(Received for publication, January 21, 1997)
From the Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900
Azotobacter vinelandii ferredoxin I
(AvFdI) is one member of a class of 7Fe ferredoxins found
in a variety of organisms that are all capable of aerobic growth.
Disruption of the fdxA gene, which encodes
AvFdI, leads to overexpression of its redox partner, NADPH-ferredoxin reductase (FPR). In this study the mechanism of
FdI-mediated regulation of FPR was investigated. Northern analysis has
shown that regulation is at the level of fpr transcription, the start site for transcription has been identified, and it is preceded by a canonical 70-type bacterial promoter. Gel mobility shift assays show that there is a putative regulatory protein in
A. vinelandii that binds specifically upstream of the
35
region. That protein is not AvFdI. A palindromic sequence
was identified as a putative binding site, and randomization of that
sequence completely eliminates binding of the putative regulatory
protein. A luciferase reporter gene was placed under control of the
A. vinelandii fpr promoter and introduced into wild type
and FdI
strains of A. vinelandii. Luciferase
activity was enhanced 7-fold in the FdI
mutant relative
to the wild type. Alteration of the palindromic sequence reduced the
luciferase levels in the FdI
strain to those of the wild
type, demonstrating that FdI regulates FPR through the palindrome and
that the reaction is an activation rather than a repression. The
identified palindrome is ~50% identical to the SoxS binding site
upstream of Escherichia coli fpr, suggesting that A. vinelandii may have a SoxS-like regulatory system and that the
function of FdI might be to specifically inactivate that system.
Azotobacter vinelandii ferredoxin I (AvFdI)1 is a small protein (Mr = 12,700) that contains two different types of [Fe-S] clusters: one [3Fe-4S]+/o cluster and one [4Fe-4S]2+/+ cluster. Sequence comparisons show that AvFdI is a member of a closely related class of 7Fe ferredoxins found in a variety of organisms that share the ability to grow aerobically (1). Despite the fact that AvFdI has been extensively characterized by x-ray crystallography (2-6) and by detailed spectroscopic (7-10) and electrochemical (1) studies, the specific cellular function(s) of the protein has yet to be determined. The initial approach to that problem involved the construction of a strain of A. vinelandii, designated LM100, that had a disruption of the fdxA gene that encodes FdI (11).
Although the FdI strain LM100 had no obvious phenotype
with respect to growth, two-dimensional gel analysis revealed that there was another small acidic protein that was dramatically increased in abundance in LM100 relative to the wild type A. vinelandii strain (11). In part this observation led Thomson to
propose that FdI might be a novel DNA-binding repressor protein (12). Recently a major advance in the field of [Fe-S] proteins has come with the recognition that a growing number of [Fe-S] proteins (e.g. iron regulatory protein (13, 14), endonuclease III
(15), Mut Y (16), Pat B (17), the FNR protein (18), and Sox R (19))
bind DNA or RNA and that many of them regulate gene expression. To
determine whether AvFdI is a member of this class of
proteins, the acidic protein that is overexpressed in the
FdI
strain LM100 was purified and characterized (20), and
the gene encoding the protein was cloned and sequenced (21). The
protein was shown to be a Mr ~29,000
NADPH-ferredoxin reductase that was designated FPR because its physical
properties and amino acid sequence showed striking similarity to the
FPR from Escherichia coli (21, 22). The A. vinelandii FPR was further shown to bind very specifically to
AvFdI, suggesting that the two proteins were likely to be
redox partners in vivo (20).
Here we report the identification of the A. vinelandii fpr
promoter and a promoter element that is responsive to fdxA
deletion, leading to the overexpression of FPR in the FdI
strain LM100.
A. vinelandii FdI was purified (9), and FdI strain LM100 was constructed (11) as described elsewhere. All chemicals were purchased from Sigma, Fisher, or Bio-Rad. All enzymes were purchased from Promega, New England Biolabs, or Life Technologies, Inc. Radioisotopes were purchased from New England Nuclear (DuPont NEN) or Amersham Corp., and film was purchased from Eastman Kodak Co. The Luciferase assay system and Primer Extension system were purchased from Promega. The Sequenase kit was purchased from U. S. Biochemical Corp.. Oligonucleotides were purchased from Midland Certified Reagent Company. All chemicals used were of reagent or molecular biology grade.
