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J Biol Chem, Vol. 273, Issue 50, 33841-33847, December 11, 1998
From the Department of Molecular Microbiology and Immunology,
School of Medicine L-220, Oregon Health Sciences University,
Portland, Oregon 97201
The expression of iron transport genes
fatDCBA in Vibrio anguillarum strain 775 is
negatively regulated by two iron-responsive repressors, the Fur protein
and the antisense RNA, RNA The marine pathogen Vibrio anguillarum strain 775 possesses a highly efficient plasmid-mediated iron uptake system that
competes with the vertebrate host fish for the iron bound to high
affinity iron binding proteins, such as transferrin and lactoferrin,
leading to the establishment of infection (1). The pJM1
plasmid-mediated iron uptake system includes an iron scavenger, the
siderophore anguibactin, and an energy-coupled iron transport system
that internalizes the ferric anguibactin complex (2). The iron
transport system includes an 86-kDa outer membrane receptor protein
encoded by the fatA gene (3, 4), a 40-kDa cytoplasmic
membrane-embedded lipoprotein that possesses periplasmic binding
domains encoded by the fatB gene (5), and two 37-kDa
integral membrane proteins encoded by the fatD and
fatC genes (6, 7). These genes are located contiguous in the
pJM1 plasmid in the order fatD, -C, -B, and -A. (Fig.
1). Genetic studies utilizing transposon
(Tn3::HoHo1) insertion mutagenesis have demonstrated that
mutations in fatD, -C, or -B affected
FatA expression, suggesting an operon organization of these genes (6,
8). A further regulatory linkage between iron transport genes and
anguibactin biosynthetic genes has also been proposed since insertion
mutations in the iron transport genes led to lowering or complete
shutoff of anguibactin biosynthesis (8).
Expression of both, siderophore biosynthesis and iron transport genes,
is regulated by positive and negative factors which function at low and
high iron level, respectively. The positive regulation is mediated by
the plasmid-encoded 110-kDa AngR protein whose gene lies immediately
downstream of fatA and by trans acting factors encoded in a
region located noncontiguous to the iron uptake region in the pJM1
plasmid (5, 8, 9, 10). The negative regulation is afforded by the
chromosomally encoded Fur protein (11, 12) and by an antisense RNA
(RNA The function of Fur in the repression of iron-regulated genes has been
described in many bacteria (16); however, the interaction of Fur with
its cognate operator has been characterized predominantly in the
Escherichia coli plasmid aerobactin system (17-19) in which the Fur protein, in the presence of divalent metal ions, binds to a
19-bp1 operator sequence in
the promoter region. The primary structure of the Fur protein (about 17 kDa) is highly conserved in a variety of bacteria. The comparison of
predicted amino acid sequence showed that V. anguillarum Fur
shares a high degree of similarity to its homolog in Vibrio species,
Vibrio cholerae (94%) and Vibrio vulnificus
(92%), and a lower degree of similarity to E. coli Fur
(76%) (11).
We report here the identification of the promoter that drives the
expression of the polycistronic transcript of the fatDCBA genes and the interaction of the promoter with the Fur protein in
vitro, to understand the regulatory mechanism of Fur in V. anguillarum. Analyses by gel retardation and DNase I footprints showed that the V. anguillarum Fur protein interacts
with the promoter region in a different manner from its counterpart
of E. coli, suggesting the existence of a different
repression mechanism in this organism.
Bacterial Strains and Plasmids--
V. anguillarum
strain 775 is a wild-type strain harboring the plasmid pJM1 (1), and
its isogenic strain 775MET11 is Fur-deficient (12). E. coli
strain MM294 harboring pRK2013 was used as a helper for conjugation
between V. anguillarum and E. coli HB101 (20) strains carrying recombinant plasmids, E. coli XL1-blue (21) for cloning. The plasmids pJHC-SC85 and pJHC-SC87 are derivatives of
pBluescript SK+ (Stratagene) containing either the 1.4-kb
BstEII-HindIII fragment from the fatD
upstream region between the sites HindIII and
SmaI in the vector (pJHC-SC85) or the 0.3-kb
XmnI-EcoRI fragment from the fatD
upstream region between the sites EcoRV and EcoRI in the vector (pJHC-SC87). The plasmid pJHC-TW95Z was constructed using
the following cloning steps: the 1.4-kb
BstEII-HindIII fragment containing the
fatD promoter region was cloned between the sites SalI and HindIII in a derivative of pBR325 (22),
which carries a kanamycin resistance gene inside the ampicillin
resistance gene (PstI site) in the opposite orientation to
the disrupted gene. The construct was then used as a vehicle to clone
the 9-kb HindIII-BamHI fragment containing the
lacZYA operon, isolated from the plasmid pPD104 (23),
between the HindIII and NcoI sites of the vector.
Cell Growth--
V. anguillarum cells were grown at
26 °C in Trypticase soy broth (Difco) supplemented with 1% (w/v)
NaCl and antibiotics, when necessary, until stationary growth phase.
The cultures were then diluted 100-fold in M9 minimal medium (1)
supplemented with 0.2% casamino acids and antibiotics, when necessary.
The cells were grown repeatedly until stationary growth phase and subsequently transferred in 100-fold dilution in the minimal medium described above without antibiotics. To generate various iron levels
for the cells, the minimal medium in this step was supplemented with
either 2 µg/ml ferric ammonium citrate for iron-rich condition or
ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA) from 0 to 15 µM for iron limitation. The cells were grown to
optical density of 0.5-1 at 600 nm and harvested to isolate total RNA or to measure the General Methods--
Conjugation to V. anguillarum
(24), Protein-DNA Interaction Assays--
V. anguillarum
Fur, purified to homogeneity, was obtained from E. Zhelenova from the
laboratory of R. Brennan. Gel mobility retardation assay was carried
out with the 310-bp HindIII-EcoRI isolated from
the plasmid pJHC-SC87 and labeled with [ Determination of the Transcription Start Site for fatDCBA mRNA
and Identification of the Iron Transport Promoter--
To determine
the transcription start of the fatDCBA mRNA, primer
extension analysis was carried out with a primer (primer 1)
complementary to the 5'-end region of the fatD transcript
(Fig. 2C). As shown in Fig.
2A, a number of primer extension products were observed only
with RNA samples harvested under iron limitation. The pattern of the
molecular species, two larger and a number of smaller bands, is
identical at all three levels of iron limitation. Using molecular
standards the two largest bands were estimated as 340 ± 10 nt,
which maps the transcription start site(s) about 300 nt upstream of the
translational initiation start site of fatD. To identify the
precise location of these two start sites, a further primer extension
experiment was performed with a primer (Primer 2, Fig.
2A) that hybridizes at the region closer to the start
site(s). The molecular weight of the two largest primer extension
products was compared with a sequence obtained using the same primer
(Fig. 2A) and located at 302 and 287 nt upstream of the
start codon of fatD.
The multiple bands detected could be generated as a result of multiple
promoters and therefore multiple transcriptional start sites or
processing products from larger molecules in vivo or in vitro. Alternatively they could be artifacts of the
detection method, generated by the stalling of reverse transcriptase at certain stem-loops in the RNA structure, which might lead to earlier stops during the reverse transcription. To clarify the presence of
multiple bands in primer extension analysis, the S1 mapping method was
applied. Radiolabeled riboprobes containing either a 302-nt
XmnI-EcoRI segment (obtained as a 378-nt fragment
using plasmid pJHC-SC87) or a 535-nt XmnI-HindIII
segment (obtained as a 609-nt fragment using plasmid pJHC-SC85) were
employed in this analysis. RNA-RNA hybrids were subjected to S1
nuclease activity. Fig. 2B shows S1-mapped transcriptional
start sites. As it was the case with the primer extension approach the
S1 mapping method detects two major transcripts whose start sites
correspond to the two major start sites identified by primer extension
analysis, confirming that these two mRNA species are indeed present
in vivo. The S1 mapping method also shows a number of minor
bands, all of which are smaller than the two major bands. The pattern
of the minor bands detected with two different riboprobes, however, are
not identical and distinguishable from bands detected by primer extension experiments. This result suggested that the minor bands observed in the two mapping methods applied here are artifacts of the
detection methods. The search of the RNA structure in the fatD upstream region, by the computer program GCG, reveals
at least four stem-loop structures (data not shown). When the location of the stem-loops was compared with that of the early stops detected in
primer extension analysis, there was no consistency between them. The
DNA sequence of the fatD upstream region is highly rich in A + T (78%). Therefore, it is likely that those minor products obtained
with both mapping methods are generated by breathing of two strands
that leads to early termination of reverse transcription or nicking by
S1. It still remains to be identified whether the two major bands are
both products of independent transcription initiation at different
sites or if the smaller band is a specific degradation product of the
larger one. According to the transcription start site mapped by the
larger band (95-nt species; Fig. 2A), we mapped a putative
Analysis of the Iron Transport Promoter-lacZ Fusion--
Since the
expression of fatDCBA mRNA is regulated by the iron
level, it is expected that the putative promoter, identified by primer
extension, is also regulated by the iron level in the cell. To assess
whether this was the case the putative promoter, called iron transport
promoter, was examined using the lacZ-reporter gene
expression to verify that fatDCBA genes are indeed driven from this promoter. The 1.4-kb BstEII-HindIII
fragment containing the putative promoter region (Fig. 1) was coupled
transcriptionally to the lacZ-reporter gene in a pBR325
derivative plasmid, resulting in plasmid pJHC-TW95Z. The fusion
construct was subsequently conjugated into the wild-type strain 775 as
well as the Fur mutant strain 775MET11. Cells harboring pJHC-TW95Z were
grown under various concentrations of iron. The Fur Binding Assay with the Iron Transport Promoter--
The
possibility that the iron regulation of the iron transport promoter is
mediated by Fur-iron complex was examined in vitro. To
determine the binding activity of the V. anguillarum Fur
protein to the promoter, a gel retardation assay was performed under
the binding reaction conditions employed for E. coli Fur
binding to the aerobactin promoter (19) with few minor modifications.
