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.     

Aminolevulinic acid dehydratase in pea (Pisum sativum L.). Identification of an unusual metal-binding domain in the plant enzyme

Aminolevulinic acid dehydratase (ALA dehydratase) catalyzes the second step of tetrapyrrole synthesis leading to the formation of heme and chlorophyll in higher plant cells. Antibodies elicited against spinach leaf ALA dehydratase were used to immunoscreen lambda gt11 cDNA libraries constructed from etiolated pea (Pisum sativum L.) leaf poly(A)+ RNAs. A set of overlapping cDNAs was characterized that encode the pea enzyme. The predicted amino acid sequence of the pea ALA dehydratase is similar to those reported for other eukaryotic and prokaryotic ALA dehydratases. The pea enzyme has an active site domain centered on lysine that is highly conserved in comparison to other known ALA dehydratases. Consistent with the previously reported requirement of Mg2+ for catalytic activity by plant ALA dehydratases, the pea enzyme lacks the characteristic Zn(2+)-binding domain present in other eukaryotic ALA dehydratases, but contains a distinctive metal ligand-binding domain based upon aspartate. Northern blot analyses demonstrated that ALA dehydratase mRNA is present in leaves, stems, and to a lesser extent in roots. Steady state levels of mRNA encoding ALA dehydratase exhibit little or no change during light-induced greening.     

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.     

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of culture conditions on production of 5-aminolevulinic acid by recombinant Escherichia coli

Aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (hemA gene). Then ALA is converted to Porphobilinogen (PBG) by the ALA dehydratase (hemB gene). For the overproduction of ALA, we used an Escherichia coli BL21(DE3) containing a hemA gene from Bradyrhzobium japonicum, which was created in our previous work. The effects of pH on the ALA synthase and ALA dehydratase were investigated. The ALA synthase and ALA dehydratase activities were dependent on the pH of the medium, with maximal activities occurring at pH 6.5 and 8.0 respectively. At pH 6.5, extracellular ALA reached 23 mM in a jar-fermenter. In addition, the effects of some nutritional factors, such as nitrogen source and the ratio of carbon to nitrogen (C/N) on the fermentative production of ALA were investigated. The highest ALA production was found with 8:1 of C/N ratio. Among various nitrogen sources, the tryptone might be a useful one for ALA production.     

Aerobic growth and respiration of a delta-aminolevulinic acid synthase (hemA) mutant of Bradyrhizobium japonicum.

Oxygen-dependent growth of the Bradyrhizobium japonicum hemA mutant MLG1 (M.L. Guerinot and B.K. Chelm, Proc. Natl. Acad. Sci. USA 83:1837-1841, 1986) was demonstrated in cultured cells in the absence of exogenous delta-aminolevulinic acid (ALA), but growth of analogous mutants of Rhizobium meliloti or of Escherichia coli was not observed unless ALA was added to the yeast extract-containing media. No heme could be detected in extracts of strain MLG1 cells as measured by the absorption or by the peroxidase activity of the heme moiety, but the rates of growth and endogenous respiration of the mutant were essentially identical to those found in the parent strain. A role for ALA in the viability of strain MLG1 could not be ruled out since the ALA analog levulinic acid inhibited growth, but neither ALA synthase nor glutamate-dependent ALA synthesis activity was found in the mutant. The data show that the cytochromes normally discerned in wild-type B. japonicum cultured cells by absorption spectroscopy are not essential for aerobic growth or respiration.     

Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.     

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III.

We have recently reported (M. Petricek, L. Rutberg, I. Schr?der, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.     

Cloning of tomato (Lycopersicon esculentum Mill.) arginine decarboxylase gene and its expression during fruit ripening.

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.     

Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules

Formation of the heme precursor δ-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°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-[¹⁴C]glutamate and 3,4-[³H]glutamate but not from 1-[¹⁴C]glutamate, and thus, operation of the C₅ pathway could not be confirmed.     

The third member of the hemA gene family encoding glutamyl-tRNA reductase is primarily expressed in roots in Hordeum vulgare

Accumulation of chlorophylls and heme is primarily controlled at the level of 5-aminolevulinate (ALA) synthesis in higher plants. ALA is formed from glutamate in three enzymatic steps in plants. Among them, the reduction of glutamyl-tRNA^Gluto glutamate-1-semialdehyde (GSA) is likely to be a regulatory point of ALA synthesis. This reaction is catalyzed by glutamyl-tRNA reductase (GTR), which is encoded by a hemA gene. We have isolated a novel isoform of a hemA cDNA clone from barley (Hordeum vulgare) that is the third member of the hemA gene family. mRNA of this isoform is accumulated primarily in roots, suggesting that the isoform is regulated in an organ-specific manner by the demand for heme synthesis rather than chlorophyll. Phylogenetic analysis was done using the deduced amino acid sequences of hemA isoforms from barley, cucumber and Arabidopsis thaliana. The results indicate that the existing gene families in these plants arose after the divergence of monocotyledonous and dicotyledonous plants.     

Effect of iron availability on expression of the Bradyrhizobium japonicum hemA gene.

Bradyrhizobium japonicum produces delta-aminolevulinic acid, the universal precursor of tetrapyrroles, in a reaction catalyzed by the product of the hemA gene. Expression of the B. japonicum hemA gene is affected by iron availability. Activity of a hemA-lacZ fusion is increased approximately threefold by iron, and RNA analysis indicates that iron regulation is at the level of mRNA accumulation. To our knowledge, this is the first example of an iron-regulated heme biosynthetic gene in prokaryotes.     

Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.     

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

Cloning and Sequence Analysis of the hemB Gene of Rhodothermus marinus

A Rhodothermus marinus gene, hemB, coding for 5-aminolevulinic acid (ALA) dehydratase (ALAD) has been cloned and sequenced. The reading frame of the hemB gene is 1020 base pairs encoding a protein of 340 amino acids with a calculated molecular mass of 37.4 kDa. The amino acid sequence shows homology with eubacterial and eukaryotic ALA dehydratases. A putative metal-binding site of the protein shows strongest homology with corresponding sites from plant ALA dehydratases that require Mg²⁺ for activity. It differs with respect to only one amino acid out of 20 from a corresponding site in pea ALAD. Received: 1 March 1999 / Accepted: 7 April 1999     

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.     

Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads.