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Journal of Bacteriology, January 2001, p. 468-475, Vol. 183, No. 2
Department of Molecular Biology and
Biotechnology, University of Sheffield, Sheffield S10
2TN,1 and Department of
Microbiology, University of Leeds, Leeds,2
England
Received 26 July 2000/Accepted 23 October 2000
The Staphylococcus aureus genome encodes three ferric
uptake repressor (Fur) homologues: Fur, PerR, and Zur. To determine the
exact role of Fur in S. aureus, we inactivated the
fur gene by allelic replacement using a tetracycline
resistance cassette, creating strain MJH010 (fur). The
mutant had a growth defect in rich medium, and this defect was
exacerbated in metal-depleted CL medium. This growth defect was
partially suppressed by manganous ion, a metal ion with known
antioxidant properties. This suggests that the fur mutation
leads to an oxidative stress condition. Indeed, MJH010
(fur) has reduced levels of catalase activity resulting from decreased katA transcription. Using a
katA-lacZ fusion we have determined that Fur functions,
either directly or indirectly, as an iron-dependent positive regulator
of katA expression. Transcription of katA is
coregulated by Fur and PerR, since in MJH010 (fur) transcription was still repressed by manganese while transcription in
MJH201 (fur perR) was unresponsive to the presence of iron or manganese. Siderophore biosynthesis was repressed by iron in 8325-4 (wild-type) but in MJH010 (fur) was constitutive. A
number of putative Fur-regulated genes were identified in
the incomplete genome databases using known S. aureus Fur
box sequences. Of those tested, the sstABCD and
sirABC operons and the fhuD2 and
orf4 genes were found to have Fur-regulated expression.
MJH010 (fur) was attenuated (P < 0.04) in
a murine skin abscess model of infection, as was double-mutant MJH201
(fur perR) (P < 0.03). This demonstrates the importance in vivo of iron homeostasis and oxidative stress resistance regulation in S. aureus.
The ability of a pathogen to
successfully colonize tissues and proliferate is limited by iron
availability in vivo. Iron, although abundant, is mostly bound to host
carrier proteins, such as transferrin and lactoferrin
(56). In addition, the limitation of metal ions, through
nonspecific host responses to infection such as hypoferremia
(6), reduces the ability of bacteria to replicate and
increases their susceptibility to clearance by the immune system. Iron,
together with manganese, is a cofactor for antioxidant defence enzymes
of the pathogen, e.g., catalase, peroxidase, and superoxide dismutase
(1). The reactivity of iron, however, means that it is
potentially toxic to bacteria. For example, the Fenton reaction between
intracellular iron and endogenously produced hydrogen peroxide produces
the deleterious hydroxyl radical (29, 30, 43, 44).
Bacteria possess a number of iron-scavenging mechanisms to overcome
iron limitation in vivo. The first of these is the secretion of
high-affinity iron(III)-chelating ligands, called siderophores, that
bind available iron and that are actively transported back into the
cell via specific surface receptors (16, 24). A second mechanism, found in the non-siderophore-producing pathogens
Neisseria meningitidis, Haemophilus influenzae,
and Actinobacillus pleuropneumoniae (16, 56),
involves direct contact between host transferrin and
bacterial-cell-wall-located transferrin-binding proteins. A third
mechanism involves importing iron(II) directly into the cell via
membrane protein FeoB (9).
Staphylococcus aureus is capable of producing siderophores,
removing iron from transferrin via cell wall transferrin-binding proteins (38, 52), and its genome encodes at least two
FeoB homologues (http://www.genome.ou.edu; http://www.tigr.org).
Siderophores aureochelin (17), staphyloferrin A (32,
35), and staphyloferrin B (19, 26) have been
isolated from different strains of S. aureus, and purified
staphyloferrin A can remove iron from transferrin (36).