The detection of Fur-DNA complex was carried out in the presence of metal ion Mn2+, as a substitution of the natural Fur
co-repressor Fe2+, both in the binding reaction and gel
electrophoresis (nondenaturing PAGE). The 302-bp
XmnI-EcoRI promoter fragment (24 pM),
obtained as a 310-bp HindIII-EcoRI fragment from
pJHC-SC87, was incubated with increasing concentrations of V. anguillarum Fur protein (30 nM to 60.8 µM) in the presence of 1 × 104-fold
weight excess of nonspecific competitor DNA poly(dI-dC) in the reaction
prior to the nondenaturing PAGE. As shown in Fig. 4, Fur protein specifically binds to the
promoter DNA fragment. The varying amounts of Fur protein in the
reaction mixture led to the detection of Fur-promoter complexes with
four different gel migrations, designated as complexes a, b, c, and d
in Fig. 4. At lower protein concentrations, 30-120 nM
monomer, only a single complex band (a) was detected, and in
higher concentrations 240-960 nM two additional complex
bands (b and c) appeared. While the complexes a
and c seem to be formed in the same high abundance in this protein
titration assay, the complex b appeared to be of lower abundance as
compared with the other two complexes, a and c. From the gel
retardation assay we were not able to estimate the number of Fur
protein binding units on the tested DNA fragment. However, it is
evident that the complexes b and c result from Fur binding at more than
two different sites in the DNA fragment. The less abundant complex b
implies an unstable nature of this complex, at least as assessed by the
gel retardation method. This could be due to either an unstable
conformation of the complex or a Fur binding site with low affinity. At
protein concentrations 960 nM and higher, the protein-DNA
interaction shows a plateau, indicating that all available Fur binding
sites are occupied by Fur. The highest retarded complex d occurred at a
60.8 µM protein concentration. It is likely that this
complex was formed as a result of nonspecific binding of Fur aggregates
in the tested conditions. Although this gel retardation assay was not a
detailed kinetic assay, we estimated the apparent binding constant
Kapp of Fur to the iron transport promoter
between 30 and 60 nM. This value is relatively high as
compared with that of E. coli Fur to the iucA
promoter (5 nM; Ref. 28) and the sodA promoter
(10-20 nM; Ref. 29).
DNA Footprinting Analysis of Fur-Iron Transport Promoter
Complex--
To locate the Fur binding sites in the 302-bp
XmnI-EcoRI fragment containing the promoter
region, DNase I footprinting assays were performed. The DNA fragment
was radiolabeled at the 3'-end of either the template or nontemplate
strand relative to the fatDCBA transcripts. The binding
reaction was carried out with 1.5 nM DNA fragment and
varying concentrations of the V. anguillarum Fur protein
(480 nM to 60 µM) under the same
binding conditions applied in the gel retardation assay. The
autoradiograms of DNase I footprint patterns on both strands are shown
in Fig. 5 and the summary of the Fur-DNA
interaction is represented in Fig. 6.
Both template and nontemplate strands showed a large interaction region with Fur that comprised about 83 and 56 nt, respectively. At low protein concentrations (480-960 nM) the template strand
reveals a region of 49 nt protected from DNase I cleavage by binding to Fur (Fig. 5, lanes 8 and 9). This primary
interaction site with Fur, designated as site I, is located directly in
the promoter region, spanning from position +10 to
The footprint pattern in the nontemplate strand was detected in the
region spanning from position The regulatory function of the Fur protein in the pJM1-encoded
iron uptake system has been demonstrated previously by isolating a
Fur-deficient isogenic strain, a null mutant that is constitutive in
both, expression of the FatB and FatA proteins, as well as in the
production of dihydroxybenzoic acid, an intermediary in the
biosynthesis of the siderophore anguibactin (5, 11). The aim of the
present study was to characterize the repression mechanism mediated by
the Fur protein in the expression of the iron transport genes, in
conjunction with the identification of the promoter(s) that drive the
transcription of these genes.
