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Journal of Bacteriology, August 2001, p. 4932-4937, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4932-4937.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Iron-Dependent Transcription of the frpB Gene of
Helicobacter pylori Is Controlled by the Fur
Repressor Protein
Isabel
Delany,1
Ana Beatriz F.
Pacheco,1,
Gunther
Spohn,1
Rino
Rappuoli,1 and
Vincenzo
Scarlato1,2,*
Department of Molecular Biology, IRIS Chiron
S.p.A., 53100 Siena,1 and Department of
Biology, University of Bologna, 40126 Bologna,2 Italy
Received 9 April 2001/Accepted 1 June 2001
 |
ABSTRACT |
We have overexpressed and purified the Helicobacter
pylori Fur protein and analyzed its interaction with the
intergenic regions of divergent genes involved in iron uptake
(frpB and ceuE) and oxygen radical
detoxification (katA and tsaA). DNase I
footprint analysis showed that Fur binds specifically to a
high-affinity site overlapping the
PfrpB promoter and to low-affinity sites located upstream from promoters within both the
frpB-katA and ceuE-tsaA intergenic
regions. Construction of an isogenic fur mutant
indicated that Fur regulates transcription from the PfrpB promoter in response to iron. In
contrast, no effect by either Fur or iron was observed for the other promoters.
| |
TEXT |
In many bacteria iron-responsive
gene expression is regulated by a transcriptional repressor called Fur
(ferric uptake regulator) (9, 12). The DNA-binding
activity of Fur is Fe dependent where Fe acts as a corepressor. Under
iron-rich conditions Fur is complexed with Fe and binds target
sequences called Fur boxes, located in the promoter regions of
iron-regulated genes, thus preventing transcription. When iron is
scarce, the Fur molecule loses the Fe corepressor and is released from
DNA, allowing transcription to occur. Fur has been extensively
characterized for Escherichia coli. Homologs of Fur have
been identified in many gram-negative bacteria and more recently in
gram-positive bacteria (7, 32).
The fur gene of Helicobacter pylori was first
cloned by virtue of the Fur titration assay and was shown to be able to
partially complement an E. coli Fur mutant in an
iron-dependent way, indicating that Fe acts as a corepressor of the
H. pylori Fur protein (4, 5). A modified Fur
titration assay with an E. coli strain expressing H. pylori Fur only was used to identify Fur binding sites in the promoter regions of the fecA2 and ribBA genes of
H. pylori (14). Furthermore, it has been shown
that transcription of both of these genes is repressed by iron
(14, 30). Recently, Fur has been shown to be necessary for
the iron-dependent regulation of the pfr gene
(6). The Fur protein has been implicated also in the regulation of genes involved in the detoxification of oxygen radicals (2, 10, 17, 18, 34).
Analysis of the annotated genomes of H. pylori (1,
29) led us to the selection of two loci as candidate targets for
Fur regulation. The structural organization of these loci is
represented schematically in Fig. 1. Each
locus is comprised of two genes that code for proteins expected to be
involved in iron uptake (frpB and ceuE) and
detoxification (katA and tsaA), that are oriented in different directions, and, therefore, that are expected to be
transcribed from divergent promoters. The FrpB protein is a homolog of
the Fe limitation-inducible outer membrane protein of Neisseria
meningitidis (27.6% amino acid identity and 49.5% similarity)
that belongs to the family of TonB-dependent receptors (25,
29). In Neisseria spp. FrpB, which is an
iron-regulated, 76-kDa outer membrane protein, functions as an
enterobactin receptor (8). The CeuE protein is a homolog
of the iron(III) ABC transporter, periplasmic iron-binding protein
(29). The other two genes, katA and
tsaA, code for the catalase (24) and alkyl
hydroperoxide reductase (AhpC) (21) proteins,
respectively, which are involved in the detoxification of oxygen
radicals in H. pylori.
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FIG. 1.
