Bradyrhizobium japonicum 6-Aminolevulinic Acid Dehydratase

JOURNAL

OF BACrERIOLOGY, Nov. 1993, P. 7222-7227 0021-9193/93/227222-06$02.00/0 Copyright C) 1993, American Society for Microbiology

Vol. 175, No. 22

Bradyrhizobium japonicum 6-Aminolevulinic Acid Dehydratase Is Essential for Symbiosis with Soybean and Contains a Novel Metal-Binding Domain SARITA CHAUHAN AND MARK R. O'BRIAN* Department of Biochemistry and Center for Advanced Molecular Biology and Immunology, 140 Farber Hall, State University of New York at Buffalo, Buffalo, New York 14214 Received 3 August 1993/Accepted 16 September 1993

The Bradyrhizobiumjaponicum hemA gene product 8-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 Mg2+-binding domain found in plant ALA dehydratases and the Zn2+-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.

Bradyrhizobium japonicum establishes a symbiotic relationship with soybean whereby the plant assimilates nitrogen fixed by the bacterium and the endosymbiont utilizes carbon fixed by the host plant. The symbiosis is manifested as nodules on soybean roots, and these specialized organs are composed of differentiated plant and bacterial cells. An increase in heme content compared with the respective asymbiotic plant and bacterial cells is among the many changes that characterize differentiated cells of a nodule (1, 21), and this phenotype accomodates the energy-intensive process of nitrogen fixation. Bacterial cytochromes allow oxidative phosphorylation in the low-02 milieu of the nodule, which drives nitrogen fixation, and plant hemoglobin (leghemoglobin) facilitates 02 diffusion to the respiring bacteroids. Evidence indicates that heme synthesis in nodules is an interactive phenomenon whereby each symbiont exerts an effect on the other. A B. japonicum mutant defective in 8-aminolevulinic acid (ALA) synthase, the enzyme which forms the heme precursor ALA from glycine and succinyl-coenzyme A, elicits nitrogen-fixing nodules on soybean (11), and expression of bacterial heme by the mutant is rescued symbiotically (20). Soybean synthesizes ALA from glutamate in nodules (20, 21), and B. japonicum bacteroids take up exogenous ALA (20); thus, we proposed that bacterial heme can be synthesized from plant-derived ALA in nodules, thereby rendering the hemA gene nonessential for symbiosis (20). The soybean ALA formation activity is induced in nodules (21), which is due, at least in part, to activation of the soybean glutamate 1-semialdehyde aminotransferase gene (22). The spatial separation of the heme pathway in nodules is reminiscent of that in yeasts and animals, in which some steps occur in the mitochondria and others occur in the cytosol.

Data supporting the hypothesis that B. japonicum heme can be formed from soybean-derived ALA do not rule out provision of other plant heme precursors to the bacterium as well, because those intermediates could, at least in principle, rescue the hemA mutant. The heme moiety itself is unlikely to be translocated because B. japonicum ferrochelatase, the enzyme which catalyzes protoheme formation from protoporphyrin IX and Fe2", is required for symbiosis (8, 9). In order to understand the interorganismic events governing heme synthesis in nodules, it is necessary to identify precisely the plant intermediates that can support B. japonicum heme formation and to determine which bacterial enzymes are required for symbiosis, as these parameters are likely to reveal points of control. Herein, we show that B. japonicum ALA dehydratase, the enzyme that utilizes ALA directly in the heme pathway, is essential for symbiosis with soybean. In addition, evidence is provided for an unusual metal-binding domain in the ALA dehydratase from B. japonicum.

MATERIALS AND METHODS Chemicals and reagents. All chemicals were reagent grade and were purchased from Sigma Chemical Co., St. Louis, Mo., or from J. T. Baker Chemical Co., Philipsburg, N.J. Purified Noble agar, yeast extract, and tryptone were obtained from Difco Laboratories, Detroit, Mich. ALA, porphobilinogen (PBG), and mesoporphyrin IX were purchased from Porphyrin Products, Logan, Utah. [co-32P]dCTP (3,000 Ci/mmol) and [ot-35S]dATP (1,185 Ci/mmol) were obtained from New England Nuclear-DuPont, Boston, Mass. Bacterial strains, media, and growth. B. japonicum MLG1 (11) and KP32 are derivatives of the wild-type B. japonicum strain 1110. These strains were routinely grown in GSY medium as described previously (10). Mutant strains KP32 and

* Corresponding author. Electronic mail address: cammrob@ubvms. cc.buffalo.edu.

