From the Department of Gastroenterology and
Hepatology, Erasmus MC University Medical Center Rotterdam, 3015 GD
Rotterdam, The Netherlands, the ¶ Department of Medical
Microbiology and Hygiene, Institute of Medical Microbiology and
Hygiene, University Hospital of Freiburg, D-79104 Freiburg, Germany,
and the
Institute for Microbiology, University of Greifswald,
D-17487 Greifswald, Germany
Received for publication, July 26, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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The production of high levels of ammonia allows
the human gastric pathogen Helicobacter pylori to survive
the acidic conditions in the human stomach. H. pylori
produces ammonia through urease-mediated degradation of urea, but it is
also able to convert a range of amide substrates into ammonia via its
AmiE amidase and AmiF formamidase enzymes. Here data are provided that
demonstrate that the iron-responsive regulatory protein Fur directly
and indirectly regulates the activity of the two H. pylori
amidases. In contrast to other amidase-positive bacteria, amidase and
formamidase enzyme activities were not induced by medium
supplementation with their respective substrates, acrylamide and
formamide. AmiE protein expression and amidase enzyme activity were
iron-repressed in H. pylori 26695 but constitutive in the isogenic fur mutant. This regulation was mediated at the
transcriptional level via the binding of Fur to the amiE
promoter region. In contrast, formamidase enzyme activity was not
iron-repressed but was significantly higher in the fur
mutant. This effect was not mediated at the transcriptional level, and
Fur did not bind to the amiF promoter region. These roles
of Fur in regulation of the H. pylori amidases suggest that
the H. pylori Fur regulator may have acquired extra functions to compensate for the absence of other regulatory systems.
The human pathogen Helicobacter pylori colonizes the
mucus layer overlaying the gastric epithelium, thereby causing
persistent gastritis, which can develop into peptic ulcer disease and
gastric carcinomas (1). H. pylori is able to survive and
colonize this hostile acidic niche, aided by the expression of its acid
resistance mechanisms (2, 3). One of the major factors contributing to
acid resistance of H. pylori is the production of ammonia by its urease enzyme, which is essential for gastric colonization in
different animal models (4-7). However, the role of urease in gastric
colonization extends beyond protection against gastric acid, because
H. pylori urease mutants are still unable to colonize the
gastric mucosa when gastric acid production is abolished with proton
pump inhibitors (4).
Ammonia is a key component of bacterial nitrogen metabolism, because it
is the preferred source of nitrogen for the synthesis of amino acids,
pyrimidines, and purines. Ammonia plays a central role in pathogenesis
and metabolism of the important human pathogen H. pylori,
because it not only serves as nitrogen source (8) but also contributes
to epithelial cell damage and apoptosis (9, 10), is involved in
chemotactic motility (11), and is required for acid resistance (2, 3).
Urea is thought to be the main source of ammonia in the gastric
environment, but H. pylori does have alternative pathways
for the production of ammonia via amino acid catabolism (12) and via
the activity of its two paralogous amidases,
AmiE1 and AmiF (13, 14).
Aliphatic amidase (AmiE, EC 3.5.1.4) and formamidase (AmiF, EC
3.5.1.49) catalyze the conversion of amide substrates to the
corresponding carboxylic acid and ammonia (13, 14).
The control of the intracellular nitrogen status is important for
living organisms, and this can be mediated by several different nitrogen regulatory systems. These include the PII
(GlnB) signal transduction protein and NtrBC two-component
regulatory system, which are widespread throughout the bacterial
kingdom (15), but alternative nitrogen regulatory systems exist
(16-18). An analysis of the H. pylori genome sequence did
not reveal the presence of any of the aforementioned nitrogen
regulatory proteins (19). The presence of nitrogen regulatory systems
is likely though, because the activity of the different
ammonia-producing enzymes seems to be balanced. The absence of urease
activity leads to higher amidase activity (13), whereas the combined
absence of urease and arginase led to higher formamidase activity (14). Conversely, the absence of arginase also led to alterations in the
activity of the amino acid deaminases (12), and thus, it is thought
that the intracellular nitrogen status of H. pylori is
controlled through yet unidentified regulatory systems.
