(Received for publication, April 14, 1995; and in revised form, June 26, 1995)
From the
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.
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 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 ()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
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 (
)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.
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.
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
(A) or ZnSO
(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.
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. Bottompanel, ALA dehydratase
activity was measured in extracts in the presence of 50 µM
ZnSO
. 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 A of 0.009.
The protein concentration of the extracts was 8 mg/ml for cultured
cells and 4 mg/ml for symbiotic bacteroids.
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.
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.