The 42-kDa cell wall transferrin-binding protein (Tpn) from S. aureus possesses glyceraldehyde-3-phosphate dehydrogenase activity
and can sequester iron from transferrin in vitro (37). The
removal of iron from transferrin was shown to be a receptor-mediated process involving primary receptor recognition of the N lobe of human
transferrin (36). The importance of these iron acquisition processes in the virulence of S. aureus is not known.
Proteins that sense the levels of intracellular ions respond
accordingly by modulating gene expression. These metalloregulatory proteins cluster into four distinct families represented by Fur (ferric
uptake regulator), DtxR (diphtheria toxin repressor), MerR, and ArsR
(42). The well-characterized DtxR and Fur proteins have
similar roles with respect to iron homeostasis (49).
S. aureus, like Bacillus subtilis (11, 12,
23, 42), encodes three Fur homologues (Fur, PerR, and Zur) and a
DtxR homologue, called MntR. Zur functions as a zinc homeostasis
regulator in S. aureus but is not important for
pathogenicity (J. A. Lindsay and S. J. Foster, submitted for
publication). We have shown that PerR functions as a peroxide stress
regulator that controls iron storage proteins and that it is necessary
for the virulence of S. aureus (M. J. Horsburgh,
M. O. Clements, H. M. Crossley, E. Ingham, and S. J. Foster, submitted for publication). In addition, PerR was found to
directly regulate the expression of Fur and to regulate its own
expression. Purified S. aureus Fur protein binds in vitro to
the promoter elements of the fhuC and sirA genes that encode homologues of iron-siderophore uptake genes
(58). Expression of S. aureus Fur from a
multicopy plasmid was shown to partially restore iron-responsive
siderophore expression in a B. subtilis fur
mutant (58). The exact role of Fur in S. aureus and its contribution to pathogenicity in vivo have not been determined.
Here we present our data that show that S. aureus Fur
functions as the major regulator of iron supply and coordinately
regulates catalase-mediated oxidative stress resistance with peroxide
stress regulator PerR.
Media and growth conditions.
S. aureus and
Escherichia coli strains and plasmids are listed in Table
1. E. coli was grown in
Luria-Bertani medium at 37°C. S. aureus was grown at
37°C with shaking at 250 rpm in brain heart infusion (BHI) broth
(Oxoid), SSD medium (34), or chemically defined metal
limitation medium (CL). The components of CL are (concentrations in
milligrams per liter are in parentheses)
Na2HPO4 (7,000), KH2PO4
(300), adenine sulfate (20), guanine-HCl (20), L-glutamic
acid (2,220), L-aspartic acid (2,220),
L-proline (2,220), glycine (2,220), L-threonine
(2,220), L-serine (2,220), L-alanine (2,220),
L-lysine-HCl (560), L-isoleucine (560),
L-leucine (560), L-histidine (440),
L-valine (440), L-arginine (330),
L-cystine (220), L-phenylalanine (190),
L-tyrosine (170), L-methionine (170), L-tryptophan (60), pyridoxal (0.8), pyridoxamine-2HCl
(0.8), D-pantothenic acid (0.4), riboflavin (0.4),
nicotinic acid (0.4), thiamine-2HCl (0.4), and biotin (0.02). CL was
treated with 20 g of Chelex-100 (Sigma) liter
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.468-475.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Staphylococcus aureus, Fur Is an Interactive
Regulator with PerR, Contributes to Virulence, and Is Necessary for
Oxidative Stress Resistance through Positive Regulation of Catalase
and Iron Homeostasis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 with
stirring at room temperature for 4 h to deplete all divalent and
trivalent metal ions. After removal of Chelex by filter sterilization, MgSO4 was aseptically added to the medium at a final
concentration of 400 µM. CLR medium was prepared as described above
but with the following metals added at 0.2 µM: calcium chloride,
copper sulfate, ferrous sulfate, manganese sulfate, nickel sulfate,
molybdenum sulfate, and zinc sulfate. Colonies from non-Chelex-treated
CL agar plates were used to inoculate a CLR preculture. Experimental 25-ml cultures in acid-washed 250-ml flasks were inoculated to a
starting optical density at 600 nm (OD600) of 0.002 prior
to growth. When included, antibiotics were added at the following concentrations: ampicillin, 100 mg liter1; kanamycin, 50 mg liter1; neomycin, 50 mg liter1;
tetracycline, 5 mg liter1; erythromycin and lincomycin, 5 and 25 mg liter1, respectively.