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B. JAPONICUM B-AMINOLEVULINIC ACID DEHYDRATASE

MLG1 were grown in the presence of kanamycin (50 jig/ml). ALA (30 jig/ml), PBG (20 jig/ml), hemin (1 jiM for broth and 15 jiM for plates), cysteine (20 jig/ml), and vitamin B12 (2 ng/ml) were added to the autoclaved media. ALA and PBG stocks were prepared in water. A 3.8 mM stock solution of commercial hemin was prepared in 0.02 N NaOH solution in 50% ethanol. Polyoxyethylene sorbitan mono-oleate (Tween 80; 0.003% [wt/vol]) was added to liquid media to prevent hemin from precipitating. Escherichia coli strains were grown at 37°C on Luria broth (LB) medium (2) with appropriate supplements. Strain RP523 is a derivative of strain C600 and carries a mutation in the gene encoding ALA dehydratase (17). Strain RP523 was grown on LB supplemented with hemin (1 jig/ml for broth and 10 jig/ml for plates), and ampicillin (50 jig/ml) was added for cells carrying plasmids. Isopropyl-3-Dthiogalactopyranoside (IPTG) (40 jig/ml) was added for induction of plasmid-borne genes. Construction of a B. japonicum expression library and isolation of the hemB gene. A B. japonicum genomic library was constructed in pBluescript II SK. To do this, B. japonicum genomic DNA was partially digested with Sau3AI, and 1- to 3-kb fragments were purified from an agarose gel by band interception. The ends were partially filled in by using dGTP, dATP, and Klenow enzyme and then were ligated into the XhoI site of pBluescript SK II (+) which had been previously digested and filled in with dCTP, d'TiTP, and Klenow enzyme. The ligation reaction was used to transform competent XL-1 Blue E. coli cells en masse by electroporation, and the resultant library contained approximately 95% recombinant clones as determined by the frequency of blue and white colonies on X-Gal plates. One hundred eighty nanograms of ligated genomic DNA yielded approximately 3 x 105 independent recombinant clones. Colonies were scraped off of LB plates, plasmid DNA was isolated from those cells as previously described, and the library was maintained in ethanol. The library was used to transform RP523 cells (a hemB E. coli strain), and complemented transformant cells were screened for those which grew in the presence of ampicillin and IPTG and in the absence of added hemin. Colonies which arose from this selection were restreaked, and plasmid DNA was isolated from them. DNA manipulations and sequencing. Routine DNA manipulations were done according to previously described methods (2). Deletions of pZEN were obtained by using an Exo-Mung deletion kit (Stratagene) according to the manufacturer's instructions or by removal of restriction fragments. Doublestranded DNA sequencing was carried out by using a Sequenase kit (version 2.0, United States Biochemicals) according to the manufacturer's instructions; the sequences of both strands were determined. The sequence was analyzed with software from the Genetics Computers Group (7). Determination of metal requirement for ALA dehydratase activity. E. coli mutant strain RP523(pZEN) was grown and induced for expression of the B. japonicum hemB gene as described above. Cell extracts were prepared as previously described (10) and dialyzed overnight against 0.1 M Tris (pH 8)-10 mM dithiothreitol-0.5 mM EDTA; the EDTA was added for chelation of metals. Various amounts of ZnSO4 or MgSO4 were added to the dialyzed extracts and preincubated for 15 min at 37°C. ALA dehydratase activity was measured in the treated extracts as described previously (8). Construction of a stable B. japonicum hemB mutant. The isolated B. japonicum hemB gene in pZEN was disrupted by inserting a 1.3-kb kanamycin resistance-encoding cassette from pUCK4 (27) into a unique StuI site. The resultant plasmid, pZXEN, could not complement strain RP523 with respect to

