Evidence for direct interaction between enzyme I(Ntr) and aspartokinase to regulate bacterial oligopeptide transport.

Bradyrhizobium japonicum transports oligopeptides and the heme precursor delta-aminolevulinic acid (ALA) by a common mechanism. Two Tn5-induced mutants disrupted in the lysC and ptsP genes were identified based on the inability to use prolyl-glycyl-glycine as a proline source and were defective in [(14)C]ALA uptake activity. lysC and ptsP were shown to be proximal genes in the B. japonicum genome. However, RNase protection and in trans complementation analysis showed that lysC and ptsP are transcribed separately, and that both genes are involved in oligopeptide transport. Aspartokinase, encoded by lysC, catalyzes the phosphorylation of aspartate for synthesis of three amino acids, but the lysC strain is not an amino acid auxotroph. The ptsP gene encodes Enzyme I(Ntr) (EI(Ntr)), a paralogue of Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. In vitro pull-down experiments indicated that purified recombinant aspartokinase and EI(Ntr) interact directly with each other. Expression of ptsP in trans from a multicopy plasmid complemented the lysC mutant, suggesting that aspartokinase normally affects Enzyme I(Ntr) in a manner that can be compensated for by increasing the copy number of the ptsP gene. ATP was not a phosphoryl donor to purified EI(Ntr), but it was phosphorylated by ATP in the presence of cell extracts. This phosphorylation was inhibited in the presence of aspartokinase. The findings demonstrate a role for a PTS protein in the transport of a non-sugar solute and suggest an unusual regulatory function for aspartokinase in regulating the phosphorylation state of EI(Ntr).

The Bradyrhizobium japonicum proline biosynthesis gene proC is essential for symbiosis.

Plant host-derived proline is proposed to serve as an energy source for rhizobia in the rhizosphere and in symbiotic root nodules. The Bradyrhizobium japonicum proC gene was isolated, and a proC mutant strain that behaved as a strict proline auxotroph in culture was constructed. The proC strain elicited undeveloped nodules on soybeans that lacked nitrogen fixation activity and plant hemoglobin. We conclude that the proC gene is essential for symbiosis and suggest that the mutant does not obtain an exogenous supply of proline in association with soybeans sufficient to satisfy its auxotrophy.

Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein.

The bacterial iron response regulator (Irr) protein mediates iron-dependent regulation of heme biosynthesis. Pulse-chase and immunoprecipitation experiments showed that Irr degraded in response to 6 microM iron with a half-life of approximately 30 min and that this regulated stability was the principal determinant of control by iron. Irr contains a heme regulatory motif (HRM) near its amino terminus. A role for heme in regulation was implicated by the retention of Irr in heme synthesis mutants in the presence of iron. Addition of heme to low iron (0.3 microM) cultures was sufficient for the disappearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein bound heme with high affinity and caused a blue shift in the absorption spectrum of heme to a shorter wavelength. A Cys(29) --> Ala substitution within the HRM of Irr (IrrC29A) abrogated both high affinity binding to heme and the spectral blue shift. In vivo turnover experiments showed that, unlike wild-type Irr, IrrC29A was stable in the presence of iron. We conclude that iron-dependent degradation of Irr involves direct binding of heme to the protein at the HRM. The findings implicate a regulatory role for heme in protein degradation and provide direct evidence for a functional HRM in a prokaryote.

Identification of a functional fur gene in Bradyrhizobium japonicum.

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.

Expression of a soybean gene encoding the tetrapyrrole-synthesis enzyme glutamyl-tRNA reductase in symbiotic root nodules.

Heme and chlorophyll accumulate to high levels in legume root nodules and in photosynthetic tissues, respectively, and they are both derived from the universal tetrapyrrole precursor delta-aminolevulinic acid (ALA). The first committed step in ALA and tetrapyrrole synthesis is catalyzed by glutamyl-tRNA reductase (GTR) in plants. A soybean (Glycine max) root-nodule cDNA encoding GTR was isolated by complementation of an Escherichia coli GTR-defective mutant for restoration of ALA prototrophy. Gtr mRNA was very low in uninfected roots but accumulated to high levels in root nodules. The induction of Gtr mRNA in developing nodules was subsequent to that of the gene Enod2 (early nodule) and coincided with leghemoglobin mRNA accumulation. Genomic analysis revealed two Gtr genes, Gtr1 and a 3' portion of Gtr2, which were isolated from the soybean genome. RNase-protection analysis using probes specific to Gtr1 and Gtr2 showed that both genes were expressed, but Gtr1 mRNA accumulated to significantly higher levels. In addition, the qualitative patterns of expression of Gtr1 and Gtr2 were similar to each other and to total Gtr mRNA in leaves and nodules of mature plants and etiolated plantlets. The data indicate that Gtr1 is universal for tetrapyrrole synthesis and that a Gtr gene specific for a tissue or tetrapyrrole is unlikely. We suggest that ALA synthesis in specialized root nodules involves an altered spatial expression of genes that are otherwise induced strongly only in photosynthetic tissues of uninfected plants.