An analysis of the genome sequence indicated that H. pylori
has a relatively limited capacity for gene regulation, and thus, it is
possible that the few regulatory proteins present regulate multiple
responses and metabolic processes (19). One well characterized regulatory protein of H. pylori is the ferric uptake
regulator (Fur), which controls intracellular iron homeostasis via
concerted expression of iron-uptake and iron-storage genes (20-23).
Because Fur has also been implicated in acid resistance of H. pylori (24) as well as in regulation of urease expression (25), we
hypothesized that Fur may also regulate the expression of alternative
ammonia-producing enzymes. Here we report that Fur regulates
transcription, expression, and activity of the AmiE amidase and
indirectly affects enzyme activity of the AmiF formamidase. The
regulation of ammonia production via the iron-regulatory protein Fur
may be an example of how H. pylori may compensate for its
relatively small regulatory capacity.
Bacterial Strains, Plasmids, Media, and Growth
Conditions--
H. pylori strain 26695 (19) and its
isogenic fur mutant (24) were routinely cultured on Dent
agar (26) consisting of Columbia agar supplemented with 7%
saponin-lysed horse blood, 0.004% triphenyltetrazolium chloride
(Sigma), and Dent-selective supplement (Oxoid, Basingstoke, United
Kingdom) at 37 °C under microaerophilic conditions (10%
CO2, 5% O2, and 85% N2). Broth cultures were grown in Brucella Broth (Difco, Sparks, MD) supplemented with 3% newborn calf serum (Invitrogen) (BBN). Ferric chloride and desferal (deferoxamine mesylate) were purchased from Sigma, filter-sterilized, and used at the indicated concentrations. To determine the effect of amide substrate on H. pylori, BBN
media were supplemented with acrylamide (Sigma) or formamide (Sigma) to
final concentrations of 5 and 100 mM, respectively. Iron
restriction was achieved by supplementing BBN with desferal to a final
concentration of 20 µM, whereas iron-repleted conditions
were achieved by supplementing desferal-treated BBN with ferric
chloride to a final concentration of 100 µM (20).
Escherichia coli DH5 Protein Analysis--
H. pylori wild-type and
fur mutant cells were grown in iron-restricted or
iron-repleted medium, centrifuged at 4000 × g for 10 min at 4 °C, and concentrated in ice-cold phosphate-buffered saline to a final A600 of 10. H. pylori cells were lysed by sonication for 15 s on ice with an
MSE Soniprep 150 set at amplitude 10. Protein concentrations were
determined with the bicinchoninic acid method (Pierce) using bovine
serum albumin as standard. Samples containing ~30 µg of protein
were separated by two-dimensional electrophoresis using a Multiphor II
electrophoresis unit (Amersham Biosciences). Isoelectric focusing was
performed on 11-cm Immobilin DryStrips (Amersham Biosciences) with a pH
range of 3-10 and subsequently separated according to molecular weight
on a ExcelGel SDS (Amersham Biosciences) with an acrylamide
concentration gradient of 12-14%. Proteins were subsequently stained
with Coomassie Brilliant Blue (27), trypsin-digested, and analyzed by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry using a Bruker Biflex III (Bruker Daltonics, Billerica,
MA). Protein identification was performed using the Mascot program (28)
and the OWL non-redundant composite protein sequence data base
(www.matrix-science.com).
