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INTRODUCTION |
The nitrogenase enzyme, which is responsible for conversion of
atmospheric nitrogen to ammonia, is found in all diazotrophs. It is
actually composed of two separately purified, oxygen-labile, metalloproteins designated the Fe protein and the MoFe protein (1-7).
The Fe protein is a homodimer of two identical subunits encoded by the
nifH. Both subunits are bridged by one 4Fe-4S metal center
and contain two nucleotide (MgATP or MgADP)-binding sites (5, 8-10).
The Fe protein serves as the obligate electron donor to MoFe protein
during catalysis in a MgATP- and reductant-dependent process (11, 12). The MoFe protein exists as an
2
2 tetramer of
about 240 kDa in size, encoded by nifD and nifK
genes, respectively (13, 14). Each 
heterodimer subunit binds two
unique metal clusters, the FeMo cofactor and the P cluster. The FeMo
cofactor that serves as the site of dinitrogen binding and reduction by the enzyme is located in the
subunit, and the P cluster, which is
positioned at the interface of
and
subunits of the heterodimer, is believed to mediate electron transfer from the Fe protein to the
FeMo cofactor (15-17). During catalysis, the Fe protein forms a
complex with the MoFe protein and transfers one electron to the MoFe
protein with concomitant hydrolysis of two ATPs per Fe-MoFe protein
interaction. It is accepted that the electron flow is from the Fe
protein cluster to the MoFe protein P cluster and then to the FeMo
cofactor, which is the substrate-binding and reduction site (13,
18-20).
Molecular evolutionary history of the nifDK, based on
comparative analysis of the amino acid sequence of NifD, NifK, NifE, and NifN peptides, suggested that the genes encoding the NifDK and
NifEN constitute a novel paralogous gene family (21). The NifEN
complex, which is required for the biosynthesis of the FeMo cofactor of
nitrogenase, is structurally analogous to the MoFe protein and provides
an assembly site for FeMo cofactor biosynthesis (22, 23). Like the MoFe
protein, the NifEN complex is also an
2
2 heterotetramer of about
200 kDa in size and contains unique metal clusters that are similar to
the P cluster of the MoFe protein. According to the model proposed by
Goodwin et al. (24), each NifEN complex [4Fe-4S] cluster
is bridged between a NifE-N heterodimer subunit interface at a position
analogous to that occupied by the P clusters of the MoFe protein. The
NifD is homologous to the NifE, and NifK is homologous to the NifN as
shown in Fig. 1. In fact homology
modeling studies showed that the predicted structure of the NifE is
very similar to the crystallographic model of NifD, and the predicted
structure of the NifN is very similar to the crystallographic model of
NifK (data not shown). The organization of these genes (nifD
followed by nifK and nifE followed by
nifN) is also conserved among diazotrophs (21, 25). However,
in Anabaena variabilis the NifEN complex is encoded by a
fused nifE-N like gene instead of two separate
nifE and nifN genes (26). In A. variabilis there are two nif clusters, the nif1 and the nif2. Whereas the
nif1 cluster carries two separate nifE and
nifN genes, the nif2 cluster has the
nifE-N gene fusion. A comparison of the nucleotide sequences
of the naturally fused NifE-N complex to that of the NifEN complex
synthesized by translating two separate genes in this organism showed
that nifE-N junction of the translationally fused
nifE-N was different from the junction of the
nifE and nifN genes of the nif1
cluster. This difference resulted in changes in the amino acid sequence
of NifE-N fusion protein encoded by the nif2 cluster
when compared with the NifE and the NifN encoded by the nif1
cluster. In Azotobacter vinelandii, the NifEN complex is
encoded by the nifE and nifN genes that are translated separately. Comparison of the amino acid sequences of the
NifEN complexes from A. variabilis to that of A. vinelandii showed that the NifEN complex encoded by the
nif1 cluster of A. variabilis is very similar to
that of the A. vinelandii. The observation that in A. variabilis the NifE-N fusion protein could function in nitrogen
fixation in a comparable manner to that of the wild type NifEN complex
implied that this complex metalloprotein is flexible and could
accommodate major structural changes and differences in the pattern of
biosynthesis and assembly (being translated as a single protein
versus being translated as two separate proteins and then
assembled as a heterodimer subunit) as long as its catalytic center is
not affected seriously by the changes. Because the MoFe protein shares
structural similarity with the NifEN complex in A. vinelandii, we were interested in testing whether the MoFe protein could be synthesized as a fusion protein by translationally fusing the nifD and the nifK genes. To test this
idea, we generated an A. vinelandii strain that encodes
NifD-K fusion protein encoded by translationally fused nifD
and nifK genes. Here we report the structural and functional
properties of the NifD-K fusion protein.