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hemin prototrophy or ALA dehydratase activity. A 2.6-kb EcoRI-KpnI fragment from pZXEN carrying the disrupted hemB gene was cloned into an EcoRI-PvuI site of pBR322 to construct pZXENOM, which was subsequently mobilized into B. japonicum 1110 as described previously (11). Replacement of the genomic hemB gene with the mutagenized one by homologous recombination was scored as KMr Tcs colonies and confirmed by Southern blot analysis. Growth of soybeans and analysis of nodules. Soybeans (Glycine max cv. Essex) were grown in a growth chamber with a 16-h light/8-h dark photoperiodicity at 25°C in vermiculite containing N-free supplemental nutrients. Germinated seedlings were inoculated with B. japonicum cultures; approximately 108 cells were used per seedling. Nitrogen fixation activity (acetylene reduction), viable cell counts, and leghemoglobin heme determinations in nodule cytosol were carried out as described previously (8). Leghemoglobin protein was discerned immunologically by Western blot (immunoblot), analysis as described previously (19). Nucleotide sequence accession number. The sequence of the Sau3AI partial digest containing the B. japonicum hemB gene (shown in Fig. 2) has been deposited in GenBank under accession number L24386.

RESULTS Complementation of an E. coli ALA dehydratase mutant with B. japonicum genomic DNA. E. coli mutant strain RP523 is defective in hemB, the gene encoding ALA dehydratase, and requires hemin for growth on LB medium (17). Strain RP523 cells were transformed with a B. japonicum genomic library in pBluescript, and complemented cells were selected as Apr hemin prototrophs. A total of 199 prototrophic colonies arose out of approximately 3 x 106 screened, and 13 of the larger colonies were picked for further analysis. Three different plasmids were found in 11 of the clones represented by pZEN, pWte5, and pWte6 (Fig. 1), and the insert DNAs overlapped each other. The other two clones carried plasmids with inserts that contained not only a 1-kbApaI-Sau3AI fragment found in pZEN, pWte5, and pWte6 but also additional DNA that was different from those plasmids and may have arisen from ligation of noncontiguous DNA during library construction; such plasmids were not studied further. Reintroduction of pZEN, pWte5, and pWte6 into strain RP523 conferred hemin prototrophy and high ALA dehydratase activity on the mutant, which confirms that the complemented phenotype of the transformants was due to the plasmids and not to a chromosomal reversion (Fig. 1). The RP523 transformants expressed much higher ALA dehydratase activity than was found in the parent strain, C600 (Fig. 1), which presumably was due to the high copy number of the pBluescript derivatives, the strength of the lacZ promoter, or both. pZEN was used for further studies. Nucleotide sequence and deduced product of the DNA that complements strain RP523. The nucleotide sequence of the 1,242-bp insert in pZEN was determined, and a large open reading frame was identified which contained two putative ATG start codons at base positions 94 and 136 (Fig. 2) and none within the vector downstream of the lac promoter in that reading frame. To determine the essentiality of these methionine codons for expression of ALA dehydratase in strain RP523, derivatives of pZEN were constructed with deletions in the 5' end of the insert which removed one or both of the codons (Fig. 3). A deletion in pZEN which removed the first ATG codon (pZENdl) resulted in a substantial increase in ALA dehydratase activity, whereas a deletion which removed

7224

CHAUHAN AND O'BRIAN

J. BAC-FERIOL.

Strain

Insert

C600

None

RP523 [pSK II (+)]

None

Hemin auxotrophy

ALAD activity 20±1

+

0

RP523 [pWte5]

74 ± 2

RP523 [pWte6]

121 ± 6

RP523 [pZENI

96 ± 4

,200bp, 'K7

FIG. 1. Complementation of E. coli RP523 with B. japonicum genomic DNA library clones. The overlapping inserts are shown with hash marks representing Sau3AI sites as determined from the nucleotide sequence. The arrow represents the open reading frame deduced from the nucleotide sequence of the insert of pZEN, and the triangle denotes the site of insertion of the Kmr interposon for construction of the B. japonicum hemB mutant KP32. ALA dehydratase (ALAD) activities in extracts of E. coli C600 and in the RP523 transformants are expressed as nanomoles of PBG formed per hour per milligram of protein and are the averages of three trials (plus or minus the standard deviation). Hemin auxotrophy was determined by the ability to grow in LB medium with or without the addition of 10 puM hemin (ferric heme hydrochloride).