Amidase, Formamidase, and Urease Enzyme Assays--
The
enzymatic activity of urease, amidase, and formamidase were determined
in fresh H. pylori lysates by measuring ammonia production
from hydrolysis of urea, acrylamide, or formamide, respectively, by
using the Berthelot reaction as described previously (13, 14, 25). The
concentration of ammonia present in the samples was inferred from a
standard NH4Cl concentration curve. Enzyme activity was
expressed as micromoles of substrate hydrolyzed per minute per
milligram of protein. Differences in enzyme activities were tested for
their statistical significance with the Mann-Whitney U test.
RNA Hybridization--
RNA was isolated from bacteria grown in
iron-restricted or iron-repleted conditions using TRIzol (Invitrogen)
according to the manufacturer's instructions. RNA was separated on 2%
formaldehyde, 1.5% agarose gels in 20 mM sodium phosphate
buffer and subsequently transferred to nylon membranes (Roche Molecular
Biochemicals) using standard protocols (25, 27). Following the
transfer, RNA was covalently bound to the membrane by cross-linking
with 0.120 J/cm2 UV light of a 254-nm wavelength. RNA was
visualized by methylene blue staining (25), and RNA samples were
normalized based on 16 S and 23 S rRNA band intensities. Internal
fragments of the amiE and amiF genes were
PCR-amplified with primers listed in Table
I. The resulting PCR fragments contained
a T7 promoter sequence on the non-coding strand and were used for the
production of antisense RNA probes labeled with DIG by in
vitro transcription using T7 RNA polymerase (Roche Molecular
Biochemicals). Northern hybridization and stringency washes were
performed at 68 °C, and bound probe was visualized with the DIG
detection kit (Roche Molecular Biochemicals) and the chemiluminescent
substrate CDP-Star (Amersham Biosciences) (25).
Recombinant DNA Techniques--
Restriction enzymes and
modifying enzymes were purchased from New England Biolabs (Beverly, MA)
and Promega (Madison, WI), and standard protocols were used for the
manipulation of DNA and transformation of E. coli (27) and
H. pylori (26). Plasmid DNA was prepared using Qiaprep spin
columns (Qiagen). PCR was carried out using Taq polymerase
(Promega).
Gel Retardation Assay--
Recombinant H. pylori Fur
protein was purified from E. coli with the pASK-IBA Streptag
system (IBA, Göttingen, Germany) as described previously (29).
DIG-labeled amiE and amiF promoter fragments were
amplified with primer combinations Amid-PrF/Amid-PrR-DIG and
Form-PrF/Form-PrR-DIG, respectively, and incubated with
increasing concentrations of recombinant Fur for 30 min at 37 °C in
binding buffer (20 mM Tris-Cl, pH 8.0, 75 mM
KCl, 1 mM dithiothreitol, 300 µg/ml bovine serum albumin,
100 µM MnCl2, 12% glycerol). Samples were
subsequently separated on a 5% polyacrylamide (37.5:1) gel in running
buffer (25 mM Tris, 190 mM glycine) for 30 min
at 200 V. The gel was then blotted onto a nylon membrane (Roche
Molecular Biochemicals), and DIG-labeled DNA was visualized using the
DIG detection kit (Roche Molecular Biochemicals) and the
chemiluminescent substrate CDP-Star.
Amidase Enzyme Activity Is Not Substrate-inducible--
In the
amidase-positive bacteria Pseudomonas aeruginosa and
Mycobacterium smegmatis, amidase activity is controlled by
substrate availability via the AmiR-AmiC and AmiA proteins,
respectively (30, 31). These proteins mediate the induction of amidase expression upon supplementation of growth medium with the amide substrate (30, 31). Although orthologs of the corresponding amidase
regulatory proteins are absent in H. pylori, the inspection of the H. pylori amiE and amiF promoters
indicated the presence of sequences resembling Furboxes, suggesting
iron-responsive regulation of these genes (22, 32).