Schematic representation of the two selected iron uptake
and detoxification loci. Arrows indicate directions of transcription.
Nomenclature is as described in the work of Tomb et al.
(29).
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In order to investigate if Fur has a role in the regulation of these
candidate loci, we purified a recombinant H. pylori Fur protein to use in DNase I footprinting experiments. The fur
gene was amplified by PCR from H. pylori chromosomal DNA and
cloned into the expression plasmid pET22b+, generating pETfur (Table 1), such that a tail encoding six
histidines was added to the fur gene. E. coli
BL21(DE3) was transformed with plasmid pETfur, and expression was
induced by adding 1 mM IPTG
(isopropyl-
-D-thiogalactopyranoside) to
exponentially growing cells. The recombinant protein was then purified
under native conditions from the cell lysate by Ni-nitrilotriacetic acid affinity chromatography as described by the manufacturer (Qiagen).
Figure 2 shows a sodium dodecyl sulfate
(SDS)-polyacrylamide gel with cell lysates from the uninduced and
induced E. coli expression culture and the purified Fur
protein.
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FIG. 2.
Expression and purification of a recombinant H.
pylori Fur protein in E. coli and
SDS-polyacrylamide gel electrophoresis analysis of protein samples. DNA
manipulations were carried out routinely as described previously
(26). Lanes 1 and 2 contain whole-cell extract of
E. coli BL21(DE3) carrying the expression plasmid pETfur
(Table 1) before and after 4 h of induction with 1 mM IPTG,
respectively. Lane 3 contains the six-His-tagged Fur protein
preparation after purification by affinity chromatography. Lane M
contains protein size standards; molecular masses are indicated
to the left. The arrow indicates the migration of the Fur protein. Fur
was purified by Ni-nitrilotriacetic acid chromatography (Qiagen),
dialyzed twice against 800 ml of buffer D (50 mM Tris-HCl [pH 8.0],
100 mM NaCl, 1 mM MgCl2, 2 mM dithiothreitol) containing
10% glycerol, and dialyzed once against 200 ml of buffer D containing
50% glycerol. Protein concentration was determined by the Bradford
method (Bio-Rad) as 1.8 mg/ml; the protein was aliquoted and stored at
 80°C.
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The H. pylori Fur protein binds to two loci involved
in iron uptake and detoxification.
DNA fragments comprising the
intergenic regions of the frpB-katA and ceuE-tsaA
divergent gene loci were cloned (Table 1), 5'-end labeled at one
extremity with [
-32P]ATP (5,000 Ci/mmol;
Amersham) and T4 polynucleotide kinase (New England Biolabs), and used
as probes in DNase I footprinting experiments with the purified Fur
protein. Protein-DNA complexes were formed in 50 µl of footprinting
buffer (10 mM Tris-HCl [pH 8], 50 mM NaCl, 10 mM KCl, 1 mM
dithiothreitol, 0.1% NP-40, 10% glycerol) containing approximately 10 ng (20,000 cpm) of the labeled probe and a 100-fold excess (1 µg) of
sonicated salmon sperm DNA as the nonspecific competitor DNA for 15 min
at room temperature. Following incubation for 1 min at room temperature
with 0.01 U of DNase I and 5 mM CaCl2, the
reaction was stopped by addition of 140 µl of stop buffer (192 mM
Na-acetate, 32 mM Na2EDTA, 0.14% SDS, 64 µg of
sonicated salmon sperm DNA per ml). Samples were phenol extracted,
ethanol precipitated, resuspended in denaturing sample buffer, and
fractionated on urea-6% acrylamide gels. Figure 3a and b show the results of DNase I
footprinting with Fur on the frpB-katA and
ceuE-tsaA probes, respectively. Addition of 0.12 µg of Fur
protein resulted in one region of DNase I protection on the
frpB-katA probe spanning positions 1 to 42 (box 1) with respect to the initiation of RNA transcription (see below) of the
frpB gene (Fig. 3a). Another region with less affinity was evident only after addition of 1 µg of the Fur protein and spans positions 57 to 91 (box 2). On addition of 1 µg of Fur protein, the ceuE-tsaA probe exhibited one region of DNase I
protection from positions 39 to 67 (box 3) upstream of the
transcription initiation site of the ceuE gene (Fig. 3b).