both codons (pZENd2) abolished enzyme activity (Fig. 3). Hence, the smallest open reading frame that confers activity begins with the second ATG at position 136, and that codon is preceded by a putative ribosome-binding site (Fig. 2). The deletion in pZENdl may have placed the translation start site closer to the lac promoter than it is in pZEN, or it may have resulted in the removal of N-terminal amino acids that decrease activity. The deduced protein encoded by the open reading frame found on pZEN was homologous to other ALA dehydratases; it shared 37 to 51% identity with the dehydratases from other bacteria (6, 12, 16), S. cerevisiae (18), plants (5, 15, 23), and animals (3, 4, 28). This sequence homology, along with complementation of the E. coli ALA dehydratase mutant (Fig. 1), strongly implies that the cloned gene encodes the B. japonicum ALA dehydratase, and it is therefore designated hemB. Evidence for a novel metal-binding domain in B. japonicum ALA dehydratase. A Zn2+-binding domain has been identified in the ALA dehydratases of animals and yeasts on the basis of four cysteines and a histidine, which is similar to the zinc-finger motif consensus (reviewed in reference 14) (Fig. 4). Bacteria also contain that motif, except that one of the conserved cysteines is missing, but at least the E. coli enzyme binds Zn2+ nevertheless (25). The corresponding domain in plant dehydratases is homologous overall to that of nonplant dehydratases, except that the four cysteines and the histidine have been replaced and the aspartate-rich region is proposed to bind Mg2+ rather than Zn2+ (5, 23) (Fig. 4). Interestingly, the B. japonicum ALA dehydratase contains neither the plant nor the nonplant metal-binding motif but rather is an exact composite of both (Fig. 4). Cysteine and histidine are present at positions 143 and 153, respectively, in the B. japonicum ALA dehydratase, which typifies the nonplant enzymes. However, Ala146, Asp-148, and Asp-156 correlate exactly with plant ALA dehydratases and the proposed Mg2+-binding domain. To determine whether Zn2+ or Mg2+ was required for enzyme activity, extracts of strain RP523 cells that overexpressed the B.

japonicum ALA dehydratase were dialyzed against EDTA to remove metals and then incubated with various concentrations of Mg2+ or Zn2+ (Fig. 5). Dialyzed extracts contained little ALA dehydratase activity, and it was not stimulated by the addition of Zn2+ up to 1 mM (Fig. 5). Conversely, addition of Mg2+ resulted in restoration of enzyme activity; hence, B. japonicum ALA dehydratase was similar to the plant enzymes with respect to the metal requirement. The simultaneous addition of equimolar concentrations of Mg2+ and Zn2+ resulted in activities similar to those elicited by the addition of Mg2+ alone (data not shown). Construction of a hemB strain of B. japonicum and characterization of soybean nodules elicited by the mutant. A stable genomic hemB mutant of B. japonicum 1110 was constructed by replacement of the wild-type gene with one disrupted with a Kmr-encoding interposon cassette (Fig. 1) (see Materials and Methods). The B. japonicum hemB mutant strain KP32 was a PBG auxotroph on minimal medium and contained no ALA dehydratase activity. Because PBG was sufficient to compensate for the hemB lesion and presumably to allow heme formation, heme synthesis genes other than hemB were not disrupted in strain KP32. PBG could be replaced by the simultaneous addition of hemin, cysteine, and vitamin B12. PBG is a precursor of the siroheme prosthetic group of sulfite reductase, which is necessary for sulfur amino acid synthesis, and of the corrin ring of vitamin B12 (24). Soybean seedlings were inoculated with strain 1110, the hemA strain MLG1, or the hemB strain KP32, and nodules from 24-day-old plants were analyzed. (Fig. 6). Mutant strain MLG1 elicited nitrogen-fixing nodules on soybean which expressed leghemoglobin (Fig. 6), as was reported previously (11), showing that the hemnA gene is not essential for symbiosis with soybean. By contrast, the nodules elicited by the hemB mutant KP32 were small and starchy in appearance, contained few viable bacteria, did not fix nitrogen, and lacked hemoglobin heme and apoprotein (Fig. 6). Thus, unlike ALA synthase, the B. japonicum ALA dehydratase is required for normal

VOL. 175, 1993

B.

GATCTCGGACATGGCTCCCTTCTGGCTGTTTCGCCCCGCAAAATCAATCGGCTGCCATCC d L g h g s L l a v s p r k i n r L p s CTTGCGGCAGGTCAAGCCGCGGTGACGGGAACCATGCGAGCTGGTATAAGAATAATTCCA