Primer extension analysis has identified, in accordance with S1 mapping
experiments, two distinct RNA species starting from the position 302 and 287 nucleotides upstream of the fatD translation start,
when the wild-type cells were grown under iron limitation. It still
remains to be determined whether these two transcripts are a result of
two independent transcription starts or the smaller transcript is a
product of a partial degradation of the larger one at a preferential
site in vivo. We presumed that, independent of the smaller
RNA species, the larger species presents the transcription start site
(+1), and according to this +1 site we mapped the Gel retardation assays showed that the V. anguillarum Fur
protein specifically binds to the 310-bp DNA fragment containing the
iron transport promoter in the presence of high amount (1 × 104-fold weight excess) of noncompetitor DNA poly(dI-dC).
The Fur-DNA complexes were formed, resulting in an early complex,
complex a, and the later complexes, complexes b and c. The successive binding of Fur to the promoter DNA was corroborated by DNase I footprinting analysis. The Fur interaction with DNA was observed in
both strands, template and nontemplate, resulting in different sensitivities to the DNase I cleavage. While the template strand showed
a large protected region by Fur, the nontemplate strand exhibited
enhanced sensitivity to the nuclease activity. These results imply that
the template strand may be providing the main contact sites to the Fur
protein, making DNase I inaccessible to those sites. Furthermore, the
contact of Fur in one strand leads to the exposure of the other with
enhanced sensitivity to DNase I. The template strand showed two
contiguous regions protected by Fur with different affinities. The
primary binding site (site I) of 42 nucleotides in size is located in
the promoter, overlapping with the The estimated molecular mass of the V. anguillarum Fur is
~16.8 kDa, similar to its homolog in E. coli. Based on
in vitro data it has been suggested that the E. coli Fur protein is active as a dimer or as a multimer (17, 18,
28) that binds to its 19-bp dyad symmetrical operator sequence, so
called Fur consensus sequence or iron box (19, 28; Fig.
7) in the promoter region. The Fur
consensus sequence has been initially identified and proposed in the
aerobactin promoter that comprises two contiguous Fur binding sites
designated as primary (site I; 31 nt) and secondary site (site II; 19 nt) due to their successive binding to the protein. In that promoter,
the primary binding site overlaps with the
Characterization of the Interaction between Fur and the Iron
Transport Promoter of the Virulence Plasmid in Vibrio
anguillarum*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
. Here we report the identification of the
promoter for the iron transport genes and studied the interaction
between the V. anguillarum Fur protein and this promoter.
The iron transport promoter was localized in a region approximately 300 base pairs upstream of fatD by both primer extension and S1
mapping analysis. High activity of the promoter was measured in
response to iron depletion in the wild-type strain when a
promoter-lacZ fusion was examined, whereas the promoter was
constitutive in the Fur-deficient strain. Gel retardation and DNase I
footprint analysis showed that Fur binds specifically to two contiguous
sites comprising the promoter region and the region downstream of the
transcription start site. The identified Fur binding sites showed a low
degree of homology to each other as well as to the consensus sequence
for the Escherichia coli Fur protein. DNase I footprints
pattern suggested a sequential interaction of Fur with these two sites
that renders a protection in the template strand and a hypersensitivity
to the nuclease in the nontemplate strand. The periodicity of the
hypersensitive sites suggested that the promoter DNA undergoes a
structural change upon binding to Fur, which might play a role in the
repression of gene expression.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Map of the iron uptake genes in V. anguillarum plasmid pJM1 and the promoter region for
fatDCBA. The iron uptake region is located in the
genetic units II and III (EcoRI-2 fragment in the physical
map; Ref. 8). The region encompassing genes fatDCBA and
angRT is preceded by insertion element ISV-A1. The relevant
restriction sites in the promoter region are shown in the subcloned
fragment: Bs, BstEII; E,
EcoRI; H, HindIII; X,
XmnI.
) (13). RNA is transcribed as a counter-transcript of the
fatB mRNA under the control of Fur (15). While the
repression mediated by RNA occurs at the post-transcriptional level,
the Fur protein seems to regulate the expression of the iron transport
genes at the transcription level. Using an RNase protection assay it
has been shown that the fatB and fatA messages are no longer negatively regulated at high iron level; and therefore, their expression is
constitutive when a null mutation is generated in the fur
gene of the 775 strain (14).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-galactosidase activity. The amounts of the antibiotics applied in V. anguillarum strains were: 60 µg/ml kanamycin. The Fur-deficient strain was grown in the presence
of 2 mM MnCl2.