Alignment of the nucleotide sequences of the Fur-protected regions
revealed 10 absolutely conserved nucleotides and 15 nucleotides
conserved in two out of three of the sequences over a 35-nucleotide
region (Fig. 3c). Analysis of the sequence of the box 1 binding site,
which shows highest affinity for the protein, reveals the presence of
overlapping sequences resembling that of the E. coli Fur box
consensus (GATAATGATAATCATTATC) at 3-nucleotide intervals
ranging from 14 matches to 12 matches out of 19 nucleotides. Figure 3c
shows in bold the nucleotides matching the consensus of the first of
the overlapping Fur boxes. Boxes 2 and 3 also contain sequences
resembling the Fur box consensus, although to lesser extents, showing
11 and 9 out of 19 matches, respectively. Therefore, as reported for
other bacteria (23, 32, 33), Fur from H. pylori
binds to sequences sharing similarity to the E. coli Fur
box.
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FIG. 3.
DNase I footprints of Fur on the
frpB-katA (a) and the ceuE-tsaA (b)
probes. Probes derived from plasmids pGemK-F and pGemC-T (Table 1) were
5'-end labeled at their EcoRI sites and incubated with
increasing microgram amounts of Fur as indicated above each lane. The
vertical bars on the right of each panel indicate the areas of DNase I
protection. Numbers indicate the distance from the initiation of
transcription of the frpB gene (a) and the
ceuE gene (b). The G+A lane is a G+A sequence reaction
on the DNA probe used as a size marker (22). (c) Alignment
of Fur binding sites. Absolutely conserved nucleotides have a black
background, and nucleotides that are conserved in two out of three
sequences have a gray background. Bold lettering indicates conservation
with the consensus E. coli Fur box
(GATAATGATAAGCATTATC), with the numbers to the left indicating numbers
of conserved nucleotides over the total number of nucleotides. A
consensus of the aligned sequences is shown below these sequences, with
uppercase letters indicating absolutely conserved nucleotides and
lowercase letters indicating nucleotides that are conserved in two out
of three sequences. Quantification of the signals from extension
products obtained was performed using a PhosphorImager and
ImageQuant software (Molecular Dynamics).
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These results showed that Fur is a DNA-binding protein that binds
specifically to the two intergenic regions of the candidate
loci.
Fur binds, in vitro, to low-affinity binding sites in both
intergenic
regions and to a high-affinity site upstream of the
frpB gene.
The divergent genes in the frpB-katA and
ceuE-tsaA loci are transcribed by
80-dependent promoters.
Total RNA was extracted
from H. pylori strain G27, and the start point of RNA
transcription was mapped by primer extension using specific primers
(27). Autoradiographs of urea-acrylamide gels in Fig.
4 (lanes 1) show the results of the
primer extension experiments. Extension of the RNA with
katA-specific, ceuE-specific, and
tsaA-specific primers gave rise to major bands placing the transcriptional start sites of the PkatA,
PceuE, and PtsaA
promoters at 55, 25, and 96 nucleotides upstream of the
katA, ceuE, and tsaA translational
start codons, respectively (Fig. 4b to d, lanes 1). Extension of RNA
with an frpB-specific primer gave rise to a cluster of faint
bands, which on longer exposure of the gel could be centered around
position 78 with respect to the translational start codon of the
frpB gene (Fig. 4a, lane 1). Nucleotide sequence analysis of
the DNA regions upstream of the four transcriptional start sites
revealed the presence of 10 hexamers with striking similarities
(maximum of two mismatches) to the consensus sequence recognized by
E(
2')70,
the major RNA polymerase in E. coli (Fig.
