©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Mutant Bradyrhizobium japonicum -Aminolevulinic Acid Dehydratase with an Altered Metal Requirement Functions in Situ for Tetrapyrrole Synthesis in Soybean Root Nodules (*)

(Received for publication, April 14, 1995; and in revised form, June 26, 1995)

Sarita Chauhan Mark R. O'Brian (§)

From the Department of Biochemistry and Center for Advanced Molecular Biology and Immunology, State University of New York, Buffalo, New York 14214

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The tetrapyrrole synthesis enzyme -aminolevulinic acid (ALA) dehydratase requires Mg 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-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-dependent enzyme of animals. These substitutions resulted in an enzyme that required Zn rather than Mg for catalytic activity, and therefore a region of the ALA dehydratase from B. japonicum, and probably from plants, was identified that is involved in Mg dependence. In addition, the data show that a change in only a few residues is sufficient to change a Mg-dependent ALA dehydratase to a Zn-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-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-dependent ALAD* can function and support bacterial tetrapyrrole synthesis within the plant milieu of root nodules.


INTRODUCTION

The bacterium Bradyrhizobium japonicum establishes a symbiotic relationship with soybean that is manifested as specialized plant organs called root nodules (reviewed in (1, 2, 3) ). B. japonicum fixes atmospheric nitrogen to ammonia that can be assimilated by the plant, and soybean fixes carbon dioxide via photosynthesis, some of which is acquired by the bacterium. The bacterium differentiates into symbiotic bacteroids during nodule development, and they reside within plant cells of mature nodules. Thus, B. japonicum functions as a specialized plant organelle that fixes N(2) at the expense of ATP generated directly by the endosymbiont, and bacterial cytochrome heme proteins increase quantitatively and change qualitatively to accommodate this energy demand.

Interestingly, studies with B. japonicum heme mutants indicate that ALA (^1)dehydratase, which catalyzes the second heme synthesis step, is the first essential bacterial enzyme for B. japonicum heme synthesis in root nodules (4) and is the subject of the current work. An ALA synthase mutant cannot synthesize the first committed tetrapyrrole precursor ALA and makes no discernible heme in culture, but the heme defect is rescued symbiotically(5) , and the mutant can establish a nitrogen-fixing symbiosis(6) . However, a lesion in hemB, the gene encoding ALA dehydratase, elicits poorly developed nodules on soybean that contain very few bacteria and cannot fix nitrogen(4) . We proposed previously that the ALA synthase (hemA) mutant is rescued in nodules by being able to take up ALA synthesized by the soybean host(5) . This hypothesis is based on the discovery of an inducible soybean ALA synthesis activity in nodules and of a bacterial ALA uptake activity(5) . Plant ALA synthesis from glutamate in nodules probably occurs by the so-called C(5) pathway (reviewed in (7) and (8) ) as evidenced by the induction of the soybean gene encoding the glutamate 1-semialdehyde aminotransferase gene gsa1(9, 10) . In addition to gsa1, the plant tetrapyrrole synthesis genes encoding ALA dehydratase (11) and coproporphorinogen oxidase (12) are also strongly induced in nodules; hence it is very likely that the soybean ALA is also incorporated into heme of the nodule-specific plant protein leghemoglobin.

ALA dehydratase is a zinc-dependent enzyme in animals, yeast, and some bacteria (reviewed in (13) ). Cysteine residues participate in Zn binding(14, 15, 16) , and these enzymes contain a cysteine-rich domain that may be involved. Plant dehydratases are localized to plastids and are needed for chlorophyll synthesis in addition to other cellular tetrapyrroles. They share 35-50% identity with the non-plant enzymes, but activity requires magnesium rather than Zn (discussed in (17) and (18) ). The peptide region in the plant enzyme that corresponds to the putative Zn-binding domain of animals lacks the cysteines and histidine residues and contains aspartate, alanine, or threonine instead(17, 18) . An alga (19) and photosynthetic bacterium (^2)contain the ``plant'' domain as well. This correlation has led to the proposal that the domain found in plants is involved in Mg binding(17, 18) , but no experimental evidence has been obtained. The B. japonicum enzyme is unusual in that it is the only known ALA dehydratase from a non-photosynthetic organism that requires Mg rather than Zn for activity and contains a putative metal-binding domain that has some residues otherwise found only in plants(4) . Phylogenetic analysis suggests that the plantlike ALA dehydratase of B. japonicum did not arise from horizontal gene transfer from the plant symbiont(11) . Several questions arise from these latter observations. (i) Are the pertinent amino acid residues found in B. japonicum and plants, but not in non-plants, involved in Mg-dependent activity? (ii) If so, would changes in these few residues be sufficient to alter the metal requirement for activity? (iii) Does the Mg-dependent ALA dehydratase of B. japonicum confer an advantage on the bacterium as a plant endosymbiont? This latter question is part of a broader issue of whether a Mg-dependent ALA dehydratase is advantageous to plants. In the current work, the B. japonicumhemB gene altered by site-directed mutagenesis encodes a dehydratase with Zn-dependent activity. The ability of the engineered enzyme to support heme synthesis in situ in culture and symbiotically is evaluated.