Strains and PlasmidsThe E. coli strains DH1
(FgyrA96 recA1 relA1 endA1 thi-1 hsdR17 supE44
) and C600 (thi-1 thr-1 leuB6 lac1 tonA21
supE44
) were used as host cells for plasmids.
The A. vinelandii strains used were OP wild type and LM100
(fdxA::Km) (11). All E. coli
strains were grown on Luria-Bertani medium at 37 °C with 300 rpm
agitation. All A. vinelandii strains were grown in 100 ml of
Burk's medium, at 30 °C and 200 rpm agitation. All plasmids were
introduced into E. coli using standard procedures; all
plasmids were introduced into A. vinelandii using
electroporation as described elsewhere (21). The luciferase fusion
reporter plasmid pXluc9 was constructed by subcloning a 400-bp PCR
derived fragment containing the fpr promoter into the
BamHI-PstI sites of pBSIIKS+, the sequence was
confirmed, and then the KpnI-HindIII fragment was
subcloned into pSP-luc+NF (Promega) to create a
translational fusion. The vector pKTXLuc7 was constructed by subcloning
the 1.9-kb XhoI liberated luciferase fusion construct from
pXluc9 into the broad range host vector pKT230 (23). The vector pXLM7
was constructed by the same strategy as for pXLuc9, except the PCR
fragment was mutagenized using the following oligonucleotides and
methods described in PCR Protocols (24). Palmut 1 (taagtaaattatcgctcgtccatcggatccgcggcactagcggcgattcactctgcg) and palmut
2 (cgcagagtgaatcgccgctagtgccgcggtaccgatggacgagcgataatttactta) randomize
the palindrome and introduce a KpnI site; XPCR 1 (ggtcaacggatccgcagggtcgcg) introduces a BamHI site at the 5
end of the fpr clone; and XPCR 2 (acgctctacctgcagattgctcattcg) introduces a PstI site between the fourth and fifth amino acids of FPR.
Whole cell RNA was extracted from A. vinelandii strains OP and LM100 as described elsewhere (25). The RNA was then LiCl2 precipitated two times to remove impurities and then quantified, and the purity was evaluated using UV-visible spectroscopy. The RNA was then denatured with deionized formamide and electrophoresed on a 1% agarose gel containing formaldehyde and EtBr and then transferred and fixed to Hybond-N+ as described elsewhere (26). The blot was then probed with a 1.2-kb gel purified fpr XhoI fragment, washed to low background using standard techniques, and exposed to Kodak XAR film for approximately 48 h.
Primer ExtensionA synthetic oligonucleotide
(ggtatcgttccagtgatgaacactgaggac) complementary to the sequence spanning
the fpr start codon was end labeled with
[-32P]ATP and annealed to whole cell RNA from
AvOP and LM100 strains. The labeled probe was extended using
avian myeloblastosis virus reverse transcriptase. Reagents were
obtained as part of the AMVRT Primer extension system (Promega), and
the reactions were carried out according to protocols therein. The
resulting cDNA was electrophoresed on a 6% polyacrylamide gel
containing 7 M urea. The parallel dideoxy sequencing
reaction was carried out using the same unlabeled oligonucleotide, pS-XF1.5Sal (21) vector template, and the Sequenase kit.
For gel mobility shift probes,
the vector pS-XF1.5Sal (21) was digested with XhoI to
liberate the 1.2-kb fpr fragment that was further digested,
and the resulting subfragments were gel purified. Aliquots of the
resulting fragments were end-labeled with [-32P]ATP
using T4 polynucleotide kinase. Cell extracts were prepared by growing 100-ml cultures on Burk's with ammonia at 30 °C and 200 rpm to mid-log phase. The cells were then harvested by centrifugation at 5 K for 10 min using a Sorvall SS-34 rotor, washed in 10 ml of
Tris-HCl, pH 7.4, resuspended in 2 ml of Tris-HCl, pH 7.4, and
sonicated on ice. The sonicate was cleared by centrifugation at 10,000 rpm at 4 °C for 30 min, transferred to a new tube, and snap frozen
in liquid nitrogen. The DNA binding reactions (10 µl) contained 60 mM KCl, 10% glycerol, 0.5 µg of poly(dI)-poly(dC), 0.005 M Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 0.3 µg/ml bovine serum albumin, approximately
0.5-1.0 fmol of labeled DNA, and 1-5 µl of cell extract. The
reactions were incubated at 25 °C for 30 min and then
electrophoresed on a 5% nondenaturing polyacryamide gel at 4 °C for
approximately 1.5 h as described elsewhere (27). The gel was fixed
on Whatman #1 paper and exposed to Kodak XAR film for approximately
12 h.
