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Journal of Bacteriology, October 1999, p. 6371-6376, Vol. 181, No. 20
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Campylobacter jejuni Contains Two Fur Homologs:
Characterization of Iron-Responsive Regulation of Peroxide Stress
Defense Genes by the PerR Repressor
Arnoud H. M.
van
Vliet,1,
Marie-Louise A.
Baillon,2,
Charles W.
Penn,2 and
Julian M.
Ketley1,*
Department of Genetics, University of
Leicester, Leicester LE1 7RH,1 and
School of Biological Sciences, University of Birmingham,
Birmingham B15 2TT,2 United Kingdom
Received 16 February 1999/Accepted 13 August 1999
 |
ABSTRACT |
Expression of the peroxide stress genes alkyl hydroperoxide
reductase (ahpC) and catalase (katA) of the
microaerophile Campylobacter jejuni is repressed by iron.
Whereas iron repression in gram-negative bacteria is usually carried
out by the Fur protein, previous work showed that this is not the case
in C. jejuni, as these genes are still iron repressed in a
C. jejuni fur mutant. An open reading frame encoding a Fur
homolog (designated PerR for "peroxide stress regulator") was
identified in the genome sequence of C. jejuni. The
perR gene was disrupted by a kanamycin resistance cassette in C. jejuni wild-type and fur mutant strains.
Subsequent characterization of the C. jejuni perR mutants
showed derepressed expression of both AhpC and KatA at a much higher
level than that obtained by iron limitation, suggesting that expression
of these genes is controlled by other regulatory factors in addition to
the iron level. Other iron-regulated proteins were not affected by the perR mutation. The fur perR double mutant
showed derepressed expression of known iron-repressed genes. Further
phenotypic analysis of the perR mutant, fur
mutant, and the fur perR double mutant showed that the
perR mutation made C. jejuni hyperresistant to
peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of
KatA expression showed that both the perR mutation and the fur mutation had profound effects on catalase activity,
suggesting additional non-iron-dependent regulation of KatA and, by
inference, AhpC. The PerR protein is a functional but nonhomologous
substitution for the OxyR protein, which regulates peroxide stress
genes in other gram-negative bacteria. Regulation of peroxide stress
genes by a Fur homolog has recently been described for the
gram-positive bacterium Bacillus subtilis. C. jejuni is the
first gram-negative bacterium where non-OxyR regulation of peroxide
stress genes has been described and characterized.
| |
INTRODUCTION |
Campylobacter jejuni is a
gram-negative, microaerophilic enteric pathogen of humans, causing
gastroenteritis. The bacterium is one of the most frequently isolated
causes of bacterial diarrhea and can therefore be considered a major
public health and economic problem (16). As a
microaerophile, C. jejuni needs to protect itself from
reactive oxygen species that are the result of aerobic metabolism. A
coordinated response of oxidative-stress genes is a necessity of the
(micro)aerobic lifestyle.
Two of the most important bacterial oxidative-stress genes in the
defense against peroxide stress inducers such as hydrogen peroxide and
alkyl hydroperoxides are catalase (KatA) and alkyl hydroperoxide
reductase (AhpC). Both of these enzymes have been identified in
C. jejuni (3, 15). In most bacteria including Escherichia coli, Salmonella typhimurium
(10), Haemophilus influenzae (18), and
Mycobacterium leprae (12), KatA and/or AhpC
expression is regulated by the OxyR regulator in response to oxidative
stress. In C. jejuni, however, AhpC and KatA expression is
transcriptionally repressed in response to increasing environmental
iron concentration (3, 31). The Fur protein usually mediates
bacterial iron-responsive gene regulation. When the intracellular
Fe2+ concentration is high, a complex consisting of a Fur
dimer and Fe2+ binds to control sequences (Fur boxes)
overlapping Fur-regulated promoters (11, 25). The presence
of Fur box-like sequences in the promoters of both ahpC and
katA indicated that iron regulation of these genes could be
carried out by Fur. Unexpectedly, we found that in C. jejuni
iron regulation of AhpC and KatA is Fur independent (31),
and therefore we hypothesized that iron regulation of AhpC and KatA is
mediated by another regulator.