MATERIALS AND METHODS

Bacteria

Escherichia coli strain BL21(DE3)(pLysS) (20) was used for overexpression of the hemB gene in the present work; it was grown in LB medium (21) supplemented with 25 µg/ml chloramphenicol, and 50 µg/ml ampicillin was added to maintain pET3c-derived plasmids in that strain. E. coli strain RP523 is hemB(22) and was grown in LB medium containing 10 µg/ml hemin. B. japonicum strain I110 is the parent strain used in the present work. Strain KP32 is an ALA dehydratase-defective derivative of strain I110 due to an insertion of a kanamycin resistance-encoding cassette within the hemB gene(4) . It requires hemin (10 µg/ml) and vitamin B (2 ng/ml) for growth in complex medium. Derivatives of strain KP32 were constructed in the current study that have either the wild type hemB gene or the altered hemB* gene (see below) integrated into the genome. To do this, DNA containing either hemB or hemB* was ligated into pBR322, and these plasmids were introduced into strain KP32 by conjugation as described previously(23) . Integration of the plasmids into the genome by single recombination events into homologous DNA was initially screened by tetracycline resistance conferred by the plasmid and then by Southern blotting analysis using hemB or pBR322 as the hybridization probe. The correct genomic structures were confirmed by nucleotide sequencing. Each derivative contained a copy of the mutant hemB gene of strain KP32 and a functional copy of either the hemB or hemB* gene introduced on the plasmid. Depending on the site of recombination, the functional gene was either downstream of B. japonicum DNA as is found in the wild type genome or of the integrated pBR322 DNA. Strains KPWt1 and KPZn1 had hemB and hemB*, respectively, downstream of genomic DNA. For strains KPWt3 and KPZn3, hemB and hemB* were downstream of integrated pBR322 DNA.

Growth of Soybeans and Analysis of Nodules

Soybeans (Glycine max cv. Essex) inoculated with a B. japonicum strain were grown in a growth chamber under a 16-h light/8-h dark regime at 25 °C. Nodules from 30-32-day-old plants were harvested for analysis. Nitrogen fixation (acetylene reduction), viable cell counts, and leghemoglobin heme determinations were carried out as described previously(24) .

Bacterial Heme Determinations

Bacterial hemes were analyzed by dithionite-reduced minus ferricyanide-oxidized absorption difference spectra using an SLM-Aminco DW2000 scanning spectrophotometer, and the features were analyzed as described previously(5) .

Construction of hemB*

An oligonucleotide 47 residues in length was synthesized that corresponded to a region of the hemB gene, except that some bases were altered to change the amino acid sequence encoded by that DNA. The sequences of the wild type DNA and the oligonucleotide were as follows: wild type, 5`-GCGACGTCGCGCTCGATCCCTTCACCAGCCACGGCCATGACGGCCTG-3`; oligonucleotide, 5`-GCGACGTCTGCCTCTGCCCCTACACCAGCCACGGCCATTGCGGCCTG-3`. The 47-mer and the M13 universal primer were used as PCR primers to amplify a region of DNA on pZEN. pZEN contains the wild type hemB gene cloned into pBluescript SKII(4) . The PCR product was denatured by heating and used with the T3 primer in a second round of PCR using pZEN as template. This product was cloned into pSKII to construct pSTAR. pZEN and pSTAR were identical except for the nucleotide changes noted above, which was confirmed by nucleotide sequencing. The result of these changes is that Ala-146, Asp-148, and Asp-156 of the wild type ALAD were each changed to cysteine. In addition, Phe-150 was changed to tyrosine. The altered gene is referred to as hemB* and its product as ALAD*.