-galactosidase assay (25), and RNA isolation (26) were
followed as described previously. The primer extension experiment was
carried out with the synthetic primers 5'-GTAGCGCACAAAGTAAAGAACGCC-3'
(primer 1) and 5'-GGTCACAGCAGGGATTTAGCAGAGC-3' (primer 2), which are
complementary to the 5'-end region of fatD. The primer was
end-labeled with T4-polynucleotide kinase (Life Technologies, Inc.) in
the presence of [
-32P]ATP and annealed to total RNA
(50 µg). Reverse transcription from the primer by avian
myeloblastosis virus reverse transcriptase (Promega) and urea-PAGE
(6%) were followed as described (21). The S1 mapping analyses were
carried out using riboprobes as described (21). The riboprobes, 690 nucleotides (nt) containing the XmnI-HindIII segment from the plasmid pJHC-SC85, and 378 nt containing the XmnI-EcoRI segment from the plasmid pJHC-SC87
were prepared by linearization of the plasmid with XmnI and
HindIII, respectively, and following transcription with T3
RNA polymerase. RNA-RNA hybridization was carried out at 42 °C
overnight and followed by digestion with S1 (Life Technologies, Inc.)
at 37 °C for 2 h. The resulting S1 digested samples were
analyzed in parallel with a known sequence marker by urea-PAGE
(6%).
-32P]dATP by
the filling in reaction of the Klenow enzyme. The labeled DNA (24 pM) was incubated with various concentrations of Fur
protein (30 nM to 60 µM), 1 µg of
poly(dI-dC), and 2 µg of bovine serum albumin in the final volume of
20 µl of reaction buffer (10 mM Bistris/boric acid (pH
7.5), 100 µM MnCl2, 1 mM
MgCl2, and 40 mM KCl) at 37 °C for 15 min.
The reaction mixture was separated by nondenaturing (nd) PAGE (5%) in
buffer (10 mM Bistris/boric acid (pH 7.5), 100 µM MnCl2) at 90 V for 3 h. DNase I
footprint assay was performed according to the method of Galas and
Schmitz (27). The 315-bp HindIII-PstI fragment
and 346-bp EcoRI-KpnI fragments were isolated
from the plasmid pJHC-SC87 and labeled at recessive 3'-end of the
fragments by the filling in reaction of the Klenow enzyme in the
presence of [-32P]dATP. The labeled DNA preparations
were incubated with various concentrations of Fur (480 nM
to 60 µM) under the same reaction conditions described in
the gel retardation assay at 37 °C for 20 min. The reaction mixture
was exposed to a partial digestion with DNase I (Boehringer Mannheim)
at 37 °C for 2 min. After stopping the reaction with 25 mM EDTA, the DNA was precipitated and analyzed by urea-PAGE
(6%).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 2.
Mapping of the transcription start sites of
fatDCBA mRNA. A, primer extension
analysis. The total RNA preparations from strain 775 grown under
various iron concentrations (lanes 1-4) were hybridized
with 32P-end-labeled primers, which are complementary to
the 5'-end region (Primer 1) and the region upstream
(Primer 2) of the fatD gene (see C).
The products from the subsequent reverse transcription starting from
the primer were analyzed in urea-polyacrylamide gel (6%). The
arrowheads indicate position of the two largest products.
The supplements to the medium were: lane 1, 2 µg/ml ferric
ammonium citrate; lane 2, none; lane 3, 3 µM EDDA; lane 4, 9 µM EDDA.
Lane M shows DNA molecular mass marker (see "Experimental
Procedures"). Lanes G, A, C, and
T represent the sequence of the fatD upstream
region obtained from the primer used in the respective experiment.
B, S1 nuclease mapping of the 5'-end. Total RNA preparations
from strain 775 grown in high iron (lanes 2, 2 µg/ml
ferric ammonium citrate) or low iron (lane 3, minimal medium
alone; lane 4, 3 µM EDDA) medium were
hybridized with two different riboprobes containing either the
XmnI-EcoRI or XmnI-HindIII
segment (see C and Fig. 1). Lanes 1, riboprobes
without S1 treatment were loaded. The gel electrophoresis was carried
out as described above. The size of the S1-generated products were
measured by comparison with known sequence marker (not shown).
C, DNA sequence of fatD 5'-end region. Only the
sequence of the nontemplate strands is shown. The translational start
of the fatD gene ATG and the location of the primer are
indicated by bold letters. The first (+1) and second
transcription start sites are indicated by small arrows and
the 
10 and 35 sequences are denoted.
10 (TAGCAT) and the 35 (CTTACA) promoter region. These sequences,
however, share a low homology to the consensus sequence of the E. coli promoter. The diversity of the sequence in the iron transport
promoter implies that this promoter might require a transcriptional
activator that locates RNA polymerase in the promoter region and
enhances its binding.
-galactosidase
activity of the cells was measured when cells were in the early
stationary growth phase, and the results are presented in Fig.
3. As the diagram shows, the
-galactosidase activity of the wild-type cells measured was negligible when the cells were grown in high iron, indicating no
promoter activity. However, the overall -galactosidase activity increased significantly under iron depletion with EDDA. The maximum increase was ~200-fold at 3 µM EDDA, and the activity
decreased slightly under high iron stress (up to 15 µM
EDDA). Such iron-responsive activity of the promoter was not observed
in the Fur mutant cells (Fig. 3). Here, the -galactosidase activity
was high under both high and low iron conditions, indicating that the
promoter is constitutive in the absence of Fur in vivo.