60 120

L a a g q a a v t g t m r a g i r i i p GCCGGAGGAAGAGTGATGGCGATCAAATACGGGCGTCCGATCGAATTGCGCGAGGTTTCG 180 a g g r v M A I K Y G R P I E L R E V S CGCCGGGATGGCGCGGCAGCCTCCCCTGCCCTCGATCTGGCCATCCGTCCGCGCCGCAAC 240 R R D G A A A S P A L D L A I R P R R N CGCAAGGCCGAGTGGGCCCGGCGGATGGTGCGCGAGAACGTGCTCACCACCGACGATCTG 300 R K A E W A R R M V R E N V L T T D D L ATCTGGCCGCTGTTCCTGATCGACGGCAACAACAAGCGCGAGCAGATCGCCTCGATGCCG 360 I W P L F L I D G N N K R E Q I A S M P GGCGTCGAGCGCCTCAGCGTCGACCAGGCCGTGCGCGAGGCCGAGCGCGCGATGAAGCTC 420 G V E R L S V D Q A V R E A E R A M K L

ACGATCCCCTGCATCGCGCTGTTTCCCTACACCGACCCGTCCCTGCGCGACGAGGAAGGC T I P C I A L F P Y T D P S L R D E E G TCGGAGGCCTGCAACCCGAACAATCTGGTCTGCCAGGCGGTACGCGCGATCAAGAAGGAA 540 S E A C N P N N L V C Q A V R A I K K E TTTCCGGAGATCGGCGTCCTCTGCGACGTCGCGCTCGATCCCTTCACCAGCCACGGCCAT 600 F P E I G V L C D V A L D P F T S H G H GACGGCCTGATCGCGGACGGCGCGATCCTGAACGACGAGACGGTCGCCGTGCTGGTGCGC 660 D G L I A D G A I L N D E T V A V L V R CAGGCCCTGGTGCAGGCCGAAGCCGGCTGCGACATCATCGCGCCCTCCGACATGATGGAC 720 Q A L V Q A E A G C D I I A P S D M M D GGCCGCGTCGCCGCGATCCGCGAGGGACTGGACCAGGCAGGGCTCATCGACGTGCAGATC 780 G R V A A I R E G L D Q A G L I D V Q I M A Y A A K Y A

S A

F Y G P

F

R D A

I

G

TCGGCCAAGACGCTGACCGGCGACAAGCGCACCTACCAGATGGACAGCGCCAACACCGAC 900 S A K T L T G D K R T Y Q M D S A N T D GAGGCGCTGCGCGAGGTCGAGCTCGACATCTCCGAGGGCGCCGACATGGTGATGGTGAAG 960 E A L R E V E L D I S E G A D M V M V K

CCGGGCATGCCCTATCTCGACGTGGTCCGCCGCGTGAAGGACACCTTTGCGATGCCGACC 1020 P G M P Y L D V V R R V K D T F A M P T TTCGCCTACCAGGTGTCCGGTGAATACGCGATGATCGCGGCGGCCGCGGGCAACGGCTGG 1080 F A Y Q V S G E Y A M I A A A A G N G W CTCGACGGCGACCGCGCGATGATGGAGAGCCTGCTCGCCTTCAAGCGCGCCGGCGCGGAT 1140 L D G D R A M M E S L L A F K R A G A D GGCGTGCTGAGCTACTTTGCCCCGAAGGCGGCGGAGAAGCTGCGGACGCAGGGGTAAGGT 1200 G V L S Y F A P K A A E K L R T Q G* 1242 CATTGTCGTCCTGGCGATGGCCGGGACCCATACCGCGTGATC

FIG. 2. Nucleotide sequence of the B. japonicum genomic fragment borne on pZEN and deduced protein product. Upper case amino acids represent the proposed hemB product based on deletion analysis described in the text and shown, in Fig. 3. An asterisk (*) denotes the termination codon. The underlined nucleotide sequence denotes the putative ribosome-binding site.

Plasmid

7225

JAPONICUM B-AMINOLEVULINIC ACID DEHYDRATASE PLANTS Soybean Pea Spinach S. martensii

.