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 bacterium Bradyrhizobium japonicum that, under iron limitation, accumulated protoporphyrin and showed aberrantly high expression of hemB, an iron-regulated gene encoding the heme synthesis enzyme delta-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.

Metals control activity and expression of the heme biosynthesis enzyme delta-aminolevulinic acid dehydratase in Bradyrhizobium japonicum.

The heme biosynthesis enzyme delta-aminolevulinic acid dehydratase (ALAD) requires magnesium or zinc for activity, depending on the organism, and the heme moiety contains iron. Thus, metals are important for heme formation in at least two different ways. Bradyrhizobium japonicum ALAD* is an engineered derivative of wild-type ALAD that requires Zn2+ for activity rather than Mg2+ (S. Chauhan and M. R. O'Brian, J. Biol. Chem. 270:19823-19827, 1995). The pH optimum for ALAD* activity was over 3.5 units lower than for that of the wild-type enzyme, and ALAD* activity was inhibited by lead and cadmium, as reported for the zinc-containing dehydratases of animals. In addition, ALAD* was significantly more thermostable than ALAD; the temperature optima are 50 and 37 degrees C, respectively. These observations strongly suggest that the metal contributes to both catalysis and structure, and this conclusion may be extrapolated to ALADs in general. Although iron did not affect the activity of the preformed protein, enzyme assays and immunoblot analysis demonstrated that the iron concentration in which the cells were grown had a strong positive effect on ALAD activity and the protein level. RNase protection analysis showed that the transcript quantity of hemB, the gene encoding ALAD, was iron dependent; thus, iron regulates hemB at the mRNA level. Induction of hemB mRNA in response to iron was rapid, suggesting that the factor(s) needed to mediate iron control was present in iron-limited cells and did not need to be synthesized de novo. ALAD protein levels and enzyme activities were similar in cells of the wild type and a heme-defective strain, indicating that control by iron is not an indirect effect of the cellular heme status. We conclude that the heme biosynthetic pathway is coordinated with cellular iron levels and that this control may prevent the accumulation of toxic porphyrin intermediates.

Transcriptional regulation of delta-aminolevulinic acid dehydratase synthesis by oxygen in Bradyrhizobium japonicum and evidence for developmental control of the hemB gene.

An increased demand for cytochromes is associated with symbiotic development and microaerobic metabolism in the bacterium Bradyrhizobium japonicum, and evidence suggests that hemB, rather than hemA, is the first essential bacterial heme synthesis gene in symbiosis with soybean. Steady-state levels of mRNA and protein encoded by hemB were strongly and rapidly induced by O2 deprivation as determined by RNase protection and immunoblot analyses, but hemH message was not induced. Oxygen limitation resulted in a greater-than-10-fold increase in the rate of hemB mRNA synthesis as determined by transcriptional runoff experiments, whereas hemH transcription was unaffected by the O2 status. Thus, hemB is a regulated gene in B. japonicum and is transcriptionally controlled by O2. Unlike the expression in parent strain I110, hemB expression was not affected by O2 in the fixJ strain 7360, and O2-limited cultures of the mutant contained quantities of hemB mRNA and protein that were comparable to uninduced levels found in aerobic cells. In addition, spectroscopic analysis of cell extracts showed that increases in b- and c-type cytochromes and the disappearance of cytochrome aa3 in response to microaerobic growth in wild-type cells were not observed in the fixJ mutant. FixJ is a key transcriptional regulator that mediates O2-dependent differentiation in rhizobia, and therefore hemB expression is under developmental control. Furthermore, the data suggest a global control of cytochrome expression and heme biosynthesis in response to the cellular O2 status.

Identification of the lrp gene in Bradyrhizobium japonicum and its role in regulation of delta-aminolevulinic acid uptake.