TABLE 1.
Strains, plasmids, and primers used in this study
Construction of strains.
The recombinant strains used in
this study were constructed by PCR using Pwo polymerase
(Roche) and standard cloning techniques (45). Derivatives
of plasmid pAZ106, an integrating plasmid conferring resistance to
erythromycin and containing a promoterless lacZ gene
(59), and plasmid pAUL-A, a temperature-sensitive integrating plasmid conferring resistance to erythromycin, were constructed (13). A plasmid for disrupting fur
was constructed by PCR amplification of two adjoining 1-kb
fur partial fragments using primers OL29 with OL31 and OL30
with OL22 (Table 1), with incorporated XhoI,
KpnI, KpnI, and EcoRI restriction
sites, respectively, on the primers. A 1.5-kb tetracycline resistance
cassette from pDG1513 (25) was amplified using primers
OL32 and OL33, which contained KpnI restriction sites.
Simultaneous ligation of the appropriately digested fur
fragments and tetracycline cassette with
SalI-EcoRI-digested pAUL-A was performed, and,
following transformation of E. coli DH5
,
tetracycline-resistant colonies were selected. Three identical clones,
pMAL17, containing a tet cassette inserted into the
fur gene were obtained. Transcriptional reporter fusions to
the fhuD2, sirA, sstA, and
orf4 genes were made by PCR amplification of suitable DNA
fragments using the primers detailed in Table 1. Typically, between 0.5 and 1 kb of upstream DNA and 0.2 and 0.5 kb of the start of the gene
were amplified using Pwo DNA polymerase (Roche). The
purified DNA fragments were digested with BamHI and
EcoRI and cloned into plasmid pAZ106 digested with the same
enzymes. Transformation of S. aureus RN4220 was performed as
described by Schenk and Ladagga (46), and phage transduction into recipient 8325-4 was performed as described by Novick
(40) using 
11 as the transducing phage. MJH010
(fur) was isolated after transduction of an integrated
S. aureus RN4220 transformant of pMAL17 into S. aureus 8325-4, selecting for Tetr Erys
colonies. Southern blotting and PCR were used in each case to verify
the location and correct integration of DNA at the chromosomal loci.
-Galactosidase assays.
Levels of -galactosidase
activity were measured as described previously (14, 15)
with the following modifications. Samples (0.1 ml) were harvested, and
cell pellets were stored at 
20°C. Thawed pellets were resuspended
in 0.5 ml of ABT buffer (60 mM K2HPO4, 40 mM
KH2PO4, 100 mM NaCl). The assay was started
with the addition of 50 µl of freshly prepared
4-methylumbelliferyl--D-galactoside (10 mg
ml1), and the assay mixture was incubated at 25°C for
60 min. The assay was stopped with the addition of 0.5 ml of 0.4 M
Na2CO3. The stopped assay mixture was then
serially diluted in a 50:50 (vol/vol) mixture of ABT and
Na2CO3 in 96-well microtiter plates (Nunc).
Fluorescence was measured using a Victor plate reader (Wallac) with a
0.1-s count time and calibrated with standard concentrations of
4-methylumbelliferone (MU). One unit of -galactosidase activity was
defined as the amount of enzyme that catalyzed the production of 1 pmol
of MU min1 OD600 unit1. The
results presented here were representative of three independent experiments, which showed less than 20% variability.
Catalase assays, H2O2 resistance, and
starvation survival.