Overexpression of B. japonicum hemB in E. coli and Antibody Production

The 5` end of the hemB gene was modified such that it could be cloned into the NdeI-BamHI sites of pET3c (20) and translated from the methionine initiation codon. To do this the upstream primer 5`-CCGCATATGGCGATCAAATACGG-3` and the downstream T3 primer complementary to the pBluescript SKII vector were employed in a PCR reaction using pNEZ as template. pNEZ has the insert of pZEN (4) in the reverse orientation. The PCR product was blunt-ended with T4 polymerase and ligated into the EcoRV site of pBluescript SKII to construct pSKEX4. The hemB coding region was removed from PSKEX4 by digestion with NdeI and BamHI and ligated into the NdeI/BamHI sites of pET3c to construct pETBJHEMB. The hemB gene encoded on pETBJHEMB was expressed in E. coli strain BL21(DE3)(pLysS) as described previously(25) , and the nearly pure 40-kDa protein recovered from the inclusion body fraction of those cells contained ALA dehydratase activity. For antibody production, 30 µg of the inclusion body extract was loaded onto a preparative 10% SDS-PAGE gel, and ALA dehydratase was excised from the gel with a razor blade. The excised fragment was homogenized by mincing with a razor blade and then forced through an 18-gauge needle several times. The sample was then used to raise antibodies in rabbits as described previously(21) . Crude antisera were affinity-purified using ALA dehydratase bound to nitrocellulose as described previously(26) .

Western Blotting

Protein extracts were run on 10-12% SDS-PAGE gels, transferred to nitrocellulose or Immobilon (Millipore) filters, and screened with antibodies raised against ALA dehydratase as described previously(21) . Cross-reactive material bound to the filter was discerned with peroxidase-conjugated goat anti-rabbit IgG and visualized by chemiluminescence using the Renaissance kit (DuPont NEN) according to the manufacturer's instructions.

ALA Dehydratase Activity and Determination of Metal Requirement

The metal requirement for B. japonicum ALA dehydratase and ALAD* was assessed in both E. coli and in B. japonicum. E. colihemB strain RP523 (22) harboring pZEN or pSTAR was grown in LB with 50 µg/ml ampicillin and 0.17 mM isopropyl-beta-D-thiogalactopyranoside. Cell extracts were prepared as described previously (27) and dialyzed against either 0.1 M Tris (pH 8) or 0.1 M MES (pH 6) with 10 mM dithiothreitol or beta-mercaptoethanol and 0.5 mM EDTA. Various amounts of ZnSO(4) or MgSO(4) were added to the dialyzed extracts and preincubated for 10 min at 37 °C. ALA dehydratase activity in extracts was measured as porphobilinogen formed from ALA in 1 h at 37 °C as described previously(24) .


RESULTS

Construction of an Altered B. japonicum hemB Gene

Animal and yeast ALA dehydratases contain four conserved cysteines and one histidine that may play a role in Zn binding, and other non-photosynthetic bacteria lack one of these cysteines (Fig. 1). The corresponding region in Mg-dependent plant ALA dehydratases is conserved overall, but the cysteines and histidine proposed to be involved in Zn binding in the animal enzymes are replaced by aspartate, threonine, or alanine. Although B. japonicum is not a photosynthetic organism, its ALA dehydratase is Mg-dependent and has 3 residues otherwise unique to plants (4) (Fig. 1). Herein, the B. japonicumhemB gene was mutagenized using a PCR technique (see ``Materials and Methods'') such that the three plant-specific amino acids, Ala-146, Asp-148, and Asp-156, were each replaced with cysteine (Fig. 1). In addition, Phe-150 found in B. japonicum was changed to tyrosine, which is found in all other dehydratases. The altered gene and its protein product were designated hemB* and ALAD*, respectively. ALAD* was identical to the mammalian dehydratase from residues 143 to 158 (Fig. 1), and it, as well as the wild type ALAD, was 40% identical to the human enzyme overall.