The heme precursor delta-aminolevulinic acid (ALA) is taken up by the dipeptide permease (Dpp) system in Escherichia coli. In this study, we identified a Bradyrhizobium japonicum genomic library clone that complemented both ALA and dipeptide uptake activities in E. coli dpp mutants. The complementing B. japonicum DNA encoded a product with 58% identity to the E. coli global transcriptional regulator Lrp (leucine-responsive regulatory protein), implying the presence of Dpp-independent ALA uptake activity in those cells. Data support the conclusion that the Lrp homolog induced the oligopeptide permease system in the complemented cells by interfering with the repressor activity of the endogenous Lrp, thus conferring oligopeptide and ALA uptake activities. ALA uptake by B. japonicum was effectively inhibited by a tripeptide and, to a lesser extent, by a dipeptide, and a mutant strain that expressed the lrp homolog from a constitutive promoter was deficient in ALA uptake activity. The data show that Lrp negatively affects ALA uptake in E. coli and B. japonicum. Furthermore, the product of the isolated B. japonicum gene is both a functional and structural homolog of E. coli Lrp, and thus the regulator is not restricted to enteric bacteria.

The Rhizobial hemA Gene Is Required for Symbiosis in Species with Deficient [delta]-Aminolevulinic Acid Uptake Activity.

Most rhizobial hemA mutants induce root nodules on their respective legume hosts that lack nitrogen fixation activity and leghemoglobin expression. However, a Bradyrhizobium japonicum hemA mutant elicits effective nodules on soybean, and we proposed previously that synthesis and uptake of the heme precursor [delta]-aminolevulinic acid (ALA) by the plant and bacterial symbiont, respectively, allow mutant rescue (I. Sangwan, M.R. O'Brian [1991] Science 251: 1220-1222). In the present work, the B. japonicum hemA mutant MLG1 elicited normal nodules on three hosts, including cowpea, a plant that is not effectively nodulated by a hemA mutant of Rhizobium sp. These data indicate that B. japonicum rather than soybean possesses the unique trait that allows normal nodule development by a hemA mutant. Cowpea expressed glutamate-dependent ALA formation activity in nodules induced by B. japonicum strains I110 or MLG1 and by Rhizobium sp. ANU240. Exogenous ALA was taken up by B. japonicum bacteroids isolated from soybean or cowpea nodules, and the kinetics of uptake were biphasic. By comparison, Rhizobium sp. ANU240 had very low ALA uptake activity. In addition, ALA uptake was observed in cultured cells of B. japonicum but not in cultured cells of three other rhizobial species tested. We suggest that the differential success of legume-rhizobial hemA symbioses is due to an ALA uptake activity in B. japonicum that is deficient in other rhizobia, thereby further validating the ALA rescue hypothesis.

A mutant Bradyrhizobium japonicum delta-aminolevulinic acid dehydratase with an altered metal requirement functions in situ for tetrapyrrole synthesis in soybean root nodules.

The tetrapyrrole synthesis enzyme delta-aminolevulinic acid (ALA) dehydratase requires Mg2+ for catalytic activity in photosynthetic organisms and in Bradyrhizobium japonicum, a bacterium that can reside symbiotically within plant cells of soybean root nodules or as a free-living organism. ALA dehydratase from animals and other non-photosynthetic organisms is a Zn(2+)-dependent enzyme. A modified B. japonicum ALA dehydratase, ALAD*, was constructed by site-directed mutagenesis of hemB in which three proximal amino acids conserved in plant dehydratases were changed to cysteine residues as is found in the Zn(2+)-dependent enzyme of animals. These substitutions resulted in an enzyme that required Zn2+ rather than Mg2+ for catalytic activity, and therefore a region of the ALA dehydratase from B. japonicum, and probably from plants, was identified that is involved in Mg2+ dependence. In addition, the data show that a change in only a few residues is sufficient to change a Mg(2+)-dependent ALA dehydratase to a Zn(2+)-dependent one. B. japonicum strains were constructed that contained a single copy of either hemB or the altered gene hemB* integrated into the genome of a hemB- mutant. Cultures of the hemB* strain KPZn3 had Zn(2+)-dependent ALA dehydratase activity that functioned in vivo as discerned by its heme prototrophy and expression of wild type levels of cellular hemes. Strain KPZn3 elicited root nodules on soybean that contained viable bacteria and exhibited traits of normally developed nodules, and the symbiotic bacteria expressed nearly wild type levels of cellular hemes. We conclude that the Zn(2+)-dependent ALAD* can function and support bacterial tetrapyrrole synthesis within the plant milieu of root nodules.

gsa1 is a universal tetrapyrrole synthesis gene in soybean and is regulated by a GAGA element.