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Fig. 1.
Alignment of the amino acid sequences
of the NifEN complexes and the MoFe protein of A. vinelandii. The NifE
(GenBankTM accession number P08293) shares homology
with the NifD ((GenBankTM accession number AAA64710)
and the NifN (GenBankTM accession number P10366)
shares homology with the NifK (GenBankTM accession
number AAA64711). The extent of shading represents the increase in
homology. Starting points of the NifE, NifD, NifN, and the NifK are
labeled and marked by arrows. Asterisks
show the stop codons that terminate the translation of NifE and NifD.
An overall homology of 29.5% is seen between the NifEN complex and the
MoFe protein.
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MATERIALS AND METHODS |
Bacterial Strains and Growth Conditions--
A.
vinelandii strains were grown at 30 °C in modified Burk's
nitrogen-free (BN
) medium (27) whenever
nitrogen-deficient medium was needed, and when nitrogen was required
BN
medium supplemented with 400 µg/ml of ammonium
acetate was used. The Escherichia coli strains were grown at
37 °C in Luria broth or 2YT (28). Ampicillin and kanamycin were used
to a final concentration of 50 and 20 µg/ml, respectively, wherever
the selection was made. The bacterial strains and plasmids used in this
study are listed in Table I.
Construction of Plasmids Carrying A. vinelandii nifD-K Gene
Fusion--
The strategy used for the construction of plasmids
carrying A. vinelandii nifD-K gene fusion is shown in
Fig. 2. Initially we constructed the
plasmid pBG1404 that carries the A. vinelandii nifD-K gene
fusion as shown in step 1. The chromosome of the wild type
A. vinelandii was isolated and was used as template to
amplify the nifD and the nifK genes by PCR
amplification (29). The primers used to amplify the nifD
gene were D5-oligo and BAMD3-oligo (Fig. 2, step 1). The
D5-oligo, 5'-GACCGGTATGTCGCGCGAAGAGGTTGAAT-3', was the 5' primer
starting from the 3rd nucleotide of the nifD gene. The
BAMD3-oligo, 5'-GGATCCGATTGCTCTGCGATCCGGCGCTGGCGGCGA-3', was the 3' primer and contained a BamHI site. This sequence
corresponded to the region of nifD that spans the codons 488 to the end of the ORF1 and
another 21 nucleotides. The two stop codons that follow the nifD ORF were changed to sense codons (TGA to GGA and TAG to
TCG), and the remaining 15 nucleotides corresponded to five sense
codons (CAG, AGC, AAT, CGC, and AGC). Thus, the DNA fragment amplified by using these primers corresponded to region of the NifD from the
second codon to the end of the open reading frame, and an extra seven
sense codon extending the ORF. Creating a BamHI site at the
3' end of this fragment also resulted in replacing the last two codons
of NifE, Glu and Arg with Gly and Ile, respectively. This fragment was
cloned into the pCR2-1 TOPO to generate plasmid pBG1285 (Fig. 2). This
plasmid was then digested with EcoRV and BamHI to
generate the nifD fragment for creating the translational fusion of nifD-K in plasmid pBG1404 (Fig. 2). The primers
used to amplify the nifK gene were BAMK5-oligo and K3-oligo
(Fig. 2, step 1). The BAMK5-oligo, 5'-
GGATCCAGCTACCCGCTGTTCCTCGATCAGGAC-3', was the 5' primer and contained a
BamHI site. This sequence corresponded to the region of
nifK that spans the codons 9-19. Introducing a
BamHI site also resulted in changing the 9th and 10th
codons, Lys and Ala, respectively, to Gly and Ser. The K3-oligo,
5'-AGCGTACCAGGTCGTGGTTGTAGTCGGT-3', was the 3' primer. This sequence
corresponded to the C-terminal region of nifK that spans the
codons 515-523. Thus, the DNA fragment amplified by using these
primers corresponded to the region of nifK starting from
the 9th codon (the initiation codon ATG and the following seven codons
were omitted) to the end of the ORF. This fragment was cloned into the
pCR2-1 TOPO to generate plasmid pBG1279 (Fig. 2). This plasmid was then
digested with BamHI and EcoRI to generate the
nifK fragment for creating the translational fusion of
nifD-K in plasmid pBG1404 (Fig. 2). The plasmid pBG1404 was
