCi). 1-ml aliquots (∼125 μg of protein) were removed at various times and added to 3 ml of quench buffer precooled on ice. The quench buffer contained 0.1 mTris, 0.1 m succinate, and 10 mm EDTA, pH 6.0. The quenched cells were collected on 0.45-μm filters presoaked in quench buffer containing 40 μm Fe-EDTA, and then counted on a LKB γ-counter. To search for mutants deregulated in iron control of heme biosynthesis, we screened a population of Tn5-induced mutants for colonies that accumulated protoporphyrin, as discerned by fluorescence under ultraviolet light. B. japonicum mutant strain LODTM5 displayed that phenotype, and the fluorescent species was purified and identified as protoporphyrin IX by the peaks of the absorption spectra of the neutral methyl ester derivative (Fig.1 A) and the free acid, and by thin layer chromatography (data not shown). No other porphyrins were detected. Protoporphyrin is the immediate precursor of protoheme (Fig.1 B), and thus the mutant was altered in some aspect of heme metabolism. In principle, protoporphyrin accumulation could be due to defects in the heme synthesis enzymes ferrochelatase or protoporphyrinogen oxidase. However, those enzyme activities were not defective in extracts of the mutant (data not shown), andhemH mRNA encoding ferrochelatase was expressed (see below). Furthermore, strain LODTM5 was not a heme auxotroph. The results show that the primary defect in strain LODTM5 is not a lesion in a structural gene of the heme biosynthetic pathway. To determine whether the mutant phenotype was iron-dependent, cells were grown in media with varying concentrations of the metal. An inverse relationship was found between the quantity of protoporphyrin produced by the mutant and the iron concentration in which the cells were grown, with the highest amount found in cells grown in media with no exogenous iron added, and none detected at high iron concentrations (Fig. 1 C). Protoporphyrin was not detected in cells of parent strain LO under any iron condition tested. When this experiment was repeated withhemH strain I110ek4, which accumulates protoporphyrin due to a defect in ferrochelatase (22Frustaci J.M. O'Brian M.R. J. Bacteriol. 1992; 174: 4223-4229Crossref PubMed Google Scholar), no decrease in the protoporphyrin level with increasing iron was observed (Fig. 1 C). Thus, the diminution observed in strain LODTM5 was not due to the nonenzymatic chelation of iron into protoporphyrin, but rather it was specific to that mutant. The effects were also metal-specific, as seen by the failure of copper, zinc, nickel, manganese, or molybdenum to abolish protoporphyrin accumulation by strain LODTM5 (Fig. 1 D). Thus, the aberration in heme metabolism was linked to iron availability, and the mutant phenotype is manifest under iron depleted conditions. The genetic locus mutated in strain LODTM5 was namedirr (iron responseregulator) based on these observations, and on those described below. The accumulation of protoporphyrin by strain LODTM5 in the absence of a heme deficiency strongly indicated an abnormally high synthesis of protoporphyrin by the heme pathway in iron-limited cells. We determined previously that hemB, the gene encoding the heme biosynthesis enzyme ALA dehydratase is regulated by iron at the mRNA level in B. japonicum wild type strain I110 and that accumulation is low in iron-limited cells (21Chauhan S. Titus D.E. O'Brian M.R. J. Bacteriol. 1997; 179: 5516-5520Crossref PubMed Google Scholar). By RNase protection and immunoblot analyses, respectively, we examined the effects of iron on hemB mRNA and protein in wild type strains I110 and LO, and in irr strain LODTM5. As observed previously, hemB mRNA and protein were very low in wild type cells grown in media with no added iron, and were elevated significantly in cells grown in iron-replete medium (Fig.2). However, mRNA and protein levels were high in strain LODTM5 even in cells grown in low iron medium, showing the loss of normal iron-dependent regulation of the gene in the mutant. Under low iron conditions, the mutant accumulated approximately 50-fold more ALA dehydratase and over 150-fold morehemB mRNA than was found in parent strain LO. This high expression of hemB by the mutant was consistent with the protoporphyrin accumulation phenotype and indicates an elevated heme pathway. Ferrochelatase catalyzes a step of the pathway subsequent to protoporphyrin formation, and therefore a regulatory system that coordinates protoporphyrin synthesis with iron availability is unlikely to control hemH, the gene encoding ferrochelatase. Consistent with this rationale, accumulation of hemHmRNA was not iron-dependent in the wild type strains, nor was its expression altered in strain LODTM5 (Fig. 2). Collectively, the results argue in favor of a role for irr in mediating iron control of hemB, and of the pathway as a whole, and support the conclusion that protoporphyrin synthesis exceeds iron bioavailability in iron-limited cells of mutant strain LODTM5. To elucidate the genetic basis of the mutation in strain LODTM5, wild type DNA corresponding to that which was mutated in the irr strain was isolated using cloned DNA flanking the Tn5 as a hybridization probe. A 3.4-kbEcoRI fragment and subclones (Fig.3 A) were tested for complementation of strain LODTM5 on the broad host range plasmid pLAFR3 (see "Experimental Procedures"). The smallest complementing clone tested was a 0.7-kb SmaI/BglII fragment containing a 492-bp open reading frame encoding a protein 163 amino acids in length (Fig. 3 A). The Tn5 was inserted into the 5′ portion of the irr gene (Fig. 3 A). Expression of the irr gene region was analyzed by measuring RNA and protein accumulation in irr + strains LO and I110 and in the mutant (Fig. 4). RNase protection analysis showed that irr accumulated in wild type strains LO and I110, but not in mutant strain LODTM5 (Fig.4 B). In addition, no mRNA corresponding to the genomic region immediately downstream of irr was detected in any strain, indicating that irr does not have another gene downstream of it encoded on the same transcript. Immunoblot analysis of whole cell protein of the wild type strains and of irrstrain LODTM5 using anti-Irr antibodies showed that the wild type strains grown in low iron medium expressed an ∼18-kDa protein that was absent in the mutant (Fig. 4 C). The results show that strain LODTM5 carries a loss of function mutation in the irrgene, and that its phenotype is due to a lesion in that gene, and not to a polar effect of the Tn5 mutagen. In addition, the transposon was inserted close to the 5′ end of the gene, thus it was important to establish that the mutation did not activate a truncated derivative of irr or a downstream gene. Finally, the data show that irr is a protein-encoding gene, and that Irr is involved in the negative regulation of hemB and of the heme biosynthetic pathway.Figure 4The irr gene is iron-regulated in wild type cells and is not expressed in mutant strain LODTM5. A, the genomic region containing the irr gene is marked by the restriction sites MluI (M),SalI (S), BglII (Bg),BamHI (B), and SacI (Sc). The triangle denotes the site of Tn5 insertion. Boxes above the restriction map denote fragments used as templates for antisense RNA probe synthesis of irr and its downstream (ds) region used in panel B. B, RNase protection analysis of the irr gene, the downstream (ds) and hemH mRNA in cells of parent strains I110 and LO and mutant strain LODTM5. The irr and downstream mRNAs were analyzed using antisense probes as shown in panel A. hemH was analyzed using an antisense probe as described previously (21Chauhan S. Titus D.E. O'Brian M.R. J. Bacteriol. 1997; 179: 5516-5520Crossref PubMed Google Scholar). Cells were grown in medium with no added iron (−) or with 6 μm FeCl3 (+). 2 μg of total RNA from cells grown in iron-limited medium were analyzed per reaction. C, immunoblot analysis of Irr protein in strains LO, I110 and LODTM5 grown in low (−) or high (+) iron media as described in panel B. 50 μg of protein were loaded onto each lane of a 15% SDS-PAGE gel and analyzed with anti-Irr antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A data base search of protein blocks (27Henikoff S. Henikoff J.G. Genomics. 1994; 19: 97-107Crossref PubMed Scopus (347) Google Scholar) suggests that Irr belongs to the GntR family of bacterial transcriptional regulators (28Reizer A. Deutscher J. Saier M.H. Reizer J. Mol. Microbiol. 1991; 5: 1081-1089Crossref PubMed Scopus (60) Google Scholar) based on homology to helix-turn-helix motif regions of members of that family (Fig. 3 B). Homology searches of individual proteins revealed that Irr shares the highest homology to the Fur protein fromPseudomonas aeruginosa (29% identity), a transcriptional regulator found in many Gram-negative bacteria involved in iron transport and other aspects of iron metabolism (29Braun V. Hantke V. Winkelmann G. Handbook of Microbial Iron Chelates. CRC Press, Boca Raton1991: 107-138Google Scholar). Bacterial Fur must be bound by iron to function (30de Lorenzo V. Wee S. Herrero M. Neilands J.B. J. Bacteriol. 1987; 169: 2624-2630Crossref PubMed Google Scholar), and consequently, it is active only under iron-replete conditions. In contrast, the phenotype of theB. japonicum irr strain indicates that it is active under iron limitation, and data (presented below) show that Irr is not detected in cells grown in iron-rich medium. Thus, Irr and Fur cannot have the same activity and therefore are not equivalent proteins. In addition, we recently isolated a B. japonicum fur gene homolog by functional complementation of and E. colimutant. 