Cell extracts were prepared by
centrifuging 1.5 ml of a log phase growing culture for 5 min in a
microcentrifuge, aspirating off the medium, adding 800 µl of
sonication buffer, (10% glycerol, 0.025 M Tris-HCl, pH
8.0, 2 mM EDTA), sonicating on ice and microcentrifuging at
4 °C for 20 min. The supernatant was transferred to a new 1.5-ml tube and stored at 20 °C. Extracts were thawed at 25 °C, and 20-µl aliquots were used to assay for luciferase activity using the
luciferase assay system from Promega, according to the manufacturer's protocol. Ten second measurements were taken on the Monolight 2010 luminometer from Analytical Luminescence Laboratory.
In previous studies we
have shown that FPR protein levels are greatly increased in
FdI strain LM100 relative to the wild type A. vinelandii strain (11, 20, 21, 28). To determine if the increase
of FPR was reflected in the fpr mRNA, total cell RNA was
isolated from wild type A. vinelandii and from the
FdI
strain LM100, and Northern analyses were performed
using a 1.2-kb DNA fragment that included the entire fpr
open reading frame as a probe. Two conclusions can be drawn from these
data, which are shown in Fig. 1. First, the levels of
fpr mRNA are greatly increased in the FdI
strain LM100, indicating that the fpr gene is not regulated
solely at the level of translation and/or protein stabilization.
Rather, the increase in FPR appears to be due to regulation at the
level of the fpr transcript. Second, only one major
transcript was observed, which was approximately 900 nucleotides in
length from both wild type A. vinelandii and from the
FdI
strain LM100 (Fig. 1). A message of this length is
only consistent with a monocistronic message originating at a promoter
proximal to the open reading frame.
Identification of the Transcriptional Start Site for the A. vinelandii fpr Gene
To identify the specific start site for
transcription, a series of primer extension experiments were carried
out. A 30-bp oligonucleotide primer that was complementary to the
region of the fpr mRNA overlapping the region encoding the
first seven amino acids of FPR was used. The radiolabeled primer was
annealed to an A. vinelandii total RNA preparation and
extended using avian myeloblastosis virus reverse transcriptase. To
determine the exact size of the product of that reaction the same
oligonucleotide primer was used to sequence a DNA template containing
the entire fpr clone (21) including 450 bp upstream of the
fpr coding region. As shown in Fig. 2 those
data clearly identified the start site for transcription as the
adenosine noted in Fig. 3. It should also be noted that
the same transcriptional start site was identified for both wild type
and FdI LM100 strains of A. vinelandii,
indicating that the same promoter is initiating transcription in both
cases. Inspection of the sequence immediately upstream of this start
site revealed a canonical
70-type promoter (Fig. 3). The identified
70 promoter has the typical 7 bp spacing between the +1 adenosine
start site and the Pribnow box, which has only a single deviation from
the consensus at a highly variant position (noted in bold
type in Fig. 3). The spacing between the Pribnow box and the
35
hexamer is the typical 17 bp; the
35 consensus also has only a single
deviation from the consensus. It should be noted that the
identification of the nearly ideal
70 promoter confirms our earlier
conclusion (21) that FPR is encoded by a non-nif gene.
A Putative Regulatory Protein Binds Upstream of the fpr Promoter, and It Is Not FdI
If, as proposed by Thomson (12), FPR expression
is being directly repressed in the wild type A. vinelandii
strain by FdI, then FdI should bind within or proximal to the promoter
shown in Fig. 3. If instead FPR expression is being regulated by
another protein in the FdI strain LM100, then that
regulatory protein should show evidence of binding the promoter region.
To determine if any protein present in A. vinelandii binds
specifically to the DNA proximal to the promoter, we undertook a series
of gel mobility shift experiments using A. vinelandii
extracts. As shown in Fig. 4, we tested a number of
overlapping DNA fragments derived from our original clone, which
extended 450 bp upstream of the fpr coding region (21).