generated as follows. The pUC18 was digested with SmaI and EcoRI to linearize the plasmid. Because the SmaI
generated blunt ends, they could be ligated with the blunt-ended
EcoRV end of the EcoRV-BamHI fragment
isolated from pBG1285 and that carries the nifD portion of
the nifD-K fusion. Therefore, the
SmaI-EcoRI-digested pUC18 was ligated with two
DNA fragments, the EcoRV-BamHI fragment isolated
from pBG1285 (as described above), and the
BamHI-EcoRI fragment that carries the
nifK portion of the nifD-K fusion. Thus, pBG1404
is a derivative of pUC18. Nucleotide sequencing of the nifD-K junction in pBG1404 showed that joining the
BamHI ends of the DNA fragments encoding the nifD
and the nifK along with two nucleotide changes introduced at
the junction because of PCR amplification resulted in creating a
nifD-K junction that differed from the wild type junction of
the nifD and the nifK genes by loss of three
amino acids and seven mismatches. A comparison of the NifD-K junction
in wild type A. vinelandii strains and in plasmid
pBG1404 is shown in Fig. 3. The plasmid
pBG1021, in which the kanamycin resistance gene was inserted to
inactivate nifDK, was described previously (30).

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Fig. 2.
Strategy for the construction of plasmid
pBG1404 that carries the junction of the translationally fused
nifD-K is shown. The plasmids PBG1285 and PBG1279
are pCR2-1 TOPO vector derivatives that carry the PCR-amplified
nifD with an extended ORF (extended by seven codons) and no
stop codon and the PCR-amplified nifK that does not have the
first eight codons, respectively. The location of the primers used for
amplifying the nifD and the nifK fragments and
the direction of transcription of these fragments are marked. To
generate the DNA fragment specifying the nifD, the plasmid
pBG1285 was digested with EcoRV (this restriction site is
located in the multicloning site of pCR2-1 TOPO vector) and
BamHI (this restriction site is part of the BAMD3 primer).
To generate the DNA fragment specifying the nifK, the
plasmid pBG1279 was digested with BamHI (this restriction
site is part of the BAMK5 primer) and EcoRI (this
restriction site is located in the multicloning site of pCR2-1 TOPO
vector). The molecular sizes of the DNA fragments specifying the
nifD and the nifK generated by restriction
digestion of the plasmids pBG1285 and pBG1279 are shown in
gray boxes. The three DNA fragments that were ligated
together to make pBG1404 were the 1510-bp
EcoRV-BamHI fragment from pBG1285 specifying the
nifD (A), the 1552-bp
BamHI-EcoRI fragment from pBG1279 that specifies
the nifK (B), and the vector backbone of pUC19
(2673-bp fragment generated by SmaI-EcoRI
digestion) (C). Curved arrows between plasmids
represent the sources of DNA fragments that were ligated together
during each plasmid construction.
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Fig. 3.
Structural changes at the translationally
fused junction of ORFs of nifD and nifK
in the plasmid pBG1404. NifDK1 represents the junction in
the chromosome of wild type A. vinelandii where
nifD and nifK are translated separately. NifD-K
represents the junction of translationally fused nifD and
nifK present in plasmid pBG1404. Asterisk
represents the stop codon of nifD on the chromosome. The
following M is the translation initiation codon for
nifK. The dashes correspond to the missing amino
acids when nifD and nifK were translationally
fused by ligating the BamHI ends of the DNA fragments
specifying the nifD and the nifK. During this
construction, the first eight amino acids of the NifK were removed, and
the ORF of NifD was extended by seven amino acids by converting the
stop codons to sense codons. These changes combined with loss of
nucleotides during the ligation of the BamHI ends of the DNA
fragments specifying the nifD and the nifK, and
two nucleotide changes that happened during the PCR amplification,
resulted in a total loss of three amino acids and seven mismatches as
shown in the figure.