JAPONICU

L L L L

V I I V

I Y T D VA L I Y T D VA L I Y T D VA L I YT D V |AL

I G V L C D V

NON-PQLANTSL Mouse Rat Human Yeast M. sociabilis B. subtilis E. coli

L L V L L V L L V L Y I L V V M V V M I V

A A A I I V M

C C C C T A S

D D D D

P Y S S P Y S S P Y Y Y P Y S S

D G H D G H D G H D G H

D G I D G I D G I D G I

V R V R V T V R

L D P F T S H G H D G L I A

D V C L C P D V C L C P D V C L C P D V C L C E D V C L C Q D T C L C E D T C F C E

Y Y Y Y Y Y Y

T T T T T T T

S S S S E

H H H H H D H S H

G G G G G G G

H H H H H H H

C C C C C C C

G G G G G G G

L L L V I L V

L L L L V V L

FIG. 4. Comparison of the metal-binding domain of B. japonicum ALA dehydratase to those of plants and nonplants. Relevant amino acid residues are boxed. The B. japonicum sequence shown corresponds to amino acids 139 to 160. Sequences from soybean (15), pea (5), spinach (23), the fern Selaginella martensii (23), mouse (4), rat (3), human (28), S. cerevisiae (18), Methanothermus sociabilis (6), Bacillus subtilis (12), and E. coli (16) ALA dehydratase metal-binding domains are shown.

nodule development and symbiosis. Because nodules elicited by the hemB mutant contained few viable bacteria, it is likely that leghemoglobin is one of many late proteins not expressed in those nodules. Therefore, the data do not directly address the role of the endosymbiont in plant hemoglobin formation, but rather the absence of plant hemoglobin is characteristic of abnormally developed nodules.

DISCUSSION In the present work, we isolated B. japonicum hemB, the encoding ALA dehydratase, and constructed a genomic hemB mutant to investigate whether expression of that gene is essential for symbiosis. The phenotype of soybean nodules elicited by the hemB strain KP32 showed clearly that B. japonicum ALA dehydratase is necessary for normal nodule development (Fig. 6), whereas expression of the bacterial hemA gene is not required (11) (Fig. 6). These data strongly indicate that B. japonicum heme synthesis in nodules cannot be supported by plant PBG or subsequent precursors in the heme pathway; hence, we postulate that traversal of heme intermediates from the host to the endosymbiont is limited to ALA (Fig. 7). From this, we also propose that B. japonicum ALA dehydratase is the first essential bacterial enzyme for B. japonicum tetrapyrrole synthesis in soybean root nodules. Previous work (20) indicated that the B. japonicum hemA gene

Deduced amino acid sequence

Acivity

DL-X29-MRAGIRIIPAGGRV&AIKYGRPIELREVSRRDGAAASPALD-

72+2

pZENd1

GGRVHAIKYGRPIELREVSRRDGAAASPALD-

474±9

pZENd2

RRDGAAASPALD-

pZEN

S S S Y K K C

0

FIG. 3. ALA dehydratase activities in E. coli RP523 transformants harboring 5' deletions of pZEN. N-terminal regions of the deduced products of pZEN and deletion clones pZENdl and pZENd2 are shown. Methionines (M) are underlined. ALA dehydratase activities in cell extracts are expressed as nanomoles of PBG formed per hour per milligram of protein and are the averages of three trials (plus or minus the standard

deviation).

J. BACrFERIOL.

CHAUHAN AND O'BRIAN

7226

150

Glu- (

ALA

(Alad)

SOYBEAN PBG

125

*3 0

fi100

'm E .->

75

50

25

< 0 0. .0

0.2

0.4

0.6

0.8

1.0

Metal concentration (mM) FIG. 5. Effect of Mg2" and Zn2+ on B. japonicum ALA dehydratase from dialyzed extracts of cells overexpressing the hemB gene. Growth of cells, induction, extract preparation, dialysis, and assays were carried out as described in the text. ALA dehydratase activity in extracts treated with Zn2+ (open circles) or Mg2+ (closed circles) is expressed as nanomoles of PBG formed per hour per milligram protein, and each point shown is the average of three trials.

mutant expresses PBG in symbiotic cells but not in culture; the present study indicates that the PBG is not provided directly by the soybean host but rather is synthesized by the endosymbiont from plant-derived ALA by the bacterial ALA dehydratase. Groups of bacteroids are surrounded by a peribacteroid membrane to form a structure termed a symbiosome. It is not yet known whether ALA formation by soybean occurs within or outside of the symbiosome, but if the latter is true, then the ALA would have to traverse the peribacteroid membrane to be metabolized by bacteroids. Herrada et al. reported a low ALA uptake activity for French bean symbiosomes and stated that soybean symbiosomes were quantitatively the same (13). However, the ALA uptake activity shown in that study was ca. 3 nmol/h/mg of protein at 25°C, which is twofold higher than the rate at which ALA is subsequently consumed by the B. japonicum ALA dehydratase (20). Therefore, the data do not