5). By contrast, the 35 hexamers showed
a high degree of sequence variability (minimum of three mismatches)
compared to the E. coli consensus sequence and among
themselves (Fig. 5). These results, taken together, suggest that the
promoters transcribing the divergent genes in both loci are transcribed
by RNA polymerase containing 80, the H. pylori homolog of the vegetative sigma factor
70 from E. coli. Previous work in
our laboratory has shown that this sigma factor shares homology,
promoter sequence recognition, and binding specificities with its
E. coli counterpart (3).
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FIG. 4.
Primer extension analysis of the promoters of the
frpB-katA and ceuE-tsaA loci. Total RNAs
isolated from H. pylori strains G27 (lanes 1 and 2) and
G27fur::kan (lanes 3 and 4)
were hybridized to the radiolabeled oligonucleotides Frp
(CTCTTAAAAACATCCAAC) (a), Kat (CACATCTTTATTAACCAT)
(b), Ceu (ACGATGAAACAAGAAGCG) (c), and Tsa
(GGCAAGTTTTGTAACTAAC) (d) and elongated with reverse
transcriptase (27). Elongated primers are indicated
by arrows. To ensure correct mapping of the extension, we sequenced in
parallel the respective cloned promoter region with the same primers
used in the primer extension reactions (not shown). The nucleotide
sequence of the sense strand upstream of the transcriptional
initiations are shown to the left of the panels, with the 10 motifs
indicated by a vertical bar and the nucleotides corresponding to +1
initiation sites indicated by bent arrows. Wild-type H.
pylori G27 and the isogenic fur mutant
G27fur:: kan were grown to
logarithmic phase in liquid cultures and then harvested or further
incubated for 15 min in the presence of 50 µM 2,2'-dipyridyl before
being harvested (lanes 2 and 4, marked by ).
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FIG. 5.
Nucleotide sequences of the promoters of the iron uptake
and detoxification genes. An alignment of sequences with respect to
their transcriptional start sites (+1) is shown. The 10 and 35
hexamers are indicated in bold. The consensus sequence for the promoter
that recognized the E. coli 70 sigma
factor is shown below. Underlined nucleotides indicate substitutions
from the consensus E. coli hexamers. It is worth
noting that the PceuE and
PkatA promoters show a TG motif at position 14
or 15 (also in bold) typical of so-called extended 10 promoters
(19).
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Mutation of the fur gene results in derepression of
PfrpB.
To study the regulation of the
PfrpB, PkatA,
PceuE, and PtsaA
promoters, we constructed an isogenic mutant strain with a mutated
H. pylori fur gene. By homologous recombination with a
suicide vector construct (pGemfur::km) (Table 1), most of the
fur coding sequence of H. pylori strain G27 was
replaced with a kanamycin resistance gene (27). Correct replacement of the wild-type sequence with the antibiotic resistance cassette was verified by means of PCR using oligonucleotides
complementary to regions flanking the insertion site. Total RNA from
the fur mutant strain
(G27fur::kan) was isolated, and
transcription from the promoters under study was investigated by primer
extension assays (Fig. 4, lanes 3). Extension of the RNA at the iron
uptake promoters gave different results. While the amount of RNA at the PfrpB promoter was increased over 10-fold in
G27fur::kan compared to that in the
wild-type strain (Fig. 4a, lane 3), suggesting that the
PfrpB promoter is derepressed in the
fur mutant, the amount of RNA at the
PceuE promoter remained unchanged in the mutant
strain (Fig. 4c, lane 3). Analysis at the detoxification promoters
PkatA and PtsaA showed
essentially the same amount of transcript resulting from both the
wild-type and fur mutant RNA preparations (Fig. 4b and d,
lanes 1 and 3). Therefore, inactivation of the fur gene
causes derepression of the PfrpB promoter,
indicating that the Fur protein of H. pylori negatively
regulates PfrpB. Under the experimental conditions used, no effect of the mutation of fur was
observed on transcription from the PceuE,
PkatA, and PtsaA
promoters, suggesting that these promoters are not Fur regulated.