In the gram-positive bacterium Bacillus subtilis, expression
of AhpC and KatA is regulated by iron (6), and the presence of a repressor regulating AhpC and KatA has been predicted
(9). Analysis of the B. subtilis genome sequence
revealed the presence of three open reading frames (ORFs) encoding Fur
homologs. One homolog was shown to function as the iron uptake
regulator (Fur) (7), one as the zinc uptake regulator (Zur)
(14), and the third, designated PerR, was responsible for
the regulation of AhpC and KatA expression (7). In B. subtilis, therefore, PerR is a functional analog of the OxyR
regulator. It was predicted that in other gram-positive bacteria
peroxide stress genes and iron uptake might also be regulated by
separate Fur homologs, as multiple Fur homologs have been described for
several other gram-positive bacteria (7).
Here we report the identification of a C. jejuni PerR
homolog and describe the effects of a perR mutation. This is
the first gram-negative organism where PerR-like regulation has been
described. A C. jejuni perR fur double mutant is also
characterized where all iron regulation was abolished, and we show that
both the perR and fur mutations influence
expression of KatA.
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MATERIALS AND METHODS |
Media and growth conditions.
C. jejuni strains were
maintained on Mueller-Hinton (MH) media (Unipath) under microaerophilic
conditions in a Variable Atmosphere Incubator (Don Whitley) containing
85% N2, 10% CO2, and 5% O2. Media were routinely supplemented with 10 µg of vancomycin and 5 µg
of trimethoprim per ml. Iron-restricted conditions were achieved by
supplementing MH media with the iron chelator deferoxamine mesylate
(desferal; Sigma Chemical Co.) to a final concentration of 20 µM.
Iron-replete conditions were achieved by adding Fe(III)SO4 to MH media at a final concentration of 40 µM. E. coli was
grown aerobically in Luria-Bertani medium (23) at 37°C.
When antibiotic selection was necessary, growth media were supplemented
with ampicillin (100 µg/ml), kanamycin (50 µg/ml), or
chloramphenicol (20 µg/ml). C. jejuni strains were tested
for resistance to the peroxide stress inducers cumene hydroperoxide
(CHP) and hydrogen peroxide by methods described previously
(3).
Bacterial strains and plasmids.
C. jejuni and E. coli strains and plasmids used in this study are listed in Table
1. The region encoding perR
and upstream and downstream sequences was amplified with primers
5'-CGC-GGTACC-TAT- TGC-TTT-GCG-TTA-TCC-TAG-A
and
5'-CGC-GGATCC-ATT-GGA-ACT-ATC-CAA-AGT-TGG-AA. These primers contain a 5' KpnI or BamHI
restriction enzyme site (underlined) for cloning in pBluescript; thus,
the polylinker HindIII and EcoRV sites were
removed during cloning, allowing subsequent insertional mutagenesis of
perR.
Recombinant DNA techniques.
Restriction enzymes and T4 DNA
ligase were purchased from Gibco BRL. All enzymes were used according
to the manufacturer's instructions. Standard protocols were used for
manipulation of DNA and transformation of E. coli (2,
23) and C. jejuni (30). Genomic DNA of
C. jejuni was prepared by the method described by Ausubel et
al. (2). Plasmid DNA was prepared with affinity columns
(Qiagen). PCR was carried out with Expand Polymerase Mix (Boehringer).
DNA sequencing was performed with an Applied Biosystems model 377 DNA
sequencing system and a Taq Dye Deoxy Terminator Cycle
Sequencing Kit (Applied Biosystems).
Protein manipulation and catalase assays.
C. jejuni
were fractionated by a technique described previously (31).
Briefly, C. jejuni cells were subjected to osmotic shock to
release the periplasm. Subsequently, the spheroplasts were disrupted by
sonication, and cytoplasm and crude membranes were separated by
ultracentrifugation. Inner membranes were solubilized in Sarkosyl, and
outer membranes were pelleted by ultracentrifugation. Samples were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and subsequently stained with Coomassie brilliant blue.