Figure 1: Region of the B. japonicum ALA dehydratase mutated to construct ALAD* and comparison to dehydratases from diverse organisms. Only ALA dehydratases in which metal dependence has been determined experimentally are shown.



ALAD* Activity Is Zn-dependent

The plasmids pZEN and pSTAR, which contained B. japonicumhemB and hemB*, respectively, were introduced into the E. colihemB mutant RP523 (22) so that the only ALA dehydratase gene expressed was that borne on the plasmid. Dialyzed extracts of the strain RP523 transformants contained very little ALAD activity in the absence of added metal (Fig. 2), and those from RP523[pZEN] showed Mg-dependent activity but lacked activity in the presence of Zn (Fig. 2). This result was observed previously for B. japonicum ALA dehydratase (4) and is consistent with studies with the pea enzyme, which is stimulated by millimolar concentrations of Mg(28) . In contrast to wild type ALA dehydratase, ALAD* activity was not affected by exogenous Mg, even up to 1 mM (Fig. 2A). However, activity of the altered enzyme was Zn-dependent (Fig. 2B), which is characteristic of animals and other non-photosynthetic organisms. Maximal activity was observed at approximately 30 µM Zn, similar to that observed for the bovine enzyme(29) , and was lower than maximal activities that could be obtained in the wild type in the presence of Mg (Fig. 2). In addition, ALAD* had maximal activity at pH 6, whereas the wild type had no activity at that pH in the presence of either metal but rather showed maximal Mg-dependent activity at pH 8 (data not shown).


Figure 2: Effect of Mg and Zn on activity of B. japonicum ALAD and the mutant ALAD*. Activity was measured in dialyzed extracts of cells that overexpressed wild type protein (closedcircles) or mutant ALAD* (opencircles) as a function of added MgSO(4) (A) or ZnSO(4) (B).



The data show that the amino acid substitutions made in B. japonicum ALA dehydratase were sufficient for both the loss of Mg-dependent activity and the acquisition of Zn-dependent activity. Therefore, one or more of the B. japonicum dehydratase residues that are conserved in plants and that were substituted to construct ALAD* must be involved in Mg dependence. This conclusion can be made strongly because the mutant ALAD* was not inactive but rather exhibited an altered metal requirement. Similarly, we conclude that one or more of the substituted cysteines found in ALAD* and in the wild type animal enzymes are involved in Zn-dependent activity.

The hemB* Gene Functions in Situ in B. japonicum Cultured Cells

B. japonicum strain KP32 is an ALA dehydratase-defective strain due to insertional inactivation of the hemB gene(4) . Wild type hemB or hemB* was cloned into pBR322 and integrated into the genome of strain KP32 by homologous, single-recombination events, resulting in a single copy of functional hemB (strains KPWt1 and KPWt3) or hemB* (strains KPZn1 and KPZn3) within the chromosome (Fig. 3). Strains KPWt1 and KPZn1 have hemB and hemB*, respectively, downstream of B. japonicum DNA as it would be in the wild type genome (Fig. 3). For strains KPWt3 and KPZn3, hemB and hemB* are downstream of the integrated pBR322 (Fig. 3). Western blot analysis showed that normal ALA dehydratase and ALAD* were expressed more strongly when the respective genes were positioned downstream of the integrated pBR322 plasmid (strains KPWt3 and KPWt1) compared with those downstream of B. japonicum DNA (Fig. 4), and this may be due to expression from regulatory elements on the vector. Extracts of B. japonicum strains KPWt1 and KPWt3, which contained the normal hemB gene, showed Mg-dependent ALA dehydratase activity, and strain KPWt3 had higher activity than KPWt1 or the wild type strain I110 (Fig. 4). Extracts of B. japonicum strains KPZn1 and KPZn3, both of which contained hemB*, had Zn-dependent ALA dehydratase activity (Fig. 4), and the lower activity in strain KPZn1 was attributed to the lesser amount of protein expressed (Fig. 4).


Figure 3: Construction of B. japonicum strains containing hemB or hemB* integrated into the genome of the hemB strain KP32. The functional gene hemB (openarrow) or hemB* (arrow with asterisk) borne on pBR322 was integrated by homologous single recombination to generate derivatives that contained the functional gene downstream of B. japonicum genomic DNA (solidline) or of integrated pBR322 (brokenline). The arrow with the blackinsert represents the disrupted, non-functional hemB gene of strain KP32.