These fragments were radioisotope labeled and incubated with extracts
from the FdI
LM100 strain of A. vinelandii
before running them out on a 4.5% nondenaturing gel. In Fig. 4 the
free DNA probe migrates fastest and is the lowest band in
each lane. The upper bands represent probes where
the migration has been retarded due to the association of protein(s)
with the DNA. Several fragments very clearly show a significant
mobility shift, indicating that some protein(s) present in A. vinelandii extracts binds very tightly to the DNA probe. Further
analysis of these data (Fig. 4) localized the specific binding to a
34-bp region of DNA just upstream of the
35 consensus of the promoter
(Fig. 5).
To confirm the importance of this region and to examine this region
further, we performed the gel shift experiment using a 60-bp
AluI to MseI fragment of DNA that extends from
the 5 end of the
35 hexamer, upstream (Fig. 5). Protein extracts
were prepared from parallel cultures of wild type A. vinelandii and the FdI
strain LM100 and harvested at
the same cell density; purified FdI was also used. As shown in Fig.
6 both wild type and LM100 extracts gave strong shifts
with the 60-bp probe, confirming that there is a protein present in
A. vinelandii that binds to this region. The data in Fig. 6
further show that the protein cannot be FdI because the protein
responsible for the shift is present in LM100, and no shift is seen
with high concentrations of purified FdI. In separate experiments DNA
fragments representing the entire 450-bp upstream region were also
tested for binding to purified AvFdI, and no binding was
detected in any of those experiments. It should be noted that although
experiments of the type shown in Fig. 6 do not give quantitative
information about the relative amounts of the DNA-binding proteins
present in wild type versus LM100, we always observe strong
shifts for extracts from both types of cells.
Inspection of the sequence (Fig. 3) of the DNA just upstream of the
35 hexamer of the promoter identified a palindromic sequence that is
a likely site for the binding of a regulatory protein. To test the
importance of this palindrome, the sequence in that region was
randomized. Fig. 7 shows that the protein present in the
A. vinelandii wild type strain and in FdI
strain LM100 that binds specifically to the DNA just upstream of the
35 consensus (complex B) no longer binds when the palindrome has been
mutated, further confirming the binding site. The other weaker binding
complexes A and C appear to persist in the mutant palindrome probes,
indicating that the binding is not specific for the palindrome.
FdI Regulates FPR Expression Indirectly through the Identified Palindrome
To try to correlate the gel shift results with the
overexpression of FPR in LM100, a luciferase reporter gene was placed
under the control of the A. vinelandii fpr promoter. This
translational fusion was then subcloned into the broad-host-range
multicopy plasmid pKT230 (29), which was then introduced into both the wild type and LM100 strains of A. vinelandii. As shown in
Fig. 8A, the luciferase activity was enhanced
about 7-fold in LM100 relative to the wild type strain, further
confirming that the up-regulation of FPR levels in LM100 is due to
regulation at the fpr promoter. This construct was then
mutated only at the palindrome by randomizing the palindromic sequence.
As shown in Fig. 8B the level of luciferase activity in
LM100 was reduced to that of the wild type when driven by a promoter
with a mutant palindrome. These data demonstrate not only that FdI
regulates FPR expression through the identified palindrome but also
that the regulation is activation rather than repression. If the gene
were regulated by a repressor protein, then disruption of the protein
DNA interaction should have led to an increase of luciferase activity
in the wild type rather than the observed result that levels of
activity declined in LM100.
Relationship between FPR Regulation in A. vinelandii and E. coli
Taken together the above data demonstrate that
fpr transcription in A. vinelandii is activated
in response to deletion of fdxA, that the activation results
from interactions at a specific palindrome, and that a putative
regulatory protein other than FdI binds to that plaindrome. Thus, the
proposal that FdI might regulate FPR expression directly by acting as a
novel repressor protein (12) is not correct. The similarity between the
E. coli FPR and the A. vinelandii FPR proteins
led us to compare the sequence of the regulatory palindrome identified
here to the region upstream of the fpr promoter in E. coli. The alignment illustrated in Fig. 9 shows
~50% identity between the palindrome identified in this study and
the parallel E. coli region. The locations of both regions relative to the promoter are also very similar. In E. coli
this region has been defined as a SoxS binding site (30), and SoxS has
been shown to activate fpr transcription in that organism (31). Further studies will therefore be directed at determining if
A. vinelandii has a SoxRS-like system and if FdI might serve as a redox sensor whose specific function would be to inactivate that
system.