Expression of plant tetrapyrroles is high in photosynthetic tissues and in legume root nodules in the form of chlorophyll and heme, respectively. The universal tetrapyrrole precursor delta-aminolevulinic acid (ALA) is synthesized from glutamate 1-semialdehyde (GSA) by GSA aminotransferase in plants, which is encoded by gsa. Immunoblot analysis showed that GSA aminotransferase was expressed in soybean leaves and nodules, but not in roots, and that protein correlated with enzyme activity. These observations indicate that GSA aminotransferase expression is controlled in tetrapyrrole formation and argue against significant activity of an enzyme other than the well described aminotransferase for GSA-dependent ALA formation. gas mRNA and protein were induced in soybean nodules, and their activation was temporally intermediate between those of the respective early and late genes endo2 and lb. A GSA aminotransferase gene, designated gsa1, was isolated and appears to be one of two gsa genes in the soybean genome. gsa1 mRNA accumulated to high levels in leaves and nodules, but not in uninfected roots as discerned with a gsa1-specific probe. Message levels were higher in leaves from etiolated plantlets than in mature plants, and expression in the former was slightly elevated by light. The expression pattern of gsa1 mRNA was qualitatively similar to that of total gsa. The data strongly suggest that gsa1 is a universal tetrapyrrole synthesis gene and that a gsa gene specific for a tissue, tetrapyrrole, or light condition is unlikely. The gsa1 promoter contained a genetic element found in numerous Drosophila melanogaster genes; the so-called GAGA element displayed single-stranded character in vitro and formed a complex with nuclear factors from nodules and leaves but not from roots. From these observations we infer that the GAGA element is involved in the transcriptional control of gsa1.

Plant delta-aminolevulinic acid dehydratase. Expression in soybean root nodules and evidence for a bacterial lineage of the Alad gene.

We isolated a soybean (Glycine max) cDNA encoding the heme and chlorophyll synthesis enzyme delta-aminolevulinic acid (ALA) dehydratase by functional complementation of an Escherichia coli hemB mutant, and we designated the gene Alad. ALA dehydratase was strongly expressed in nodules but not in uninfected roots, although Alad mRNA was only 2- to 3-fold greater in the symbiotic tissue. Light was not essential for expression of Alad in leaves of dark-grown etiolated plantlets as discerned by mRNA, protein, and enzyme activity levels; hence, its expression in subterranean nodules was not unique in that regard. The data show that soybean can metabolize the ALA it synthesizes in nodules, which argues in favor of tetrapyrrole formation by the plant host in that organ. Molecular phylogenetic analysis of ALA dehydratases from 11 organisms indicated that plant and bacterial enzymes have a common lineage not shared by animals and yeast. We suggest that plant ALA dehydratase is descended from the bacterial endosymbiont ancestor of chloroplasts and that the Alad gene was transferred to the nucleus during plant evolution.

Bradyrhizobium japonicum delta-aminolevulinic acid dehydratase is essential for symbiosis with soybean and contains a novel metal-binding domain.

The Bradyrhizobium japonicum hemA gene product delta-aminolevulinic acid (ALA) synthase is not required for symbiosis of that bacterium with soybean. Hence, the essentiality of the subsequent heme synthesis enzyme, ALA dehydratase, was examined. The B. japonicum ALA dehydratase gene, termed hemB, was isolated and identified on the basis of its ability to confer hemin prototrophy and enzyme activity on an Escherichia coli hemB mutant, and it encoded a protein that was highly homologous to ALA dehydratases from diverse organisms. A novel metal-binding domain in the B. japonicum ALA dehydratase was identified that is a structural composite of the Mg(2+)-binding domain found in plant ALA dehydratases and the Zn(2+)-binding region of nonplant ALA dehydratases. Enzyme activity in dialyzed extracts of cells that overexpressed the hemB gene was reconstituted by the addition of Mg2+ but not by addition of Zn2+, indicating that the B. japonicum ALA dehydratase is similar to the plant enzymes with respect to its metal requirement. Unlike the B. japonicum hemA mutant, the hemB mutant strain KP32 elicited undeveloped nodules on soybean, indicated by the lack of nitrogen fixation activity and plant hemoglobin. We conclude that the hemB gene is required for nodule development and propose that B. japonicum ALA dehydratase is the first essential bacterial enzyme for B. japonicum heme synthesis in soybean root nodules. In addition, we postulate that ALA is the only heme intermediate that can be translocated from the plant to the endosymbiont to support bacterial heme synthesis in nodules.

Analysis of the Bradyrhizobium japonicum hemH gene and its expression in Escherichia coli.