N-terminal amino acid sequences were determined from proteins
transferred to Fluorotrans membranes (Flowgen Laboratories) by using
Edman degradation on an ABI 476 sequencer (Applied Biosystems).
Catalase activity was measured by a technique described by Beers and
Sizer (4). Briefly, C. jejuni cells were
disrupted by sonication, and insoluble particles were pelleted by
ultracentrifugation for 10 min at 100,000 × g. The
soluble fraction was subsequently used for quantitative analyses. The
protein concentration was measured as described by Bradford
(5). Catalase activity was measured by monitoring the
enzymatic breakdown of hydrogen peroxide at 240 nm, by using 50 mM
phosphate buffer with a hydrogen peroxide concentration of 19.6 mM.
| |
RESULTS |
C. jejuni contains a second Fur homolog.
To
identify putative regulators of oxidative-stress genes, we screened the
C. jejuni genome sequence (24) for OxyR and Fur homologs. This analysis did not reveal the presence of an OxyR homolog
but did reveal the presence of an ORF encoding a second Fur homolog
approximately 70 kb from fur. The region containing this
second Fur homolog was amplified from NCTC 11168 by PCR, and the
nucleotide sequence was confirmed to be identical to the sequence
determined by the C. jejuni genome project. The 1.4-kb region amplified contained three ORFs (Fig.
1). The product of the one complete ORF,
designated PerR, showed significant homology with bacterial Fur
homologs. An alignment of the C. jejuni PerR protein with
the B. subtilis PerR and the C. jejuni and
B. subtilis Fur proteins is shown in Fig.
2. The identity between all Fur and PerR
sequences was 17%. However, the identity between the two Fur proteins
was 34%, and that between the two PerR proteins was 32%. The ORF
upstream of perR encodes a putative transketolase B
(tktB) homolog, and the downstream ORF (orf3) did
not have any significant homology with sequences deposited in the
GenBank and EMBL databases (Fig. 1). The predicted perR gene
is 408 bp long, encoding a protein of 15,926 Da with seven cysteine
residues and eight histidine residues (Fig. 2). A putative ribosome
binding site (AAGGA) was identified 10 bp upstream of the
perR gene. There is 86 bp between tktB and
perR, and this intergenic region has a very high A+T content
of 90.6% and includes a putative stem-loop structure overlapping with
the stop codon of tktB. This putative stem-loop structure
has a stem of 11 bp and a loop of 5 bp and has a free energy of 
8.6
kcal. Downstream of perR a small putative stem-loop
structure is found with a stem of 5 bp, a loop of 3 bp, and a free
energy of 2.2 kcal. The predicted transcriptional organization of the
perR genomic region and the putative stem-loop structures
present suggest that perR is transcribed as a monocistronic messenger, unlike C. jejuni fur (8, 29) and thus
probably has its own promoter. The PCR primers used to amplify the
perR region from C. jejuni NCTC 11168 gave
similar-sized products with four other C. jejuni strains,
indicating that the organization of the perR region is
conserved in other C. jejuni strains (data not shown).
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FIG. 1.
Schematic diagram representing the genomic region
containing the perR gene. The position and orientation of
the inserted antibiotic resistance cassette in perR are
shown. Only partial ORFs of tktB and orf3 are
indicated. Ev, EcoRV; H3, HindIII.
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FIG. 2.
Alignment of C. jejuni (Cj) and B. subtilis (Bs) PerR and Fur proteins. Boxed residues are identical
in the PerR and Fur proteins. Asterisks and dots indicate identical
residues and conservative substitutions, respectively, in all four
proteins.
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PerR is the repressor of C. jejuni AhpC and KatA
peroxide stress genes.
The perR gene was mutated to
determine whether it was the repressor of the ahpC and
katA genes in C. jejuni. By using the unique
internal EcoRV site (Fig. 1), a kanamycin resistance
cassette was inserted into the perR gene, and the disrupted
perR gene was subsequently introduced into the C. jejuni NCTC 11168 genome by allelic exchange (17).