2I. Hamza and M. R. O'Brian, GenBankTM accession no. AF052295. A B. japonicum fur strain expresses ALA dehydratase normally and does not accumulate protoporphyrin under any growth condition (data not shown). Consistent with those observations, protoporphyrin was undetectable inE. coli fur strain H1780 (31Hantke K. Mol. Gen. Genet. 1987; 210: 135-139Crossref PubMed Scopus (192) Google Scholar) grown in low or high iron media (data not shown). The results indicate that irr is a novel gene. Strain LODTM5 displayed a mutant phenotype only under iron limitation, suggesting that Irr function is restricted to those cells. Thus, it is likely that iron either modulates the activity of constitutively expressed Irr protein, or that the metal regulates expression of irr in some way. This problem was addressed by measuring irrmRNA and protein in cells grown in high or low iron media (Fig. 4). Immunoblot analysis showed that Irr accumulated in wild type cells grown in low iron medium, but was undetectable in those from iron-replete medium (Fig. 4 C). This pattern was consistent with the mutant phenotypes (see Figs. 1 and 2 and below) and shows that Irr is present and active only in iron-limited cells. Unlike protein expression, however, RNase protection analysis showed that iron had only a moderate negative effect on irr mRNA levels, with a substantial quantity found even in iron replete cells (Fig.4 B). This 3-fold difference was consistent with transcriptional run-off assays demonstrating that the rate ofirr mRNA synthesis was 4-fold greater in iron-limited cells compared with those grown in iron-rich medium (data not shown). The discrepancy between irr mRNA and protein strongly indicates post-transcriptional control of the irr gene by iron in additional to transcriptional regulation. Addition of iron to cells grown under iron deprivation results in the rapid induction of hemB mRNA (21Chauhan S. Titus D.E. O'Brian M.R. J. Bacteriol. 1997; 179: 5516-5520Crossref PubMed Google Scholar), thus B. japonicum responds quickly to a change in iron availability. If the assertion that Irr mediates negative control of hemBexpression by iron is correct, then we expect a rapid loss of Irr activity upon an increase in iron availability. To address this, we monitored Irr levels in response to iron. The addition of 6 μm FeCl3 iron to cells grown in low iron medium resulted in the rapid loss of Irr protein that was observed by 5 min after addition of the metal, and almost no protein remaining after 30 min (Fig. 5). This disappearance reflects a very high turnover rate for a bacterial protein (32Gottesman S. Maurizi M.R. Microbiol. Rev. 1992; 56: 592-621Crossref PubMed Google Scholar, 33Mosteller R.D. Goldstein R.V. Nishimoto K.R. J. Biol. Chem. 1980; 255: 2524-2532Abstract Full Text PDF PubMed Google Scholar). Bacterial (34Schweder T. Lee K.-H. Lomovskaya O. Matin A. J. Bacteriol. 1996; 178: 470-476Crossref PubMed Scopus (281) Google Scholar, 35Yura T. Nagai H. Mori H. Annu. Rev. Microbiol. 1993; 47: 321-350Crossref PubMed Scopus (400) Google Scholar) and eukaryotic (36Iwai K. Klausner K.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (193) Google Scholar, 37Guo B. Brown F.M. Phillips J.D. Yu Y. Leibold E.A. J. Biol. Chem. 1995; 270: 16529-16535Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) regulatory proteins with short half-lives are specifically targeted for proteolysis under the appropriate conditions, and the observations for Irr strongly suggest a specific mechanism for its degradation. We postulate that this post-transcriptional control permits a fast response to an increase in iron availability, and that it can account for the discrepancy betweenirr mRNA and protein in iron replete cells. Iron is an essential nutrient involved in many biologic functions, but is also toxic in high concentrations. A regulatory mechanism that couples protoporphyrin synthesis with iron levels should also integrate systems necessary for iron homeostasis. Therefore, we examined the effects of iron availability on high affinity iron uptake activity in parent strain LO and mutant strain LODTM5. Wild type cells grown in iron-replete medium lacked iron transport activity using 0.05 μm59Fe (as ferric citrate) as a tracer, but uptake was induced in cells grown in low iron medium (Fig.6 A). This is a common response by bacteria to efficiently scavenge the metal when it is limiting. However, this activity was severely diminished in mutant strain LODTM5 (Fig. 6 B), indicating that iron transport is under positive control in B. japonicum by a mechanism that involves Irr. In addition, it is plausible that the iron uptake defect in strain LODTM5 resulted in an iron deficiency that contributed to the protoporphyrin accumulation phenotype. Because iron limitation repressedhemB in wild type cells (Fig. 3) (21Chauhan S. Titus D.E. O'Brian M.R. J. Bacteriol. 