Iron-dependent transcription of PfrpB is
Fur mediated.
To study the transcriptional response of the Fur
regulatory protein to various concentrations of iron, we isolated RNAs
from liquid cultures of the wild-type and
G27fur::kan strains that had been grown
to logarithmic phase and subsequently incubated for a further 15 min in
the presence or absence of 50 µM 2,2'-dipyridyl to deplete iron.
Primer extension analysis of these RNAs showed that, in the wild-type
strain, a 15-min treatment of cells with 2,2'-dipyridyl led to an
increase (10-fold) in the amount of transcripts from the
PfrpB promoter (Fig. 4a, compare lane 1 with lane 2); thus, depletion of iron from the growth medium resulted in
induction of the promoter. The amount of transcript at the PfrpB promoter in the
G27fur::kan mutant was unaffected by
the depletion of iron with 2,2'-dipyridyl (Fig. 4a, lanes 3 and 4) and
was comparable to the amount in the wild-type strain treated for 15 min
with 2,2'-dipyridyl (lane 2). Primer extension analyses carried out on
the PkatA, PceuE,
and PtsaA promoters (Fig. 4b, c, and d,
respectively) revealed no significant differences in the amounts of RNA
in either the wild-type or the mutant strain under either of the growth
conditions. Furthermore, a 15-min treatment with 100 µM
Fe2SO4 to increase the iron
concentration in wild-type or fur mutant cells had no effect
on the amount of RNA transcribed from the PceuE, PkatA, and PtsaA
promoters (data not shown).
These results showed that transcription from the
P
frpB promoter is derepressed in the wild-type
strain on depletion
of iron from the growth medium, that treatment with
50 µM 2,2'-dipyridyl
is sufficient to fully derepress the promoter to
a level comparable
to that in the
fur mutant, and that this
iron regulation is Fur
mediated, as the response to iron is not present
in the
fur mutant.
In addition, no iron regulation was
observed for any of the P
ceuE,
P
katA, and P
tsaA promoters.
In conclusion, we have characterized the Fur protein of
H. pylori to be a DNA-binding protein that binds to sequences
resembling
the
E. coli consensus Fur box. We have
characterized the promoter
of an
frpB gene predicted to be
involved in iron uptake as iron
regulated and demonstrated that Fur
represses transcription from
this promoter in an iron-dependent fashion
by binding directly
to the promoter sequences and, probably, denying
access to RNA
polymerase (
11,
13). Furthermore, we found
two low-affinity
sites whose functions in vivo remain unclear. The
biological function
of these low-affinity binding sites may be in the
regulation of
genes in response to other environmental signals.
Accordingly,
Fur has been shown to respond to environmental signals
other than
iron concentration, such as in
Salmonella
enterica, where Fur
was shown to mediate induction of acid
tolerance genes in response
to acid shock (
15). In
H. pylori, where regulators are few,
it will be interesting
to discover the full extent of the role
of this
protein.
| |
ACKNOWLEDGMENTS |
We thank C. Mallia for editing the manuscript and G. Corsi for artwork.
This work has been supported partially by EU-TMR grant FMRX-CT980164,
Chiron, and MURST. I.D. is the recipient of an EU-TMR fellowship
(FMRX-CT980164), and A.B.F.P. was supported by a CAPES fellowship (Brazil).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology, IRIS, Chiron S.p.A., Via Fiorentina 1, 53100 Siena, Italy. Phone: 39 0577 243565. Fax: 39 0577 243564. E-mail:
enzo_scarlato{at}chiron.it.
Present address: Biophysics Institute, University of Rio de
Janeiro, Rio de Janeiro, Brazil.
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Journal of Bacteriology, August 2001, p. 4932-4937, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4932-4937.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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