To determine whether amidase and formamidase activity were
substrate-inducible or iron-regulated, we determined the effect of
substrate supplementation and varying iron-availability on amidase and
formamidase activity of H. pylori strain 26695. The highest
concentrations of amidase substrates that still allowed growth of
H. pylori 26695 were 5 mM acrylamide and 100 mM formamide (data not shown). Unlike other bacterial
amidases, supplementation with these concentrations of amide substrates
did not result in the induction of amidase or formamidase enzyme
activity (Fig. 1). However, changing iron
availability had a pronounced effect on amidase activity, which was
high in iron-restricted conditions but was almost absent in
iron-repleted conditions (Fig. 1A). In contrast, formamidase
activity was not changed in iron-restricted conditions when compared
with iron-repleted conditions (Fig. 1B). Thus, we conclude
that amidase and formamidase activity in H. pylori 26695 is
not substrate-inducible but that amidase activity is regulated by iron
availability, whereas formamidase activity seems constitutive.
Amidase Expression and Activity and Formamidase Activity Are
Regulated by Fur--
The AmiE (HP0294) protein was previously
identified as a protein of approximately 45 kDa with a pI of 6.4 (33).
A protein of similar molecular mass and pI was identified when
comparing two-dimensional protein profiles for the identification of
Fur- and iron-regulated proteins of H. pylori 26695 (Fig.
2). Wild-type cells expressed this
protein when grown in iron-restricted conditions but not in
iron-repleted conditions. This iron-repression was absent in the
fur mutant strain (Fig. 2), suggesting that iron regulation
was mediated by Fur. Subsequent identification of the protein by mass
spectometry confirmed that this iron- and Fur-repressed protein was
indeed AmiE (13, 14). Because the AmiF protein has not been identified
on two-dimensional gels yet (33), we were unable to compare AmiF
protein expression levels.
To assess whether the effect of Fur and iron on AmiE at the protein
expression level was also present at the enzyme activity level, we
determined amidase activity in lysates of H. pylori 26695 and its isogenic fur mutant grown in iron-restricted and iron-repleted conditions (Fig. 3). As
control, we also determined formamidase activity in both strains and
medium conditions. Amidase activity displayed identical regulation as
observed at the protein expression level. In wild-type cells, amidase
activity was high at iron-restricted conditions and absent in
iron-repleted conditions (p < 0.01), whereas in the
fur mutant, activity was always high, independent of iron
availability (Fig. 3A, p = 0.56).
Surprisingly, formamidase activity was also affected by the
fur mutation. Formamidase activity did not differ between
cells grown in iron-restricted and iron-repleted conditions but
differed significantly between the wild-type and fur mutant
cells (Fig. 3B, p < 0.01). In wild-type cells, formamidase activity was low but present, whereas formamidase activity was increased almost 3-fold in the fur mutant (Fig.
3B). These results were reproduced with a second
independently constructed H. pylori 26695 fur
mutant (data not shown), indicating that the increase in formamidase
activity is not caused by a secondary mutation.
Fur Mediates the Regulation of amiE but Not amiF at the
Transcriptional Level--
Regulation via iron and Fur is usually
mediated at the transcriptional level (32). The observed iron- and
Fur-responsive regulation of AmiE expression was indeed reflected at
the mRNA level as demonstrated by Northern hybridization (Fig.
4). There was no amiE mRNA
detected in the wild-type strain under iron-repleted conditions, but
the transcription of a 1-kilobase mRNA was clearly apparent in
iron-restricted conditions. In contrast, in the fur mutant,
amiE mRNA was present irrespective of the iron
availability of the medium (Fig. 4). However, the effect of the
fur mutation on formamidase activity is not mediated at the
transcriptional level, because the small changes in the levels of
amiF mRNA observed on Northern hybridizations (Fig. 4)
did not correlate with the changes in the enzyme activity observed
(Fig. 4).