1997; 179: 5516-5520Crossref PubMed Google Scholar), the high expression of the gene in strain LODTM5 cannot be an indirect consequence of the iron transport defect, which should render the cell iron-deficient. We showed previously that hemB is expressed in iron-replete cells of a heme-defective strain (21Chauhan S. Titus D.E. O'Brian M.R. J. Bacteriol. 1997; 179: 5516-5520Crossref PubMed Google Scholar), therefore the elevatedhemB level in the irr mutant is not an indirect result of a heme deficiency created by iron limitation. The present findings suggest that Irr has dual activities to positively affect iron transport and negatively regulate hemB, and that the control of heme biosynthesis is coordinated with iron homeostasis. In the present study, we provide evidence for coordination of a bacterial heme biosynthetic pathway with iron availability by a regulatory mechanism involving the newly described protein Irr. Irr accumulates in response to iron limitation by iron-dependent regulation of the irr gene to attenuate the heme pathway, thereby preventing protoporphyrin synthesis from exceeding iron availability. The phenotype of the irrmutant is important because it demonstrates that copious protoporphyrin accumulation is a consequence of uncoupling the heme pathway as a whole from iron availability, and that it can result from a single genetic lesion. Because this phenotype is not normally observed in cells, it is reasonable to extrapolate from the present work that iron control of heme biosynthesis is a general regulatory phenomenon found in other organisms. Humans with iron deficiency anemia show a 5-fold increase in blood protoporphyrin, reaching approximately 2 × 10−4 mol/mol of heme (38Garrett S. Worwood M. Acta Haematol. 1994; 91: 21-25Crossref PubMed Scopus (45) Google Scholar). This elevated level is modest by comparison with the B. japonicum irr mutant, which accumulates approximately 15 mol of protoporphyrin/mol of heme under iron limitation (by recalculation of the data in Fig. 1 D). Thus, iron may regulate the heme pathway in erythrocytes to prevent severe porphyrin accumulation under iron deficiency. In bacteria, numerous genes have been recently identified that encode proteins that, like Irr, have low but significant homology to Fur, but are distinct from bona fide fur genes in those organisms (GenBankTM accession nos. Z82044, D84432, D90909, U76538,P32692, U58365, and U25731). Most of those genes were identified by whole genome sequencing and their functions are unknown. However,fur-like genes of Vibrio cholerae (39Camilli A. Mekalanos J.J. Mol. Microbiol. 1995; 18: 671-683Crossref PubMed Scopus (235) Google Scholar) andP. aeruginosa (40Wang J. Mushegian A. Lory S. Jin S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10434-10439Crossref PubMed Scopus (153) Google Scholar) were shown to be expressed, and theP. aeruginosa mutant does not have afur-defective phenotype. From this, it is pertinent to ask whether bacteria other than B. japonicum contain Irr homologs that mediate iron control of heme synthesis. The defective iron uptake activity in mutant strain LODTM5 indicates a link between control of the heme pathway and iron homeostasis. The proposed roles for Irr are physiologically sensible because it allows the cell to maximize accessibility to exogenous iron when the metal is limiting as well as attenuate the heme biosynthetic pathway. By analogy, the iron regulatory proteins in mammalian erythroid cells regulate iron transport and a heme biosynthesis gene enzyme (reviewed in Refs. 14Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1132) Google Scholar and 15Rouault T.A. Klausner R.D. Trends Biochem. Sci. 1996; 21: 174-177Abstract Full Text PDF PubMed Scopus (232) Google Scholar), and it will be important to determine whether that control is sufficient to modulate synthesis by the pathway as a whole. Furthermore, the identification of regulators of iron homeostasis such as bacterial Fur (29Braun V. Hantke V. Winkelmann G. Handbook of Microbial Iron Chelates. CRC Press, Boca Raton1991: 107-138Google Scholar) and Aft1 from Saccharomyces cerevisiae (41Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Crossref PubMed Scopus (289) Google Scholar) may provide the tools necessary to assess the relationship between iron transport and heme biosynthesis in those organisms. We thank Dr. K. Hantke for E. colistrains.
