(Received for publication, April 17, 1995; and in revised form, July 5, 1995)
From the
Azotobacter vinelandii ferredoxin I (AvFdI)
controls the expression of another protein that was originally
designated Protein X. Recently we reported that Protein X is a
NADPH-specific flavoprotein that binds specifically to FdI (Isas, J.
M., and Burgess, B. K.(1994) J. Biol. Chem. 269,
19404-19409). The gene encoding this protein has now been cloned
and sequenced. Protein X is 33% identical and has an overall 53%
similarity with the fpr gene product from Escherichia
coli that encodes NADPH:ferredoxin reductase. On the basis of this
similarity and the similarity of the physical properties of the two
proteins, we now designate Protein X as A. vinelandii NADPH:ferredoxin reductase and its gene as the fpr gene.
The protein has been overexpressed in its native background in A.
vinelandii by using the broad host range multicopy plasmid,
pKT230. In addition to being regulated by FdI, the fpr gene
product is overexpressed when A. vinelandii is grown under
N-fixing conditions even though the fpr gene is
not preceded by a nif specific promoter. By analogy to what is
known about fpr expression in E. coli, we propose
that FdI may exert its regulatory effect on fpr by interacting
with the SoxRS regulon.
Azotobacter vinelandii ferredoxin I (AvFdI) is
a small protein, M = 12,700 that has been
characterized extensively by x-ray crystallography (1, 2, 3, 4, 5) and by
detailed spectroscopic (6, 7, 8, 9) and electrochemical (10) studies. The protein contains two types of
[Fe-S] clusters: one [3Fe-4S]
cluster and one [4Fe-4S]
cluster with pH 7.8 reduction potentials of -425 mV and
-647 mV, respectively. Sequence comparisons show that AvFdI is a member of a closely related class of 7Fe
ferredoxins found in a variety of organisms(10) . Given the
enormous amount of information that is available concerning these
proteins, it is surprising that their specific cellular functions have
not as yet been determined.
One approach that was taken to this
problem involved the disruption of the A. vinelandii fdxA gene
that encodes FdI(11) . The resulting A. vinelandii strain, designated LM100, had no obvious phenotype with respect to
cell growth(11) . However, two-dimensional gel electrophoresis
analysis of LM100 revealed that there was another small acidic protein
that was dramatically overproduced in the FdI strain
when compared to the wild-type. This protein was given the trivial name
Protein X(11, 12) . The observation that the synthesis
of Protein X was ``repressed'' by FdI further led to the
proposal that FdI might be a novel DNA binding repressor
protein(13) . Thus, FdI appears to have both an electron
transfer and a regulatory function. In order to gain insight into both
of these functions, we previously reported the purification and
characterization of Protein X(14) . It was shown to be a M
29,000, NADPH-specific flavoprotein whose
physical properties and NH
-terminal amino acid sequence
showed striking similarity with the NADPH:ferredoxin reductase from Escherichia coli(14) . Protein X was further shown to
bind FdI specifically, suggesting that the two proteins were likely to
be redox partners in vivo(14) .
Here we report the cloning and sequencing of the A. vinelandii gene encoding Protein X and the overexpression of the protein product.
Figure 1: Construction of pS-XF1.5Sal. Plasmid pS-XF1.5Sal is an A. vinelandii DNA-Bluescript II hybrid which contains approximately 1.5 kb of A. vinelandii genomic DNA including the fpr gene. Its construction is described in the text. Restriction enzymes used were: S, SalI; X, XhoI; A, AsmI; N, NcoI; E, EcoRV; BglII; and P, PstI.
Figure 2: Sequencing strategy used to establish the nucleotide sequence of the fpr gene. Details are as described in the text.
Figure 3: DNA sequence of the coding strain of the fpr gene and flanking regions. This sequence has been given the Genome Sequence Data Base Accession number L36319. Amino acids are numbered from the ATG codon. No consensus nif promotor sequences [GCTGG-8bp-CTGCA] (21) were found upstream of the start site for the fpr gene. Two putative promoter elements and the ribosome binding site are denoted by brackets. Downstream of the FdI coding region there is a predicted stem loop structure, denoted by (>) that probably acts as a transcription termination signal.