Specific Binding of Fur to the amiE Promoter but Not to the amiF
Promoter--
The Fur protein normally functions by
metal-dependent binding to a binding sequence (Furbox)
located in the promoter region of the regulated gene (32). An analysis
of the sequence directly upstream of the amiE and
amiF genes had already indicated the presence of putative
Furboxes (Fig. 5A). To confirm
that amiE and amiF transcription was indeed
differentially regulated by Fur, we performed gel retardation assays
using recombinant H. pylori Fur (29) and DIG-labeled
amiE and amiF promoter regions. The addition of
recombinant H. pylori Fur with the metal cofactor Mn2+ to the amiE promoter region shifted the
mobility of the amiE promoter, consistent with binding of
Fur to this promoter (Fig. 5B). Gel retardation was
dependent on the presence of the Mn2+ metal cofactor (data
not shown). To check sequence specificity, we also used an internal
fragment of the amiE gene whose mobility was not affected by
Fur (data not shown). Finally, as predicted from the Northern
hybridization experiments but despite the presence of Furbox-like
sequence, the mobility of the amiF promoter was not affected
by Fur (Fig. 5B).
Many species of the genus Helicobacter colonize the
acidic gastric mucosa of humans and animals, and in this respect, they represent unique pathogens (1). Colonization is dependent on acid
resistance, and although this process is multifactorial, the production
of high levels of ammonia is essential to allow initial infection as
well as subsequent colonization. Acid resistance of H. pylori has long been considered to be solely based on unregulated production of large amounts of urease, but recent studies have shown
that acid resistance of H. pylori is based on
multifactorial, interactive, and probably well regulated processes (3,
25, 34-36). In these processes, metal-responsive regulatory proteins play an important role with the NikR protein regulating urease expression (25, 34) and the Fur protein regulating iron homeostasis, acid resistance (20-24), and amidase- and formamidase-mediated ammonia
production (this study) (Fig. 6).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MCR (Invitrogen) was grown aerobically in Luria-Bertani medium at 37 °C (27). For antibiotic selection, growth media were supplemented with ampicillin, kanamycin, or chloramphenicol to final concentrations of 100, 20, and 10 µg/ml, respectively.
Oligonucleotide primers used in this study
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amidase and formamidase activity in H. pylori is not substrate-inducible, but amidase activity is
iron-repressed. A, amidase activity in H. pylori grown in iron-repleted (+Fe) and iron-restricted
( Fe) BBN medium without (black bars) and with
acrylamide (white bars) supplemented to a final
concentration of 5 mM. B, formamidase activity
in H. pylori grown in iron-repleted (+Fe) and
iron-restricted (
Fe) BBN medium without (black
bars) and with formamide (white bars) supplemented to a
final concentration of 100 mM. Graphs represent three
independent experiments, and error bars denote means ± S.D. Statistical evaluations of the comparison of enzyme activities
using the Mann-Whitney U test are given.
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Fig. 2.
Iron-regulated expression of the
H. pylori AmiE protein is mediated by
Fur. Protein profiles of H. pylori 26695 wild-type and
fur mutant cells grown in iron-restricted ( Fe)
and iron-repleted (+Fe) conditions were compared on
two-dimensional protein gels. The relevant part of the protein gel is
magnified for each gel, and the iron- and Fur-repressed AmiE protein is
circled. The estimated molecular mass and pI are
indicated.
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Fig. 3.
Effect of varying iron availability on the
activity of amidase (A) and formamidase
(B) activity in H. pylori 26695 wild-type and fur mutant strains. Enzyme
activities were compared in lysates of cells grown in iron-repleted
conditions (black bars) and iron-restricted (white
bars) conditions, and their respective enzyme activities were
determined. Graphs represent a minimum of five independent experiments,
and error bars denote ± S.D. Statistical evaluation of
the comparison of enzyme activities using the Mann-Whitney U
test are given.
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Fig. 4.