STRAIN

PROPERTY 1110

MLG1

KP32

Genotype

wild type

hemA

hemB

Nitrogenase activitya

12.0 ± 2.2

12.1 ± 1.4

0

Viable cell countsb

2x109

NDe

2x103

Leghemoglobin hemec

87± 1

78±3

0

FIG. 7. Proposed mechanism for B. japonicum heme formation in soybean root nodules based on the present study and previous work (20). The figure distinguishes plant from bacterial phenomena in nodules, and no higher-order structure is implied. The following B. japonicum and soybean genes shown have been isolated: hemA, encoding ALA synthase (11); hemB, encoding ALA dehydratase; hemH, encoding ferrochelatase (8); Gsa, encoding glutamate 1-semialdehyde aminotransferase (22); and Alad, encoding soybean ALA dehydratase (15).

demonstrate that ALA uptake across the peribacteroid membrane would be the limiting step of bacteroid heme synthesis. It should be kept in mind that the influx of a committed precursor to a stable molecule such as heme need not be as great as the uptake of dicarboxylates (e.g., see reference 26) that serve as carbon and energy sources and which are rapidly consumed. The putative metal-binding domain of the B. japonicum ALA dehydratase was found to be a structural composite of plant and nonplant domains that bind Mg2' and Zn2+, respectively. The B. japonicum enzyme activity required Mg2+ (Fig. 5), and thus it was similar to the plant dehydratases in that regard. The reasons for the anomalous structural and functional properties of the B. japonicum ALA dehydratase are not known, and acquisition of additional sequences may show that other enzymes are similar to the dehydratase described herein. We note, however, that the sequences in hand (Fig. 4) are from taxonomically diverse organisms, which supports the argument that the ALA dehydratase from B. japonicum is unique. It is plausible that a Mg2+-binding enzyme confers an advantage on the bacterium as a plant endosymbiont; this idea, although speculative, is particularly attractive because it would provide a means of control of an essential enzyme in a symbiotic context. This unanswered question is part of the broader issue of why plants differ from other organisms in the metal component of the ALA dehydratase holoenzyme. Finally, the unusual structure of the putative metal-binding domain of the B. japonicum enzyme may help elucidate which amino acids are crucial for metal binding in the more conventional domains of other dehydratases. ACKNOWLEDGMENTS

Leghemoglobin apoproteind

FIG. 6. Phenotype of soybean nodules elicited by B. japonicum strains 1110, MLG1, or KP32. Footnotes: a, average activity expressed as millimoles of ethylene formed per hour per gram of nodule (fresh weight) (plus or minus the standard deviation of three trials); b, CFU per gram of nodule (fresh weight); c, nanomoles of heme in plant nodule cytosol per gram of nodule (fresh weight) (plus or minus the standard deviation of three trials); d, protein discerned immunologically as cross-reactive bands on polyacrylamide gels by Western blot analysis using anti-leghemoglobin antibodies. e, not determined.

We thank Sharon Cosloy for E. coli RP523. This work was supported by National Science Foundation grant IBN-9204778 to M. R. O'Brian. REFERENCES 1. Appleby, C. A. 1984. Leghemoglobin and Rhizobium respiration. Annu. Rev. Plant Physiol. 35:443-478. 2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman, and K. Struhl. 1987. Current protocols in molecular Biology. Wiley Interscience, New York. 3. Bishop, T. R., L. P. Frelin, and S. H. Boyer. 1986. Nucleotide

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4. 5.

6.

7.

8. 9. 10.

11.

12.

13.

14.

15. 16.