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Transformation of A. vinelandii, Growth Curve, and Western
Blotting--
Because the plasmids pBG1404 and pBG1021 are
derivatives of the pUC18, they are not capable of replicating in
A. vinelandii. However, the very high recombination
efficiency of A. vinelandii strains allows rescuing of the
regions that are homologous to the chromosome from non-replicative
plasmids. Therefore, these plasmids could be used to deliver the
nifD-K fusion or the disrupted nifD-K (due to the
insertion of the kanamycin resistance gene) to the chromosome of wild
type A. vinelandii via homologous recombination. The
transformation of A. vinelandii Trans was carried out
as described previously (31-33). The resulting transformants were
selected either on BN+ medium containing 5 µg/ml
kanamycin or on BN
medium. Growth characteristics of the
transformants were determined by growth curve analysis as described
previously (34). ECL Western blotting analysis system (Amersham
Biosciences) was used to determine the presence of the NifD-K fusion
protein in the cell lysates of A. vinelandii strains
carrying the wild type nifDK operon and the
nifD-K translational fusion. Anti-MoFe protein antibody was a gift from Prof. Barbara K. Burgess, Department of Molecular Biology
and Biochemistry, University of California, Irvine.
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RESULTS AND DISCUSSION |
It was shown previously that the NifE-N fusion protein in
A. variabilis is functional in biological nitrogen
fixation (26, 34). As mentioned above, the NifEN complex is a
metalloprotein that resembles the MoFe protein in many aspects. For
example, both the MoFe protein and the EN complex are seen as
2
2
tetramers. It was also shown that the NifEN has two identical
[4Fe-4s]2+ clusters similar to the P clusters found in
the MoFe protein. The extent of amino acid sequence homology between
the components of these two metalloproteins (between NifD and NifE as
well as between NifK and NifN) is also significant (Fig. 1). The
observation that the NifE-N fusion protein could support biological
nitrogen fixation as efficiently as the wild type NifEN complex implied that this metalloprotein could accommodate major structural changes and
differences in the pattern of their biosynthesis (either translated as
separate NifE and NifN proteins and assembled or translated as a fusion
protein in which the NifE and NifN are encoded by a single gene) and
still remain functional. Therefore, it was interesting to investigate
whether the MoFe protein that shares structural similarity with the EN
complex could also be synthesized as a fusion protein. To test this
idea, we employed a strategy to construct of A. vinelandii
strain that can synthesize the NifD-K fusion protein.
Construction of an A. vinelandii BG1304 Carrying the nifD-K Gene
Fusion--
The plasmid pBG1404 (Fig. 2) was employed in the
construction of an A. vinelandii strain carrying the
nifD-K gene fusion. As mentioned under "Materials and
Methods," one of the advantages of A. vinelandii is that
it has a high efficiency recombination system that allows the delivery
of mutated genes to the chromosome by homologous recombination (33, 35,
36). The plasmid pBG1404, which is a derivative of pUC18 and possessed
ColE1 replicon, could not replicate in A. vinelandii.
Because it carried portions of wild type nifD and
nifK genes that flanked the nifD-K gene fusion junction, it could undergo homologous recombination with identical regions of the nifD and nifK on the A. vinelandii chromosome and cause rescuing of the nifD-K
gene fusion or the kanamycin resistance gene onto the chromosome.
Construction of the A. vinelandii strain BG1065 in which the
kanamycin resistance gene interrupted the nifDK expression
on the chromosome was described previously (30). We observed that the
crude extracts from A. vinelandii strain BG1065 contained only the NifD, when these extracts were subjected to SDS-PAGE, Western
blotting, and probing with anti-MoFe protein antibody. This strain
could grow on BN+ agar containing 5 µg/ml kanamycin (Fig.