Taken together, these results strongly suggested that the promoter
within the tested area is activated by iron limitation and repressed in
high iron levels. Such iron regulation in a promoter region may be
achieved by a complex of the repressor Fur protein with iron, as
characterized in many other iron-regulated genes in Gram-negative
bacteria.
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Fig. 3.
-Galactosidase activities of wild-type and
Fur mutant strain harboring a promoter-lacZ fusion
(pJHC-TW95Z) grown under various iron levels. The wild-type (775)
and the Fur mutant (775MET11) cells were grown in minimal medium with
either 2 µg/ml ferric ammonium citrate (Fe) or various
concentrations of EDDA as indicated (in micromolar). The
-galactosidase activity is expressed in Miller units (Ref. 26). The
results shown are representative of three independent
experiments.
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Fig. 4.
Gel mobility retardation assay of
Fur-promoter complexes. The 302-bp
XmnI-EcoRI fragment containing the
fatDCBA promoter was obtained as a 310-bp
EcoRI-HindIII fragment from the plasmid pJHC-SC87
and 32P-labeled at both ends. The labeled DNA fragment (24 pM) was incubated with increasing concentrations of
V. anguillarum Fur in the presence of 1 µg nonspecific
competitor poly(dI-dC) in the binding reaction for 10 min at 37 °C
prior to 5% nondenaturing PAGE. Both binding reaction and
nondenaturing PAGE were carried out in the presence of 100 µM MnCl2. Protein-DNA complexes showing
different gel mobilities are indicated with a, b,
c, and d in the figure. Fur concentrations in
each lane were as follows: lanes 1 and 1', Fur omitted;
lane 2, 30 nM; lane 3, 60 nM; lane 4, 120 nM; lane
5, 240 nM; lane 6, 480 nM;
lane 7, 960 nM; lane 8, 1.9 µM; lane 9, 3.8 µM; lane
10, 7.6 µM; lane 11, 15.2 µM; lane 12, 30.4 µM; lane
13, 60.8 µM.
32 relative to the
transcription start site, overlapping with the 10 and 35 region of
the promoter. In the region between position +10 and 7 the protection
was observed in only a few nucleotides. It is possible that protected
nucleotides in this region are the result of the paucity of the DNase I
footprints. At higher concentrations of protein (>1.9 nM)
a protected region appeared in the downstream region of the
transcription start site from position +17 up to +50. This secondary
binding site (site II, 36 nt) is a few nucleotides smaller than the
primary binding site I (42 nt), indicating that Fur binds these two
sites either with different multimeric forms or in a different manner.
The protection pattern in sites I and II (82 nt) was maintained in the
presence of higher protein amounts (Fig. 5, lanes 2-6).
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Fig. 5.
DNase I footprint analysis of Fur-promoter
complexes. The 302-bp XmnI-EcoRI fragment
containing the fatDCBA promoter was obtained as a 315-bp
HindIII-PstI fragment or as a 346-bp
EcoRI-KpnI fragment from pJHC-SC87 for
32P labeling at the 3'-end of template or nontemplate
strand, respectively. In each case, 1.5 nM labeled DNA
fragment were incubated with different concentrations of Fur in the
presence of 1 µg of nonspecific competitor poly(dI-dC) in the binding
reaction for 10 min at 37 °C and were subjected to partial digestion
with DNase I and subsequent urea-PAGE (6%). The Fur interaction sites
were mapped by comparison with a known sequence marker and the size of
the fragments are shown on the left-hand side of each
autoradiogram in base pairs. The location of the Fur interaction sites
(I, II, I', and II') are
represented with the positions relative to the transcription start site
(+1). Fur concentrations in each lane were as follows: lanes
1, Fur omitted; lane 2, 60.8 µM;
lane 3, 30.4 µM; lane 4, 15.2 µM; lane 5, 7.6 µM; lane
6, 3.8 µM; lane 7, 1.9 µM;
lane 8, 960 nM; lane 9, 480 nM.
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Fig. 6.
Summary of Fur interaction with the iron
transport promoter DNA. The top and bottom strands represent the
nontemplate and template strand, respectively. The protected
nucleotides are shaded, and the hypersensitive sites are
indicated by vertical arrows. The transcription start site
(+1) and the direction of transcription of iron transport genes are
denoted by a horizontal arrow. The numbers
indicate nucleotides relative to the transcription start site.