Figure 4: Expression of ALA dehydratase protein and metal-dependent activity in cultured cells of hemB and hemB* strains of B. japonicum. Toppanel, extracts of cultured cells (20 µg) were separated by SDS-PAGE and immunoblotted using anti-ALA dehydratase antibodies. Middlepanel, ALA dehydratase activity was measured in extracts in the presence of 1 mM MgSO(4). Bottompanel, ALA dehydratase activity was measured in extracts in the presence of 50 µM ZnSO(4). The activities are averages of triplicate trials, and the standard deviations were less than 10%.



Two lines of evidence showed clearly that ALAD* functioned in situ in B. japonicum cultured cells. First, strains KPZn1 and KPZn3 grew in medium in the absence of exogenous heme, whereas the hemB strain KP32 required exogenous hemin (heme hydrochloride) for growth, even in complex medium (data not shown). Strain KPZn1 showed a significant lag phase in growth that was missing in strain KPZn3 and in strains containing a wild type hemB gene (data not shown). We assume that this was due to the low ALA dehydratase activity in that strain. Second, strain KPZn3 had wild type levels of cellular hemes, as did KPWt1 and KPWt3 (Fig. 5A), showing that ALAD* could support heme synthesis. Spectral analysis showed that KPWt3 synthesized c-, b- and a-type cytochrome hemes as discerned by the features at 552, 561, and 603 nm, respectively, as was observed for strains containing wild type hemB (Fig. 5A). Strain KPZn1 showed little cytochrome heme in culture, indicating that the low level of ALA dehydratase activity in that strain resulted in heme formation sufficient for viability but not for wild type levels of heme expression. The very low heme requirement for normal growth of B. japonicum cultured cells has been documented previously(27) . Nevertheless, a higher level of ALAD* expression in strain KPZn3 compensated for a lower enzymatic activity; thus we conclude that the Zn-dependent ALAD* can function in situ in B. japonicum cultured cells to support heme synthesis.


Figure 5: Reduced minus oxidized absorption spectra of extracts of cultured cells (A) or symbiotic bacteroids (B) of B. japonicumhemB and hemB* strains. Extracts were reduced and oxidized with a few grains of dithionite and ferricyanide, respectively. The relevant absorption peak or shoulder wavelength is indicated on the diagram. The verticalbar represents a DeltaA of 0.009. The protein concentration of the extracts was 8 mg/ml for cultured cells and 4 mg/ml for symbiotic bacteroids.



ALAD* Supports Heme Synthesis in Symbiotic Root Nodules

B. japonicum ALA dehydratase is unusual in that it is Mg-dependent(4) , a trait otherwise confined to photosynthetic organisms. This observation led us to ask whether the Mg-dependent enzyme confers an advantage on the bacterium as a plant endosymbiont. Since the hemB* strain KPZn3 had wild type levels of cytochrome heme in culture, it was tested symbiotically in soybean, along with the hemB strains KPWt3 and I110 and with the hemB strain KP32. Strain KP32 formed small nodules that were undeveloped in appearance; they contained few viable bacteria and did not fix nitrogen; thus ALA dehydratase is necessary for symbiosis (4) (Fig. 6). However, strain KPZn3 formed nodules that appeared normal, contained viable bacteria, and fixed nitrogen, similar to those induced by strains I110 or KPWt3 (Fig. 6). Both the viability and nitrogen fixation activity in nodules elicited by strain KPZn3 indicated that bacteroids expressed heme, and absorption spectra demonstrated this directly (Fig. 5B). The cytochrome heme pattern of strain KPZn3 bacteroids was qualitatively the same as that found in strains KPWt3 and I110, and the quantities of c-type (551.5 nm) and b-type (561 nm) hemes were slightly less in the hemB* strain. From this, we conclude that ALAD* can function within plant cells of soybean root nodules to support bacterial heme biosynthesis.