Differential effect of Fur on amiE
and amiF transcription. RNA was isolated
from H. pylori 26695 wild-type and fur mutant
cells, grown in iron-restricted ( Fe) and iron-repleted
(+Fe) conditions, and subjected to Northern hybridization
with amiE- and amiF-specific probes. Top
panel, staining of transferred RNA for comparison of RNA amounts;
middle panel, hybridization with the
amiE-specific probe; lower panel, hybridization
with the amiF-specific probe. rRNA species and hybridizing
RNAs are defined on the right-hand side.
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Fig. 5.
The Fur protein binds specifically to the
amiE promoter but not to the amiF
promoter. A, identification of putative Furboxes
in the promoters of the amiE and amiF genes. The
Furbox consensus sequence is given on the top with the
identified amiE and amiF sequences
boxed. Residues identical to the Furbox consensus sequence
(32) are underlined. The distance of the putative Furboxes
to the ribosome binding site and start codon are also indicated.
B, gel retardation assay of the amiE and
amiF promoter regions and increasing amounts of recombinant
H. pylori Fur. The unbound promoters are indicated by
PamiE and PamiF, and the retarded fragment is indicated
as Fur-PamiE, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Schematic representation of the role of Fur
in ammonia production of H. pylori and the
role of ammonia in H. pylori metabolism and
virulence.
Under physiological conditions, the optimal pH for H. pylori growth lies between 4 and 6 (1). As a result, the colonization pattern of H. pylori varies with the level of acid production within the host stomach, and these different colonization patterns are associated with different long term outcomes of infection (37, 38). As such, H. pylori acid resistance is very relevant for the clinical outcome of disease and may also offer clues for therapy. H. pylori produces large amounts of ammonia through urea degradation and actually requires an acidic environment to survive in the presence of urea because of alkalinization of the medium to toxic levels at a neutral pH (39). The presence of alternative pathways for the production of ammonia is likely to have evolved for situations where either urea is not available where ammonia production is required at neutral pH conditions or when toxic concentrations of amides are encountered in the natural niche of H. pylori.
Amidase enzymes are often present in environmental bacteria where they function in the degradation of toxic amides in the environment and are of interest for waste disposal. The function or natural substrate(s) of the H. pylori amidases are not yet known, and thus, it is difficult to predict their exact function in H. pylori metabolism. Although it is difficult to envisage high levels of amides being produced intracellularly in H. pylori, recent reports of possibly toxic or carcinogenic concentrations of acrylamide in food have raised concerns for public health (40). The acrylamide can be produced after Strecker degradation of asparagine or methionine in the presence of dicarbonyl compounds via the Maillard reaction (41, 42). Of special interest in the gastric environment may be the route via methionine, because this reaction has a requirement for ammonia as produced by H. pylori (41, 42). Furthermore, although it is possible that the amidases function in protection against toxic amides, our preliminary data indicate that the production of the AmiE amidase does not increase protection against toxic concentrations of acrylamide in a disc assay (data not shown).
Regulation of amidase expression was so far only studied in P. aeruginosa and in M. smegmatis where amidase expression is induced upon supplementation with amide substrate (30, 31). We have demonstrated here that amidase and formamidase activity is not substrate-induced in H. pylori but that amidase activity is Fur- and iron-repressed. This unexpected type of regulation may be explained by either a role for amidase in siderophore synthesis or by a link between amide availability and iron availability. Amidases like AmiE and AmiF can form hydroxamates via an acyl transferase reaction using hydroxylamide as acceptor molecule (14). Hydroxamates are an important class of siderophores, and in siderophore-producing bacteria, the biosynthesis of siderophores is usually iron-regulated (43). H. pylori lacks orthologs of bacterial siderophore biosynthesis genes (19) but may use amidase-mediated formation of hydroxamates as an alternative route to produce siderophores. However, the toxicity of hydroxylamine makes it unlikely that H. pylori is able to safely produce the quantities of hydroxylamine necessary to scavenge sufficient iron from the gastric environment.