Catalase activity was assayed
spectrophotometrically at 240 nm as described by Beers and Sizer
(5), and protein was measured by the method of Bradford
(7) using bovine serum albumin (fraction V; Sigma) as the
standard. Hydrogen peroxide resistance assays were carried out as
described by Watson et al. (54) with the following
modifications. Cells, grown to exponential phase
(OD600 = 0.5) in CLR, were washed and diluted into
phosphate-buffered saline (PBS) to an OD600 of 0.2. Following challenge with 7.5 mM H2O2, the cells
were diluted in PBS containing catalase at 10 mg ml1 and
serially diluted in PBS and viability was assessed by overnight growth
on BHI agar. Comparative starvation survival experiments were performed
in glucose-limiting CDM medium with shaking at 250 rpm, at 37°C, as
described by Watson et al. (54).
Detection of siderophore activity. Siderophore activity in culture supernatants from cells grown in SSD medium was assayed by the liquid chrome azurol S (CAS) assay described by Schwyn and Neilands (47). Dilutions of supernatants were mixed with equal volumes of CAS shuttle solution. After 30 min of incubation at room temperature, the absorbance at 630 nm was determined using SSD medium as a blank and deferroxamine mesylate (Sigma) as a reference standard. Activity was measured as micromoles of deferroxamine equivalents per OD600 unit of the culture.
Virulence testing of strains in a murine skin abscess model.
S. aureus strains were grown to stationary phase in BHI
(time, 15 h), harvested by centrifugation, and washed twice in
PBS. The cell numbers were adjusted to 5 × 108 CFU
ml1, and then 200 µl of cell suspension was injected
subcutaneously into female 6- to 8-week-old BALB/c mice. After 7 days,
the mice were euthanized with CO2 and skin lesions were
aseptically removed and stored frozen in liquid nitrogen. The lesions
were weighed, chopped, and homogenized in a miniblender in 2.5 ml of
cold PBS. After 1 h of incubation on ice the lesions were
homogenized again before serial dilution of the suspension, and the
total number of bacteria were determined by growth on BHI agar. The
statistical significance of the percent recovery of strains was
evaluated by using Student's t test and the Mann-Whitney U
test, with a 5% confidence limit.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of an S. aureus fur mutant. A homologue of the B. subtilis fur gene was identified in the S. aureus 8325 genome database (http://www.genome.ou.edu). To determine the role of fur in S. aureus, we introduced a tetracycline resistance cassette into the fur gene using allelic replacement to disrupt the chromosomal copy, creating strain MJH010.
MJH010 (fur) has a growth defect.
The growth yield
of MJH010 (fur) was found to be much reduced compared to
that of 8325-4 (wild type) after growth to stationary phase in complex
media such as BHI broth (OD600 = 4.5 and 9.5, respectively) (data not shown). This phenotype was partially recovered in rich medium when a mutation in perR was introduced,
producing strain MJH201 (fur perR) (OD600 = 7.8). The growth rates of MJH010 (fur) and MJH201 (fur
perR) in chemically defined metal-depleted medium (CL) were
greatly reduced compared to that of 8325-4 (wild type) (Fig.
1A). This reduced growth rate was
improved in CLR, which contains 0.2 µM concentrations of a range of
divalent metal ions (data not shown), or CL containing micromolar
concentrations of manganese (Fig. 1B). In contrast, the addition of 20 µM iron sulfate did not improve the growth of MJH010 (fur)
and further impaired the growth of MJH201 (fur perR) (Fig.
1C).
|
), MJH010
(fur) (
), and MJH201 (fur perR) (
) in CL
medium (no added metals except magnesium) (A), CL with 20 µM
manganese chloride (B), and CL with 20 µM iron sulfate (C).
MJH010 (fur) has reduced oxidative stress
resistance.