4) but could not grow on BN
agar, because this strain was unable to produce any functional MoFe
protein (Fig. 4). This strain was then transformed with pBG1404 that
carries the uninterrupted nifD-K gene fusion. The resulting transformants were selected on BN
agar. These
transformants could grow on BN
agar only when the
nifD-K translational fusion junction disrupted by the
kanamycin resistance gene present on the chromosome of the A. vinelandii strain BG1065 was replaced by the nifD-K
gene fusion junction present on the pBG1404. Homologous recombination that would lead to this event would also cause the loss of the kanamycin resistance. One of these transformants that could grow on
BN
agar, but was unable to grow on BN+ agar
containing 5 µg/ml kanamycin, was designated A. vinelandii strain BG1304 (Fig. 4). This result confirmed that the
nifD-K gene fusion junction present on the pBG1404 was
rescued on to the chromosome of A. vinelandii strain BG1304
by homologous recombination and also that the kanamycin resistance gene
was lost during this process. Nucleotide sequence of the
nifD-K junction present on the chromosome of A. vinelandii BG1304 was further verified by PCR amplification and
nucleotide sequencing of the DNA specifying nifD-K junction.
The sequence of the nifD-K junction present on the
chromosome of A. vinelandii BG1304 is shown in Fig.
5.

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Fig. 4.
Translationally fused NifD-K can support
growth of A. vinelandii on nitrogen
deficient medium. The chromosome of A. vinelandii
BG1065 carries the kanamycin resistance gene that disrupts
synthesis of the MoFe protein. This strain can grow on BN+
agar supplemented with kanamycin but cannot grow on nitrogen-deficient
BN agar. Neither A. vinelandii Trans (wild
type) nor A. vinelandii BG1304 that carries the
translationally fused nifD-K are able to grow on
BN+ agar supplemented with kanamycin because they do not
have kanamycin resistance gene. However, both strains are capable of
growing on BN agar. These results indicated that the
A. vinelandii BG1304, a derivative of A. vinelandii BG1065, had lost the kanamycin marker that was
originally present on the chromosome of A. vinelandii BG1065
during homologous recombination between the nifD and
nifK regions on the chromosome and the plasmid pBG1404 and
gained the nifD-K gene fusion.
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Fig. 5.
Nucleotide sequence of the junction of the
translationally fused nifD and nifK
genes in the chromosome of A. vinelandii
BG1304. PCR amplification of the nifD-K junction
present on the chromosome of A. vinelandii BG1304 was
carried out using internal primers corresponding to middle regions of
the nifD and NifK. Nucleotide sequence was
determined using dideoxy nucleotide sequencing technique. The location
of C-terminal region of the nifD and N-terminal region of
the nifK is labeled. The BamHI site at the
junction of the NifD-K fusion is shown in boldface.
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The NifD-K Fusion Could Support Biological Nitrogen
Fixation--
The observation that the A. vinelandii BG1304
could grow on BN
agar plates indicated that the MoFe
protein encoded by the nifD-K translational fusion is
functional in biological nitrogen fixation. To analyze further whether
the MoFe protein encoded by the nifD-K gene fusion is
comparable with the wild type MoFe protein in its ability to
participate in biological nitrogen fixation, we compared the growth
rates of A. vinelandii Trans (which expresses the wild type MoFe protein encoded by separate nifD and
nifK genes) with that of the A. vinelandii BG1304
(which expresses the MoFe protein encoded by the nifD-K gene
fusion) on nitrogen-deficient medium. Single colonies from each strain
were inoculated into BN
medium, and growth rate was
assessed using a Klett-Summerson Colorimeter (Klett Manufacturing Co.
Inc., New York) as described previously. These experimental results
showed that compared with A. vinelandii Trans, A. vinelandii BG1304 was a slow-growing strain on nitrogen-deficient
medium (Fig. 6). These results suggested that although the MoFe protein encoded by the nifD-K gene
fusion junction is capable of supporting the biological nitrogen
fixation, its efficiency is not as good as that of the wild type MoFe
protein.

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Fig. 6.
The A. vinelandii
BG1304 is a slow growing strain on nitrogen-deficient
medium. The growth rate of the A. vinelandii
(AV) BG1304 was compared with that of the wild type strain
A. vinelandii Trans and also with the A. vinelandii BG1065 in which the MoFe protein synthesis is disrupted
by the insertion of a kanamycin resistance gene. All the strains were
grown from single colonies, and the medium used was nitrogen-deficient
BN liquid medium. The A. vinelandii BG1065 did
not grow on BN liquid medium. A. vinelandii
BG1304 was capable of growing on BN liquid medium,
although it took longer time to reach stationary phase suggesting that
its growth rate was slower than that of the A. vinelandii
Trans.