Fig. 4compares the sequence of the A. vinelandii fpr gene product reported here to that of the previously published E. coli fpr gene product(19) . When these two
sequences are optimally aligned, 33% of the amino acids are identical
and the overall similarity is 53%. In E. coli, the fpr gene product was originally designated dA1 and was proposed to be
a NADPH:ferredoxin reductase based on its similarity to the spinach
enzyme(19) . We have previously compared the physical
properties of the protein products from E. coli and A.
vinelandii and have shown that both proteins have molecular
weights of 29,000, that both contain noncovalently bound FAD, that
both are specific for NADPH, and that both catalyze the
ferredoxin-dependent reduction of cytochrome c(14) .
On this basis, and with the additional sequence information shown in Fig. 4, we now designate the A. vinelandii fpr product
(which was formerly given the trivial name Protein X(11) ) as A. vinelandii NADPH:ferredoxin reductase
(ferredoxin-NADP
reductase, EC 1.18.1.2).
Figure 4:
Comparison of homologous NADPH:ferredoxin
reductases. A, compares the A. vinelandii (Av) FPR sequence reported here to the published (36) E. coli (Ec) FPR sequence. The program
used was GAP from the Genetics Computer Group, Inc., Madison, WI.
Identical residues (), strongly (:) and weakly (.) conserved
amino acids are indicated.
As shown in Fig. 4, the similarity of the A. vinelandii protein to the E. coli NADPH:ferredoxin reductase occurs over the entire length of the proteins to yield products of similar molecular weight. A recent structural study of an extended family of flavoprotein reductases further identified six sequence motifs that are highly conserved among all members of the family and are involved in FAD and NADPH binding(20) . As shown in Fig. 5, all six of these regions are also highly conserved when the E. coli and A. vinelandii sequences are compared, confirming that the proteins are likely to be closely related functionally.
Figure 5:
Conserved regions that characterize the
ferredoxin reductase family. The regions are numbered according to (22) with the fingerprint regions for the A. vinelandii and E. coli proteins aligned in lines 1 and 2. The consensus sequence for the whole family is shown in line 3 with capital letters representing residues
that are always conserved and two stacked uppercase letters indicating positions where only two residues are allowed. The
first and second sequence bind the isoalloxazine and phosphate groups
of FAD, respectively. FMN-containing proteins have a different sequence
in the second region. The third, fourth, and fifth regions are involved
in NADP binding. NAD
proteins have a
different sequence in the fourth region. Where structures are
available, the aromatic residue in the sixth region is stacked against
the flavin (23) .
Figure 6: A. vinelandii contains only one copy of the NADPH:ferredoxin reductase gene. Southern analysis of A. vinelandii genomic DNA digested with the enzymes shown and probed with a DNA fragment containing the fpr gene. For EcoRV, there is a single site within the fpr coding region. For all other enzymes there are no sites within the fpr coding region.
Figure 7:
NADPH:ferredoxin reductase is up-regulated
when cells are grown under N-fixing conditions. Western
blot analysis showing reaction with anti-A. vinelandii NADPH:ferredoxin reductase antibodies after separation of
cell-free extracts by SDS-polyacrylamide gel electrophoresis. A.
vinelandii strain LM100 is shown grown on
NH
(a) and under
N
-fixing conditions (b).
In spite of obvious up-regulation of the
NADPH:ferredoxin reductase under N-fixing conditions (Fig. 7), the restriction fragments shown in Fig. 1, and
the sequence shown in Fig. 3, which includes the fpr gene that codes for A. vinelandii NADPH:ferredoxin
reductase, are not found in the 50 kb of A. vinelandii DNA from the nif region that has been mapped to date. All nif genes so far sequenced from Azotobacter and Klebsiella contain a consensus nif promotor sequence
and a nifA binding site (e.g.(21) ). This nif promotor sequence is not found immediately
upstream of the fpr gene nor is a nifA binding site
found within 400 bp upstream of the fpr initiation codon (Fig. 3). This situation is reminiscent of the situation with
respect to the regulation of cytochrome d in A.
vinelandii(28) . Cytochrome d is involved in
maintaining the low intracellular O
concentrations that are
required for N
fixation. The gene encoding cytochrome d is not located in the nif region of the chromosome, and
it is not preceded by a nif specific promoter or nifA
binding sequence. However, like the fpr gene (Fig. 7),
the cytochrome d gene is up-regulated when the cells are grown
under N
-fixing conditions(28) .