Complementation analysis showed that the Bradyrhizobium japonicum hemH gene was both necessary and sufficient to rescue mutant strains I110ek4 and I110bk2 in trans with respect to hemin auxotrophy, protoporphyrin accumulation, and the deficiency in ferrochelatase activity. The B. japonicum hemH gene was expressed in an Escherichia coli T7 expression system and yielded a 39-kDa protein, which was consistent with the predicted size of the deduced product. The overexpressed protein was purified and shown to contain ferrochelatase activity, thereby demonstrating that the hemH gene encodes ferrochelatase. When expressed from the lac promoter, the B. japonicum hemH gene was able to complement the enzyme activity of a ferrochelatase-defective E. coli mutant, and it also conferred hemin prototrophy on those cells. These latter findings confirm the identity of the hemH gene product and demonstrate that B. japonicum ferrochelatase can interact with the E. coli heme synthesis enzymes for heme formation in complemented cells.

Expression of the soybean (Glycine max) glutamate 1-semialdehyde aminotransferase gene in symbiotic root nodules.

Extracts of soybean (Glycine max) root nodules and greening etiolated leaves catalyzed radiolabeled delta-aminolevulinic acid (ALA) formation from 3,4-[3H]glutamate but not from 1-[14C]glutamate. Nevertheless, those tissue extracts expressed the activity of glutamate 1-semialdehyde (GSA) aminotransferase, the C5 pathway enzyme that catalyzes ALA synthesis from GSA for tetrapyrrole formation. A soybean nodule cDNA clone that conferred ALA prototrophy, GSA aminotransferase activity, and glutamate-dependent ALA formation activity on an Escherichia coli GSA aminotransferase mutant was isolated. The deduced product of the nodule cDNA shared 79% identity with the GSA aminotransferase expressed in barley leaves, providing, along with the complementation data, strong evidence that the cDNA encodes GSA aminotransferase. GSA aminotransferase mRNA and enzyme activity were expressed in nodules but not in uninfected roots, indicating that the Gsa gene is induced in the symbiotic tissue. The Gsa gene was strongly expressed in leaves of etiolated plantlets independently of light treatment and, to a much lesser extent, in leaves of mature plants. We conclude that GSA aminotransferase, and possibly the C5 pathway, is expressed in a nonphotosynthetic plant organ for nodule heme synthesis and that Gsa is a regulated gene in soybean.

The Escherichia coli visA gene encodes ferrochelatase, the final enzyme of the heme biosynthetic pathway.

An Escherichia coli mutant with a disrupted visA gene was defective in ferrochelatase activity but expressed wild-type levels of protoporphyrinogen oxidase activity. The visA coding region was placed under the transcriptional control of T7 RNA polymerase in an E. coli expression system, and the product was expressed as a 38-kDa protein. The overexpressed protein was purified to near homogeneity and was found to contain ferrochelatase activity. The data show that the visA gene encodes ferrochelatase, and we propose that it be renamed hemH to reflect that conclusion.

Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene.

A Tn5-induced mutant of Bradyrhizobium japonicum, strain LORBF1, was isolated on the basis of the formation of fluorescent colonies, and stable derivatives were constructed in backgrounds of strains LO and I110. The stable mutant strains LOek4 and I110ek4 were strictly dependent upon the addition of exogenous hemin for growth in liquid culture and formed fluorescent colonies. The fluorescent compound was identified as protoporphyrin IX, the immediate precursor of protoheme. Cell extracts of strains LOek4 and I110ek4 were deficient in ferrochelatase activity, the enzyme which catalyzes the incorporation of ferrous iron into protoporphyrin IX to produce protoheme. Mutant strain I110ek4 could take up 55Fe from the growth medium, but, unlike the parent strain, no significant incorporation of radiolabel into heme was found. This observation shows that heme was not synthesized in mutant strain I110ek4 and that the heme found in those cells was derived from exogenous hemin in the growth medium. The putative protein encoded by the gene disrupted in strain LORBF1 and its derivatives was homologous to ferrochelatases from eukaryotic organisms. This homology, along with the described mutant phenotype, provides strong evidence that the disrupted gene is hemH, that which encodes ferrochelatase. Mutant strain I110ek4 incited nodules on soybean that did not fix nitrogen, contained few viable bacteria, and did not express leghemoglobin heme or apoprotein. The data show that B. japonicum ferrochelatase is essential for normal nodule development.

Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules.

Formation of the heme precursor delta-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 60 degrees C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate-dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[(14)C]glutamate and 3,4-[(3)H]glutamate but not from 1-[(14)C]glutamate, and thus, operation of the C(5) pathway could not be confirmed.