An alternative possibility is that there may be a link between the availability of iron and amide substrates. Both the urease- and amidase-enzymatic reactions lead to the production of ammonia, but although the urease reaction results in alkalinization of the environment (39), the amidase reaction is pH-neutral (13, 14). Amidase-generated ammonia is probably not sufficient for acid resistance of H. pylori (44) but may still be used to form urea through the previously suggested urea cycle of H. pylori (12, 45), and thus, amidase activity may be important when urea availability is low. Alternatively, because ammonia also plays an important role in nitrogen metabolism, the pH-neutral production of ammonia by both amidases may allow the production of sufficient intracellular concentrations of ammonia without alkalinization of the cellular environment.
Finally, a coupling between iron availability and substrate availability is supported by studies on the function and secretion of the H. pylori vacuolating cytotoxin VacA (46, 47). Firstly, the VacA protein has been suggested to function as a urea permease, promoting urea diffusion from epithelial cells (46). Secondly, VacA is present in outer membrane vesicles that are thought to deliver pro-inflammatory proteins to the epithelial cells but only in iron-repleted conditions (47). Combined, this would result in high urea release in iron-repleted conditions but low urea release in iron-restricted conditions. It is under these conditions where urea availability is low that amidase activity may be an alternative source of ammonia and, as such, make iron-repression of amidase physiologically relevant.
Surprisingly, the amiE and amiF genes were differentially regulated by Fur. The amiE gene is regulated at the transcriptional level by Fur, whereas the fur mutation only affects enzyme activity of AmiF but not amiF transcription (Figs. 4 and 5). The mechanism behind the increased formamidase activity in the fur mutant is currently unknown. We hypothesize that this increase may be the result of the altered intracellular environment caused by the pleiotropic effects of the fur mutation, by changes in availability of a yet unknown enzyme cofactor, or by altered stability or conformation of the formamidase enzyme. We have also tested a second independent fur mutant in H. pylori strain 26695, which contains a promoterless chloramphenicol cassette in fur (23). This independent fur mutant also displayed derepressed amidase activity and increased formamidase activity (data not shown), thus the observed effect on formamidase activity is unlikely to result from a secondary mutation or polar effects of the antibiotic cassette inserted in the fur gene. The Fur protein showed specific binding to the amiE promoter but not to the amiF promoter, despite both promoters having sequences resembling Furboxes (Fig. 5A). This again demonstrates the limitations of Furbox predictions that are based solely on sequence similarity (48).
In conclusion, we have identified a novel type of gene regulation for
bacterial amidases, which is mediated by Fur at the transcriptional and
enzyme activity level (for AmiE) and at the enzyme activity level (for
AmiF). The diverse roles of the Fur regulatory protein in metabolic and
pathogenic processes of H. pylori indicate that this
bacterium is able to use several intricately linked mechanisms to
survive and thrive in the gastric mucosa and is able to sense and cope
with the variable conditions and multiple stresses occurring there
despite its relatively limited range of regulatory proteins.
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ACKNOWLEDGEMENTS |
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We thank Theo Hoogenboezem for technical assistance with protein sequencing and David J. Kelly for providing the H. pylori 26695 fur mutant.
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FOOTNOTES |
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* This work was supported by in part by Grants 901-14-206 and DN93-340 from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (to A. H. M. v. V. and J. G. K.), respectively, and Grant Ki201/9-1 of the Deutsche Forschungsgemeinschaft (to M. K.).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 should be addressed: Dept. of Gastroenterology and Hepatology, Rm. L-455, Erasmus MC-University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Tel.: 31-10-4635944; Fax: 31-10-4632793; E-mail: a.h.m.vanvliet{at}erasmusmc.nl.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M207542200
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ABBREVIATIONS |
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The abbreviations used are: AmiE, aliphatic amidase; AmiF, formamidase; Fur, ferric uptake regulator; BBN, Brucella Broth supplemented with 3% newborn calf serum; BSA, bovine serum albumin; DIG, digoxigenin.
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