B. JAPONICUM 8-AMINOLEVULINIC ACID DEHYDRATASE

sequence of rat liver f-aminolevulinic acid dehydratase cDNA. Nucleic Acids Res. 14:10115. Bishop, T. R., Z. I. Hodes, L. P. Frelin, and S. H. Boyer. 1989. Cloning and sequence of mouse erythroid 8-aminolevulinate dehydratase cDNA. Nucleic Acids Res. 17:1775. Boese, Q. F., A. Spano, J. M. Li, and M. P. Timko. 1991. Aminolevulinic acid dehydratase in pea (Pisum sativum L). Identification of an unusual metal-binding domain in the plant enzyme. J. Biol. Chem. 266:17060-17066 Brockl, G., M. Berchtold, M. Behr, and H. Konig. 1992. Sequence of the 5-aminolevulinic acid dehydratase-encoding gene from the hyperthermic methanogen Methanothermus sociabilis. Gene 119: 151-152. Devereaux, J., P. Haeberli, and 0. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387-395. Frustaci, J. M., and M. R. O'Brian. 1992. Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene. J. Bacteriol. 174:4223-4229. Frustaci, J. M., and M. R. O'Brian. 1993. Analysis of the Bradyrhizobium japonicum hemH gene and its expression in Escherichia coli. Appl. Env. Microbiol. 59:2347-235 1. Frustaci, J. M., I. Sangwan, and M. R. O'Brian. 1991. Aerobic growth and respiration of a 8-aminolevulinic acid synthase (hemA) mutant of Bradyrhizobium japonicum. J. Bacteriol. 173:1145-1150. Guerinot, M. L., and B. K. Chelm. 1986. Bacterial 8-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis. Proc. NatI. Acad. Sci. USA 83:1837-1841. Hansson, M., L. Rutberg, I. Schroder, and L. Hederstedt. 1991. The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III. J. Bacteriol. 173:2590-2599. Herrada, G., A. Puppo, and J. Rigaud. 1992. A-Aminolevulinate uptake by Rhizobium bacteroids and its limitation by the peribacteroid membrane in legume nodules. Biochem. Biophys. Res. Commun. 184:1324-1330. Jordan, P. M. 1990. The biosynthesis of 5-aminolevulinic acid and its transformation into coproporphyrinogen in animals and bacteria, p. 55-121. In H. A. Dailey (ed.), Biosynthesis of heme and chlorophyll. McGraw-Hill Publishing Co. New York. Kaczor, C. M., and M. R. O'Brian. Unpublished observations. Li, J. M., C. S. Russell, and S. D. Cosloy. 1989. The structure of the

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Escherichia coli hemB gene. Gene 75:177-184. 17. Li, J. M., H. Umanoff, R. Proenca, C. S. Russell, and S. D. Cosloy. 1988. Cloning of the Escherichia coli K-1 2 hemB gene. J. Bacteriol. 170:1021-1025. 18. Myers, A. M., M. D. Crivellone, T. J. Koerner, and A. Tzagoloff. 1987. Characterization of the yeast HEM2 gene and transcriptional regulation of COX5 and CORI by heme. J. Biol. Chem. 262:16822-16829. 19. O'Brian, M. R., P. M. Kirshbom, and R. J. Maier. 1987. Bacterial heme synthesis is required for the expression of the leghemoglobin holoprotein but not the apoprotein in soybean root nodules. Proc. Natl. Acad. Sci. USA 84:8390-8393. 20. Sangwan, I., and M. R. O'Brian. 1991. Evidence for an interorganismic heme biosynthetic pathway in symbiotic soybean root nodules. Science 251:1220-1222. 21. Sangwan, I., and M. R. O'Brian. 1992. Characterization of 8-aminolevulinic acid formation in soybean root nodules. Plant Physiol.

98:1074-1079. 22. Sangwan, I., and M. R. O'Brian. 1993. Expression of the soybean (Glycine max) glutamate 1-semialdehyde aminotransferase gene in symbiotic root nodules. Plant Physiol. 102:829-834. 23. Schaumburg, A., H. A. W. Schneider-Poetsch, and C. Eckerskorn. 1992. Characterization of plastid 5-aminolevulinate dehydratase (ALAD; EC 4.2.1.24) from spinach (Spinacia oleracea L.) by sequencing and comparison with non-plant ALAD enzymes. Z. Naturforsch. Teil C 47:77-84. 24. Scott, A. I., and P. J. Santander. 1991. The biosynthesis of vitamin B12 p. 101-138., In P. M. Jordan (ed.), Biosynthesis of tetrapyrroles. Elsevier, New York. 25. Spencer, P., and P. M. Jordan. 1993. Purification and characterization of 5-aminolevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain. Biochem. J. 290:279-287. 26. Udvardi, M. K., G. D. Price, P. M. Gresshoff, and D. A. Day. 1988. A dicarboxylate transporter on the peribacteroid membrane of soybean nodules. FEBS Lett. 231:36-40. 27. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268. 28. Wetmur, J. G., D. F. Bishop, C. Cantelmo, and R. J. Desnick. 1986. Human 8-aminolevulinic acid dehydratase: nucleotide sequence of a full-length cDNA clone. Proc. Natl. Acad. Sci. USA 83:77037707.