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The MoFe Protein of A. vinelandii BG1304 Is a 120-kDa
Protein--
SDS-PAGE analysis followed by Western blotting and
probing with anti-MoFe protein-antibody was employed to determine the
molecular size of the MoFe protein components encoded by the A. vinelandii BG1304. The wild type MoFe protein is made of two
separately purified proteins, the
subunit or the NifD with a
molecular mass of 55 kDa and the
subunit or the
nifK with a molecular mass of 59 kDa. When the
nifD and nifK were fused translationally (as in A. vinelandii BG1304), the resulting fusion protein should
have a molecular mass of 114 kDa. The A. vinelandii BG1304
and A. vinelandii Trans were grown on BN
liquid medium to early stationary phase, and the cells were collected by centrifugation. The cells were resuspended in sample loading buffer,
and lysis was carried out by heating for 10 min at 95 °C. The
samples were applied on SDS-polyacrylamide gel, and after electrophoresis the proteins from the gels were transferred to nylon
membranes (Schleicher & Schuell) by using Bio-Rad Gel Transfer Chamber.
ECL Western blotting analysis system was used to determine the
molecular sizes of the components of the MoFe protein in both samples.
Fig. 7A shows the results of
Coomassie Blue staining of the SDS-polyacrylamide gel to which samples
were applied. In the A. vinelandii BG1304 sample a new band
corresponding to the molecular mass of about 120 kDa was visible. This
band was missing in the sample, of A. vinelandii Trans. Fig.
7B shows the results of Western blotting analysis using
anti-MoFe antibody as probe. In the sample A. vinelandii
BG1304, a band corresponding to the molecular mass of about 120 kDa was
visible suggesting that the MoFe protein of this strain is composed of
translationally fused NifD and NifK proteins. This is the new 120-kDa
protein band that was also found in the SDS-polyacrylamide gel lane
carrying the sample from A. vinelandii BG1304 after
Coomassie Blue staining (Fig. 7A). The Western blotting
analysis using anti-MoFe antibody as probe showed a band of ~60 kDa
that corresponded to the NifD and NifK proteins (which could co-migrate
on the gel under these experimental conditions) in the lane containing
the sample from of A. vinelandii Trans. This was expected
because the NifD and NifK are synthesized separately in this strain.
Taken together, these results confirmed that the MoFe protein of
A. vinelandii BG1304 is indeed a NifD-K fusion protein.

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Fig. 7.
The MoFe protein synthesized by
translationally fused nifD-K in A. vinelandii BG1304 is a large protein.
A shows the Coomassie Blue-stained SDS-polyacrylamide gel.
Each lane contained 0.5 mg of total protein. A band corresponding to
120 kDa is visible in the lane 2, which was loaded with cell
lysate from A. vinelandii BG1304. This molecular size
corresponds to the size of translationally fused NifD-K. This band is
missing in the lane 1, which was loaded with cell lysate
from A. vinelandii Trans. B shows the results of
Western blot analysis after being probed with anti-MoFe antibody.
A. vinelandii Trans shows a band of about 60 kDa molecular size
(lane 3) that corresponds to co-migrating and subunits (or NifD and NifK) of the MoFe protein. A. vinelandii BG1304 shows a band of about 120 kDa molecular size
(lane 4) that corresponds to the NifD-K fusion
protein.