Since the impaired growth phenotype of MJH010
(fur) could be rescued by adding manganous ion, a metal ion
with known antioxidant properties (3, 7, 22, 48), we
hypothesized that the mutant had an oxidative stress defect. To test
this further, MJH010 (fur), MJH201 (fur perR),
MJH001 (perR), and 8325-4 (wild type) were assayed for
resistance to 7.5 mM H2O2 and for levels of
catalase activity after growth in CLR medium. Both MJH010
(fur) and MJH201 (fur perR) were significantly
more sensitive to H2O2 than 8325-4 (wild type)
(Fig. 2A), and the catalase-specific
activity measured in MJH010 (fur) was found to be sixfold
lower than that in 8325-4 (wild type). After growth in CLR medium, with
either 20 µM manganese or 20 µM iron sulfate added, the levels of
catalase in MJH010 (fur) were 30- and 3-fold less than that
in 8325-4 (wild type), respectively (Fig. 2B). MJH001 (perR)
showed the expected increase in catalase activity under all
conditions. Catalase levels in MJH201 (fur perR) were found
to be lower than those in 8325-4 (wild type) and MJH001
(perR) in each of the growth conditions tested, revealing a
loss of response to the metal ions in the growth medium. These results
suggest that Fur acts as a positive regulator of catalase expression.
|
), and MJH001
(perR) (
). (B) Total catalase activities of washed, lysed
stationary-phase cells after growth in CLR medium (white bars), CL with
20 µM manganese chloride (grey bars), or CL medium with 20 µM iron
sulfate (black bars).
The effect of Fur on katA expression.
Expression
of katA in MJH206 (fur katA-lacZ) was reduced
compared to that in MJH006 (katA-lacZ) during growth in CLR
or CLR with 20 µM iron sulfate (Fig. 3A
and B). Growth in CLR with 20 µM manganese added, a concentration
that ensures PerR-mediated repression (M. J. Horsburgh, et
al., submitted), virtually eliminated all transcription of
katA in MJH206 (fur katA-lacZ) (Fig. 3B). Expression of katA in MJH306 (fur perR katA-lacZ)
was found to be uniformly low in each of the media tested (Fig. 3C).
These results support the function of Fur as a positive regulator of katA transcription.
|
The effect of Fur on control of the PerR regulon. Since Fur apparently regulates katA, which was shown to be a member of the PerR regulon (M. J. Horsburgh et al., submitted), we investigated the effect of Fur on the expression of other known PerR-regulated genes. The fur mutation was transduced into lacZ fusion strains of some of the known PerR-regulated genes. MJH002 (ahpC-lacZ), MJH202 (fur ahpC-lacZ), MJH003 (bcp-lacZ), MJH203 (fur bcp-lacZ), MJH007 (mrgA-lacZ), MJH207 (fur mrgA-lacZ), MJH008 (perR-lacZ), and MJH208 (fur perR-lacZ) were grown in CLR medium to determine whether there was any Fur-regulated expression. In each case expression was at a higher level in the fur background than in the wild-type background (data not shown), in contrast to expression of katA, which was reduced in MJH206 (fur katA-lacZ). The increased expression of these PerR-regulated genes was at levels similar to that observed when katA was inactivated (M. J. Horsburgh et al., submitted). A similar observation was made for B. subtilis, where inactivation of katA or ahpC increases expression of the PerR regulon, possibly due to an intracellular accumulation of peroxide (2, 10).
Fur regulation of siderophore production.
Since Fur is a
known regulator of iron homeostasis in many bacteria, we examined the
production of siderophores in MJH010 (fur). Siderophore
biosynthesis was found to be constitutive in MJH010 (fur),
whereas production was strongly repressed by iron and partially
repressed by manganese in 8325-4 (wild type) (Fig. 4). Levels of siderophore production in
MJH001 (perR) and 8325-4 (wild type) were the same in all
conditions tested (data not shown).
|
Fur regulates iron uptake proteins in S. aureus.
A
search of the incomplete S. aureus 8325-4 genome
(http://www.tigr.org and http://www.genome.ou.edu) revealed a large
number of genes with homology to iron-regulated proteins from other
bacteria. Many of these were preceded by sequences with homology to the putative Fur box previously identified for sirA
(28) and to part of the fhuC promoter
region protected by Fur (Fig. 5A)
(58). To investigate whether these genes were regulated by
Fur, lacZ fusions were constructed to monitor
transcription from the promoter regions of sirA,
fhuD2, sstA, and orf4, creating
strains MJH011 (fhuD2-lacZ), MJH211 (fur
fhuD2-lacZ), MJH012 (sirA-lacZ), MJH212 (fur sirA-lacZ), MJH013 (orf4-lacZ), MJH213
(fur orf4-lacZ), MJH014 (sstA-lacZ),
and MJH214 (fur sstA-lacZ).
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The importance of Fur in vivo.
The pathogenicities of
MJH010 (fur), MJH201 (fur perR), and 8325-4 (wild
type) in a murine skin abscess model of infection were tested (Fig.
7). The mean percentages of recovery for
the strains and Student's t test P values are as
follows: 8325-4 (wild-type), 143%; MJH010 (fur), 45.7%,
P < 0.04; MJH201 (fur perR), 38.9%, P < 0.03.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The S. aureus genome contains three fur homologues and one dtxR homologue, which encode Fur, PerR, and Zur and MntR, respectively. To date Zur has been shown to regulate zinc homeostasis (J. A. Lindsay and S. J. Foster, submitted), and PerR functions as a manganese-dependent repressor of a regulon of proteins required for oxidative stress resistance and iron storage (M. J. Horsburgh et al., submitted). The role of Fur in S. aureus as a regulator of iron-siderophore gene transcription was proposed by Xiong et al. (58) on the basis of purified Fur protein binding to Fur box sequences located in the promoters of the fhuC and sirA genes.
Our results demonstrate that S. aureus Fur functions as an iron-dependent transcriptional repressor of genes encoding iron uptake proteins. Fur mediates iron-dependent repression of the putative hydroxamate siderophore uptake gene, fhuD2, and the putative iron-siderophore transport operons sirABC and sstABCD. In addition orf4, which encodes a putative metal transporter, was also Fur regulated. As with many other bacteria, iron represses siderophore biosynthesis in S. aureus; significantly, this iron-dependent repression was abolished in MJH010 (fur). We propose that Fur is the primary regulator of iron uptake in S. aureus. A search of the incomplete S. aureus genomes with the two known S. aureus Fur box sequences identified many genes likely to be members of the Fur regulon (Fig. 5), and of those tested all were confirmed to be Fur regulated. Heinrichs et al. (28) demonstrated that the sirABC operon Fur box was sufficient for Fur-dependent regulation in E. coli.
In S. aureus, expression of the oxidative stress resistance enzymes, catalase, alkyl hydroperoxide reductase, thiol-dependent peroxidase (Bcp), and thioredoxin reductase (TrxB), is controlled through manganese-dependent PerR-mediated transcriptional repression (M. J. Horsburgh et al., submitted; 31). The S. aureus fur mutant was found to have low levels of catalase activity that were repressed by manganese, but not iron, in a PerR-dependent manner. In a perR mutant catalase levels are increased during growth in high iron in a Fur-dependent manner but are no longer repressed by manganese. This demonstrates that both Fur and PerR regulate the transcription of katA, with Fur acting, either directly or indirectly, as an iron-responsive activator of transcription. PerR acts as a manganese-dependent transcriptional repressor, and the PerR regulon is induced during growth in elevated levels of iron (M. J. Horsburgh et al., submitted). While the induction of katA in response to high iron levels was mediated by Fur, no such induction of other PerR genes was observed since in the fur mutant background the expression of other PerR-regulated genes was increased.
An explanation for this regulation in S. aureus is that elevated iron produces significant oxidative stress through formation of deleterious hydroxyl radicals via the Fenton reaction. An increased level of catalase through peroxide-induced, PerR-mediated derepression of katA (M. J. Horsburgh, et al., submitted) coupled with iron-Fur-mediated induction of katA will effectively reduce the Fenton reaction by lowering the intracellular level of hydrogen peroxide. In addition to this, the increased levels of iron produce derepression of PerR-regulated iron storage protein ferritin and ferritin-like Dps protein MrgA (M. J. Horsburgh et al., submitted), allowing this excess iron to be more safely stored and further limiting hydroxyl radical formation. Elevated concentrations of manganese repress katA transcription in a PerR-dependent manner. The antioxidant properties of manganese complexes have been demonstrated clearly (3, 7, 22, 48). Lactobacillus plantarum has been shown to accumulate high intracellular levels (30 µM) of manganese and does not require iron (3). Treponema pallidum has also been suggested to utilize manganese but not iron for growth (41). The sensitivity of MJH010 (fur) to hydrogen peroxide is likely to be exacerbated by the unregulated uptake of iron into the cell, which has been shown to confer sensitivity in an E. coli fur mutant (51).