3 up to position +49. The footprints
within this region contain three areas that show hypersensitivity to
DNase I cleavage. In each case, two hypersensitive nucleotides are
flanked by protected nucleotides. These three hypersensitive sites
appeared periodically spaced by about 15-16 nucleotides, which means a
periodicity of longer than one helical turn of B-DNA. As is the case of
the template strand, the footprints of the nontemplate strand revealed
two Fur interaction sites (I' and II') that are distinguishable in
affinity for Fur. The primary interaction at site I' observed at
protein concentrations 480-960 nM reveals protection and
hypersensitive sites from position 6 to position +13 (Fig. 5,
lanes 8 and 9) within the site I region in the
opposite strand. The secondary interaction site (site II') with two
hypersensitive sites was detectable at 1.9 µM Fur in the
downstream region of site I'. The site II' is located in the
complementary region of site II. The temporal and spatial coincidences
of interaction sites suggested that the footprints at sites I and I' or
at site II and site II' feature the same event with different
consequences. The sensitivity of this strand was enhanced at higher
concentrations of protein. The upstream region of the site I' revealed
extremely high sensitivity in a large area of about 80 nt. The
hypersensitive sites appeared only in the nontemplate strand, and the
main protection was observed in the template strand. These data
suggested that the mutimeric forms of Fur bind mainly to the template
strand and change the conformation of the strand upon binding that
causes a stronger exposure of the less interactive nontemplate strand to the DNase I nucleolytic attack. The enhanced sensitivity in the
upstream area could be generated likewise by a conformational change of
the DNA, e.g. DNA bending, presumably constrained by protein-protein interaction. Experiments to explore these possibilities are currently being carried out.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
10 and 35
promoter region. This putative promoter, however, shows a high
divergence in the 10 and 35 sequence from the promoter consensus
sequence. Since the genes fatDCBA are expressed at high level under iron stress, we presumed that a transcriptional activator functioning under iron stress is involved in the stimulation on the
binding of RNA polymerase to the 35 and 10 promoter region. The
activity of the promoter was monitored by using a promoter fusion to
the lacZ reporter gene. The -galactosidase activity representing the promoter activity was seen in high level in response to iron depletion in the wild-type cells (Fig. 3). This corresponds to
the appearance of the fatDCBA transcripts in the early stage of the iron limitation when low iron stress was applied (Fig. 2). In
addition to the high activity, the promoter identified seems to possess
high strength by itself, although it lacks sequence homology to the
10 and 35 consensus sequence. In the absence of positive
regulators, this promoter was still able to drive the transcription at
a low level under iron
stress.2 Moreover, as shown
in the Fur-deficient strain, the promoter is active under high iron in
the absence of the negative regulator. All these data imply a crucial
role of Fur as a repressor acting at the promoter to control the
expression of the genes fatDCBA at the transcription
initiation level.
10 and 35 region. The
preferential protection sequences were observed predominantly in the
35 region. Unexpectedly, the secondary Fur binding region was mapped
in the downstream region of the transcriptional start site, protecting
36 nucleotides.
35 region and the spacer
and the secondary site with the 10 region. As reported by de Lorenzo
et al. (19), a 19-bp symmetry dyad has been found in a
number of promoters of iron-regulated genes, e.g. promoters
for iucA, fhuA, and fepA. However, no
significant degree of sequence homology was found among them, except
that base pair A:T in few positions of this 19-base pair operator is conserved. Our results show that V. anguillarum Fur
interacts with a much larger DNA segment than its homolog in E. coli. The active polymeric state of V. anguillarum Fur
has not been identified. However, as proposed for E. coli
Fur (17), a dimer of the about 17-kDa (monomer) protein could interact
with a 19-bp DNA segment. Therefore, it is plausible that two dimers
(or a tetramer) of V. anguillarum Fur form a complex with
each site, site I (42 nt) and site II (36 nt), as an active unit in DNA
binding. To learn more about the DNA element involved in Fur binding, a
search for an analog of E. coli Fur consensus has been
attempted with the sequence of the V. anguillarum Fur
binding sites I and II. A comparison of Fur binding sequences is
presented in Fig. 7. The highest similarity to the Fur consensus
sequence was observed in a region of 20 nucleotides around the 10
region in site I. The other portions of the binding sites share a low
similarity to the E. coli Fur consensus sequence. Furthermore, the characteristic of the Fur consensus sequence, a dyad
symmetry, was found, although imperfect, only in the region with the
highest similarity. In site I, the Fur protection was observed
preferentially upstream of the 10 region, but rather weak in the 10
region. It is unlikely, therefore, that the analog to the E. coli consensus sequence actively participates in Fur binding in
the V. anguillarum system. As depicted in Fig. 7, the V. anguillarum Fur binding sites both in the promoter region
and downstream of the transcription start are highly rich in A + T. The
sequence comparison among those sites shows no significant homology.