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Next we verified whether the nifD-K gene fusion was
integrated downstream to the nifH promoter in such a way
that the transcription of the fusion gene was regulated by the
nifH promoter, as seen in the case of wild type A. vinelandii. A PCR amplification of the nifD-K fusion
gene using the forward primer corresponding to the C-terminal sequence
of the nifH and the reverse primer corresponding to the
nifK C-terminal sequence, and chromosomal DNA of A. vinelandii BG1304 as template, resulted in generating a DNA band
corresponding to 2.6 kb, the molecular size expected only if
nifD-K fusion gene was integrated downstream to the
nifH. This PCR product was subjected to nucleotide sequence
analysis, and the location of integration and presence of the
nifD-K fusion junction was further confirmed. This result
suggested that the nifD-K fusion gene was integrated
downstream to the nifH promoter. To confirm that the
expression of the nifD-K fusion gene was under the
transcriptional control of the nifH promoter, we grew
A. vinelandii BG1304 in Burk's medium supplemented
with nitrogen. A. vinelandii Trans grown under similar
conditions was used as the control. Crude extracts from A. vinelandii BG1304 and A. vinelandii Trans were then
subjected to SDS-PAGE, Western blotting, and probing with anti-MoFe
protein antibody. No bands corresponding to the MoFe protein were
detected either in the crude extracts from A. vinelandii
BG1304 or in the crude extracts from A. vinelandii Trans.
These results supported the idea that the nifD-K fusion gene
in the chromosome of A. vinelandii BG1304 is under the
transcriptional control of the nifH promoter.
The MoFe protein encoded by A. vinelandii BG1304 (or the
NifD-K fusion protein) has many structural differences when compared with the wild type MoFe protein encoded by A. vinelandii
Trans. The wild type MoFe protein is visualized by using the RasMol
program in Fig. 8A. This
figure shows one
and
subunit of the MoFe protein depicted as
ribbon diagram and the other
and
subunit of the MoFe protein
depicted as line diagram. The Ala-481 at the C terminus of the NifD and
the Pro-13 at the N terminus of the NifK are shown. In the NifD-K
fusion protein, both these residues were connected with the amino acid
sequence that included
481-
491, and nine newly inserted residues
(GSQSNRIQL). The Fig. 8B shows the NifD-K fusion protein
with these newly inserted residues that join the C terminus of NifD to
the N terminus of NifK. The structural changes include the following:
(a) conversion of two stop codons that terminate translation
of the NifD into sense codons and thus extend the NifD by seven new
amino acids; (b) removal of the first eight amino acids of
the NifK resulting in removing the translation initiation codon of the
NifK, where translation is normally initiated to synthesize NifK;
(c) conversion of the 9th and 10th amino acids of NifK from
Lys and Ala to Gly and Ser, respectively; and (d) a change
in the ORF of the NifD-K fusion junction caused by joining the
BamHI ends of the DNA fragments encoding nifD and
nifK along with two nucleotide changes introduced during PCR
amplification. As shown in Fig. 3, these changes resulted in an overall
loss of three amino acids and seven mismatches at the fusion junction of the NifD and the NifK, generating a NifD-K fusion protein that was
smaller in size when compared with the wild type MoFe protein. Moreover, the biosynthesis and assembly of the MoFe protein encoded by
A. vinelandii BG1304 is also significantly different from
that of the wild type MoFe protein encoded by A. vinelandii
Trans, because in the first case a single large NifD-K fusion protein is synthesized, and in the second case the smaller NifD and NifK are
synthesized separately and then assembled together. The extent of amino
acid changes that occurred during the construction of NifD-K fusion
protein is a total of 10 amino acids. It is interesting to note that
the NifD-K fusion protein was functional despite all these amino acid
changes and could support biological nitrogen fixation. The reduced
efficiency of the NifD-K fusion protein to support growth on
nitrogen-deficient medium when compared with the wild type MoFe protein
is not surprising considering the major structural changes that were
introduced during the construction of the fusion protein. Nevertheless,
the fact that the NifD-K fusion protein is functional points out that
the MoFe protein is also flexible, similar to the NifE-N (34) regarding
the amino acid composition and nature of biosynthesis and assembly as
long as the catalytic sites are unaffected.

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Fig. 8.
The structure of the MoFe protein and
the NifD-K fusion protein as visualized in RasMol program.
A shows the wild type MoFe protein. One and subunit
is shown as the ribbon diagram and the other and subunit is
shown as the line diagram. The and subunits are labeled. The
location of the Ala-481 residue at the C terminus of the subunit
and the location of the Pro-13 residue at the N terminus of the subunit are marked by arrows. B shows the NifD-K
fusion protein. The residues joining the Ala-481 of the subunit
with the Pro-13 of the subunit are shown by the arrow,
and the newly inserted residues in the NifD-K fusion protein are
listed.
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