Further analysis
of the regions upstream of the fpr gene shows that two
putative 70 type E. coli consensus promotor elements are
present (Fig. 3). The data in Fig. 3also show an
excellent ribosome binding site (29) for A. vinelandii NADPH:ferredoxin reductase 12 bases upstream from the initiation
codon. Downstream of the coding region there is a predicted stem-loop
structure denoted by the arrows (Fig. 3) which probably
acts as a rho-independent transcription termination
signal(30) . Thus, it is unlikely that additional genes are in
the same operon downstream of fpr.
Fig. 8shows the SDS-gel electrophoresis separation of cell-free extracts of wild-type A. vinelandii compared to the overproduction strains trans/pKTR1.2 and trans/pKTF1.2. Clearly there has been an overproduction of the NADPH:ferredoxin reductase in both trans/pKTF1.2 and trans/pKTR1.2. The fpr gene product overexpressed in A. vinelandii is indistinguishable in terms of activity and crystallization properties from the protein purified from A. vinelandii strain LM100. However, the amount of protein is much greater for the strain that has the gene in the same orientation as the kanamycin promoter on the vector. This observation is in contrast to the result obtained for the overexpression of FdI using the same pKT230 system(17) . In that case, the protein was being maximally expressed from its own promoter such that the levels of overexpression were the same regardless of the orientation of the insert. The data in Fig. 8show that something is preventing the maximal expression of the NADPH:ferredoxin reductase from its own promoter in trans/pKTR1.2. These data therefore suggest that unlike the situation with respect to FdI, expression from the fpr promotor may be tightly regulated, by a factor(s) whose concentration is limited.
Figure 8:
Overproduction of A. vinelandii NADPH:ferredoxin reductase in A. vinelandii using the
broad host range multicopy plasmid pKT230. Western blot analysis
showing reaction with anti-A. vinelandii NADPH:ferredoxin
reductase antibodies after separation of cell-free extracts by
SDS-polyacrylamide gel electrophoresis. A. vinelandii strains
are: a, wild-type trans; b, trans/pKTR1.2; and c, trans/pKTF1.2. All were grown under N-fixing
conditions.
Second, in our work, we encountered the fpr gene product while working on AvFdI. In that context, we have shown that the NADPH:ferredoxin reductase binds specifically to AvFdI and that it can mediate electron transfer between NADPH and FdI(14) . In E. coli, the fpr gene product also mediates electron transfer between NADPH and ferredoxin(26) . However, it should be noted that the only ferredoxin that has so far been isolated from E. coli is a [2Fe-2S] protein (32) that is very different from the 7Fe-containing AvFdI(1, 2, 3, 4, 5) . Given the extent of similarity of the E. coli and A. vinelandii fpr gene products throughout their entire length (Fig. 4), the specificity of the A. vinelandii fpr product for FdI(14) , and the known multiplicity of different types of ferredoxins in a number of organisms, it may be that one natural redox partner of NADPH:ferredoxin reductase in E. coli is an as yet unrecognized FdI-type ferredoxin.
The final way in
which this protein was identified is through studies of oxygen toxicity
in E. coli. In that case, mutants sensitive to methyl
viologen, a superoxide radical propagator, were isolated, and the gene
involved was cloned (33) and shown to be identical with the E. coli fpr gene(19) . Subsequently, Fridovich and
co-workers (34) showed that the gene product, NADPH:ferredoxin
reductase, was part of an O protection system that was
controlled by the SoxRS regulon. SoxR is proposed to be an
[Fe-S] protein(35) . In response to superoxide it is
converted into an activator of the soxS gene, and SoxS then
activates the transcription of a number of other proteins including the fpr gene product. Fridovich and co-workers (34) proposed that reduced ferredoxins or flavodoxins might
control the system by reducing SoxR which would then turn off the
activation. Thus, it is proposed that when SoxR is oxidized, the soxS gene is activated causing NADPH:ferredoxin reductase to
be overexpressed. It then reduces flavodoxin and ferredoxin, which in
turn reduce SoxR, which then deactivates the SoxRS
system(34, 35) . It is important to note that in
A. vinelandii the fpr gene product initially came to our attention
because it was overexpressed in strains that do not synthesize
FdI(11, 12) .It is not overexpressed in
strains that do not synthesize Fld(12) . Further studies will
therefore be directed at determining how FdI exerts its regulatory
effect on fpr and examining the possibility that it might act
through a SoxRS-like regulon in A. vinelandii.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].