However, the base pair A:T was found in a number of positions conserved
in the sequence of 5'-ATnnnn(A/T)T/A)n(A/T)(A/T)nn(T/A)nnA(T/A)n-3'. The predicted secondary structure of Fur homologs, including V. anguillarum and E. coli Fur, reveals a number of
-helices in conserved positions (30, 12). Although there was no
helix-turn-helix DNA binding motif found, it has been suggested that
-helices in the N-terminal region of E. coli Fur may
interact with the major groove of the DNA upon sequence-specific
recognition (30, 31). However, it is difficult to employ the binding
mode of E. coli Fur for the V. anguillarum Fur,
since the sequence-specific protein-DNA interactions through the major
groove generally require a high conservation of DNA sequence, but the
DNA sequences involved in the binding to V. anguillarum Fur
show a high diversity without any pattern. Therefore, an alternative
binding mode could be considered for the V. anguillarum Fur
system that is based on low sequence specificity, e.g.
recognition and binding to a specific DNA structure in the primary
binding sequence.
View larger version (23K):
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Fig. 7.
Comparison of Fur binding sequences. The
sequences of the template strand are shown in the V. anguillarum Fur binding sites; each binding site is divided into
two operator sequences (19-21 nucleotides) to maximize the alignment
with the E. coli Fur consensus sequence. The identical
nucleotides to the E. coli Fur consensus sequence are shown
in bold characters, and the repeated nucleotides are
shaded. The numbers indicate the nucleotide
position relative to the transcription start site.
The DNase I footprinting pattern shows different characteristics in the template (site I and II) and nontemplate strand (site I' and II'). While the template strand with sites I and II features protection by Fur, the complementary strand with site I' and II' contains at least two types of hypersensitive sites: periodic hypersensitive sites spaced by 15-16 nucleotides appeared in site II' and a cluster of hypersensitive sites in the upstream region of site I' (Fig. 6). Such hypersensitivity to DNase I is observed when protein-DNA interactions induce a conformational change of DNA, e.g. a curve in the DNA axis whereby the hypersensitive sites are at the outer face of the helical groove (32, 33). Hypersensitivity with periodicity larger than one B-helical repeat was detected when a right-handed superhelix DNA was formed by a multimeric protein, as is the case for the prokaryotic histone-like protein, H-NS (H1; Ref. 34). It is noteworthy that many proteins forming such multimeric complexes with DNA are categorized as histone-like and interact with DNA with low sequence specificity (34, 35). Interestingly, the H-NS protein binds to certain promoters with low sequence specificity and deforms the DNA structure, which results in shutting down of the expression of certain metabolic genes (33). Taken together, the binding mode of V. anguillarum Fur resembles those suggested for the prokaryotic DNA-binding protein II with histone-like properties (34, 35).
We interpret the results from DNase I footprints and gel retardation
assay as follows. V. anguillarum Fur recognizes the DNA structure given by A + T-rich sequences and binds as two dimers (tetramer) primarily to the promoter region (site I), the major interaction occurring with the template strand. This step could correspond to complex a, detected in the gel retardation assay. At
higher concentrations of Fur, a further tetramer binds to the secondary
binding site II, the downstream region of the transcription start site.
The complex formed here seems to be an intermediate and unstable
(complex b) and, therefore, undergoes further conformational changes
caused by protein-protein interaction between the sites I and II. The
protein-protein interaction seems to accompany the formation of the
superhelix in the whole Fur binding segment (80 bp), and such a
distortion may enhance sensitivity to the DNase I in the outer face of
the DNA helix. This complex with superhelicity (complex c) seems to
render the repression mechanism by Fur in which the distortion of the
DNA helix in the promoter region leads to a deteriorated positioning of
the 10 and 35 region. Such a structural change is probably no
longer recognizable by RNA polymerase, as shown in the mercury
resistance operon when the MerR protein was bound to its operator
located in the promoter in the absence of metal ion Hg2+
(36). Alternatively the presence of Fur bound in the promoter region
could hinder the formation of the closed or open complex of RNA
polymerase and the promoter as is the case for the H-NS mediated
repression system (32).
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ACKNOWLEDGEMENTS |
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We thank Ekaterina Zheleznova and Richard Brennan for providing purified V. anguillarum Fur protein.
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FOOTNOTES |
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* This work was supported by United States Public Health Grant AI 19018 from the National Institutes of Health (to J. H. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence and reprint requests should be addressed:
Dept. of Molecular Microbiology and Immunology, Oregon Health Sciences
University, Mail Code L-220, 3181 SW Sam Jackson Park Rd., Portland, OR
97201-3098. Tel.: 503-494-7583; Fax: 503-494-6862; E-mail:
crosajor{at}ohsu.edu.
The abbreviations used are: bp, base pair(s); kb, kilobase(s); EDDA, ethylenediamine-di-(o-hydroxyphenylacetic) acid; nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
2 S. Chai, T. J. Welch, and J. H. Crosa, unpublished results.
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REFERENCES |
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Abstract
Introduction
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Discussion
References
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