The Fur protein of S. aureus, like that of E. coli, has been shown to bind a number of metals in vitro (4, 58). The role of Fur in vivo has been demonstrated to be predominantly iron homeostasis; however, some manganese regulation has been observed in E. coli for a limited number of Fur-dependent loci (4, 21). The Fur-dependent loci tested here were not found to display significant manganese regulation (data not shown), further suggesting that the major role of Fur in S. aureus is iron uptake. The consensus putative S. aureus PerR box element (M. J. Horsburgh et al., submitted) and the consensus putative S. aureus Fur box element bear a striking similarity in terms of modular composition (Fig. 5B) when interpreted as arrays of three repeats of 6 bp using the method of Escolar et al. (21).
The dual regulation of catalase synthesis by PerR and Fur has been shown in Campylobacter jejuni, where both of these proteins function as iron-responsive repressors of catalase expression (53). However, it is not clear whether in C. jejuni this repression of activity is due to the direct or indirect regulation of katA transcription by both PerR and Fur. Similarly, in S. aureus it is not yet clear if the positive regulation of katA transcription by Fur is direct or indirect.
Until recently, Fur was believed to function solely as a repressor of transcription. Indeed Escolar et al. (21) discuss the fact that there have been reports of Fur-positive regulation (4, 39), but as yet there is no confirmation of this activity at the DNA level. A recent report by Dubrac and Touati (20) has confirmed that in E. coli the sodB gene, encoding the iron-containing superoxide dismutase, is positively regulated, in part or in whole, posttranscriptionally by Fur in an iron-dependent manner; no obvious Fur box is located in the sodB promoter region. A deletion analysis demonstrated that an AT-rich region was required for positive regulation by Fur; however, no Fur-DNA binding analysis at this promoter was undertaken, and the authors do not exclude an indirect effect. We note that the S. aureus katA gene does not have an obvious Fur box similar to the E. coli sodB promoter region.
The importance of S. aureus Fur as a central regulator of iron homeostasis was confirmed by the reduced virulence of MJH010 (fur) in a murine skin abscess model of infection. The reduced virulence is unlikely to be merely a consequence of the reduced levels of catalase since a katA mutant is not attenuated, at least in this model of infection (M. J. Horsburgh et al., submitted). Instead, the reduced growth rate and the unregulated uptake of iron into the cell in MJH010 (fur) coupled with a diminished ability to prevent toxic hydroxyl radical formation by catalase-mediated dismutation of hydrogen peroxide may be more significant.
This study has begun to reveal the complex interplay between metal ion homeostasis and stress resistance in S. aureus. Both of these mechanisms are important for pathogenesis, and their regulation will be crucial as part of the host-pathogen interaction. It is the adaptive ability of S. aureus that enables it to be such a versatile and successful pathogen.
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ACKNOWLEDGMENTS |
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We thank the BBSRC (M.J.H.) and the Royal Society (S.J.F.) for funding this research.
We thank the S. aureus Genome Sequencing Project (8325) and B. A. Roe, Y. Qian, A. Dorman, F. Z. Najar, S. Clifton, and J. Iandolo. Preliminary sequence data of S. aureus (COL) were obtained from The Institute For Genomic Research website at http://www.tigr.org.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, England. Phone: 44 0114 222 4411. Fax: 44 0114 272 8697. E-mail: S.Foster{at}sheffield.ac.uk.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, January 2001, p. 468-475, Vol. 183, No. 2
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.2.468-475.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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