Figure 6: Properties of soybean nodules elicited by the hemB* strain KPZn3, hemB strains KPWt3 and I110, and hemB strain KP32. Nitrogen fixation activity is expressed as micromoles of ethylene formed from acetylene per h per g of nodule, fresh weight. Leghemoglobin is expressed as nanomoles of plant nodule heme per g of nodule, fresh weight. Leghemoglobin is measured in this study as a plant marker of mature nodules. Viable bacterial cell counts are expressed as colony-forming units per g of nodule, fresh weight. For strains KPWt3 and KPZn3, over 99% of the colonies formed were tetracycline-resistant, which is conferred by the integrated pBR322. Nodule weight per plant is expressed as grams of nodule, fresh weight, per plant. Nodules from strain KP32 could not be easily separated from the roots; thus the weight given includes non-nodule root tissue. ALA dehydratase is discerned by immunoblotting using anti-ALA dehydratase antibodies as described in Fig. 4. ND, not determined due to insufficient bacterial mass in nodules.




DISCUSSION

In the present work, a modified B. japonicum ALA dehydratase was constructed that showed an altered metal requirement for activity. A change in four proximal amino acids was sufficient for the simultaneous loss of Mg dependence and the acquisition of activity that was Zn-dependent. These results directly ascribe a region in the B. japonicum ALA dehydratase involved in Mg-dependent activity, and they make strong inferences concerning ALA dehydratases in general. First, one or more of the residues in the wild type B. japonicum enzyme that was changed, namely Ala-146, Asp-148, Phe-150, and Asp-156, must be involved in Mg-dependent activity; otherwise ALAD* would either retain that metal dependence or be inactive. Second, three of these amino acids, Ala-146, Asp-148, and Asp-156, are conserved in plant ALA dehydratases but not in the Zn-dependent enzymes; therefore it is likely that this region is involved in Mg-dependent activity in plants as well. Third, some or all of the three cysteines engineered into ALAD* and conserved in the non-plant enzymes are probably involved in Zn-dependent activity. This conclusion is noteworthy because, although extended x-ray absorption fine structure spectroscopy analysis implicates cysteines in Zn binding(15) , mammalian ALA dehydratases have eight cysteines, none of which have been directly assigned to play a role in Zn binding. Finally, we reiterate the striking conclusion that a change in only a few amino acid residues is sufficient to change a Mg-dependent ALA dehydratase to a Zn-dependent one.

The altered enzyme ALAD* was able to function in vivo to support heme biosynthesis in cultured cells, as seen by the heme prototrophy and expression of cellular hemes in cells that contained a single copy of hemB* integrated into the genome (Figs. 5A). Although a higher level of ALAD* protein was required compared with wild type ALAD to obtain normal levels of cytochrome heme expression (Fig. 5), the data demonstrate that the Zn-dependent enzyme can function within those cells. Interestingly, the hemB* strain KPZn3 elicited soybean nodules that fixed nitrogen, and symbiotic bacteroids expressed almost normal levels of cytochrome heme (Fig. 5B), whereas the hemB strain KP32 could not establish a symbiosis with soybean (4) (Fig. 6). Thus, the plant milieu in nodules permits the functioning of the Zn-dependent ALAD* in symbiotic bacteroids as part of a heme biosynthetic pathway. It is plausible that differences in symbiotic heme synthesis between the hemB* strain KPZn3 and those containing the normal hemB gene could be found in plants of different age or in plants grown under different environmental conditions. However, although there may be conditions where the plantlike dehydratase of B. japonicum confers an advantage as a plant symbiont, these possibilities do not alter the conclusion that the Zn-dependent dehydratase can function in nodules. It would be interesting to learn whether an engineered plant ALA dehydratase with a Zn-dependent activity could function in chloroplasts to support chlorophyll synthesis in photosynthetic tissues of transgenic plants.


FOOTNOTES

*
This work was supported by National Science Foundation Grant MCB-9404818 (to M. R. O'B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, 140 Farber Hall, State University of New York, Buffalo, NY 14214. Tel.: 716-829-3200; Fax: 716-829-2725; cammrob{at}ubvms.cc.buffalo.edu.

(^1)
The abbreviations used are: ALA, -aminolevulinic acid; ALAD, ALA dehydratase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; MES, 2-(N-morpholino)ethanesulfonic acid.

(^2)
K. Indest and A. J. Biel, GenBank U14593[GenBank].


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