Mutants were obtained with the antibiotic resistance cassette inserted
in either orientation at the same frequency. This is in contrast with
the C. jejuni fur gene (31). Correct allelic
exchange of the wild-type perR allele with the mutated copy
was confirmed by PCR (data not shown). The C. jejuni perR
mutant was designated AV63. In addition to the perR mutant, we also constructed a perR fur double mutant by mutating
perR in C. jejuni AV42, which is a fur
mutant with a chloramphenicol resistance cassette inserted in the same
orientation as fur (31). The presence of mutated
fur and perR alleles was confirmed by PCR (data
not shown). The perR fur double mutant, named AV67, was
viable and showed the same growth characteristics as a fur mutant (31). The perR mutant showed the same
growth characteristics as the wild-type strain (data not shown).
Protein profiles and subsequent protein sequencing were used to
determine whether PerR was indeed the iron-responsive repressor
of
ahpC and
katA expression. Bacteria were grown
under iron-restricted
and iron-replete conditions and fractionated into
periplasm, cytoplasm,
and outer membranes. This approach has been used
previously to
identify members of the
fur regulon of
C. jejuni (
31). These
protein profiles following
separation by SDS-PAGE are shown in
Fig.
3; proteins previously identified as Fur
repressed are indicated,
and these are all still iron repressed in the
perR mutant but
not in the
fur mutant or the
double mutant. This shows that the
perR mutation does not
notably affect Fur-regulated protein expression.
Three highly expressed
proteins with molecular sizes of approximately
25, 26, and 55 kDa can
be seen in the periplasmic and cytoplasmic
fractions of the
perR mutant and
perR fur mutant. Expression of
these three proteins is iron repressed in the wild type and
fur mutant (Fig.
3). All three proteins were identified by
N-terminal
amino acid sequencing. The N-terminal amino acid sequence of
the
55-kDa protein (MKKLTNDFG) was identical to that of
C. jejuni KatA (
15). Surprisingly, the N-terminal
amino acid sequence
of both the 25- and the 26-kDa proteins
(MIVTKKALDF) was identical
to that of AhpC (
3).
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FIG. 3.
Protein profiles of fractionated C. jejuni
wild type and mutants grown in low-iron and high-iron media, following
separation by SDS-PAGE. Iron-repressed proteins are indicated on the
left, with PerR-regulated proteins boxed. (A) Cytoplasmic fraction; (B)
periplasmic fraction; (C) outer membrane fraction.
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The perR mutation makes C. jejuni
hyperresistant to peroxide stress.
The perR mutation
derepressed the katA and ahpC promoters, leading
to a very high level of expression of KatA and AhpC. In order to test
whether this led to expression of functional enzymes, the level of
resistance of wild-type C. jejuni to peroxide stress was
compared to those of the perR, fur, and
perR fur mutants. The results obtained with two levels of
the peroxide stress inducers hydrogen peroxide (cleared by KatA) and
CHP (cleared by AhpC) are shown in Fig.
4. The perR mutation induces
hyperresistance to both of these peroxide stress inducers, showing that
in a perR background both overexpressed enzymes are
functional. The fur mutation alone did not have a major
effect on resistance of C. jejuni to the peroxide stress
inducers.
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FIG. 4.
Effects of perR and fur mutations
on oxidative-stress resistance of C. jejuni wild type and
mutants to CHP (A) and hydrogen peroxide (B). Resistance is expressed
as the zone of growth inhibition after overnight growth on agar plates.
Resistance was measured against 3% (open bars) and 10% (closed bars)
CHP and H2O2. The error bars represent data
from three separate plates. The perR mutation derepresses
expression of AhpC and KatA and results in the perR mutants
showing hyperresistance to CHP and H2O2. The
fur mutation on its own slightly decreases resistance to
both CHP and H2O2, whereas the fur
perR double mutant is more resistant to CHP and
H2O2. The perR mutants did not show
an inhibition zone with 3% H2O2 (asterisk).
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The derepression of KatA expression by the
perR mutation was
determined by direct enzyme assay. KatA synthesis was measured
directly
by spectophotometric detection of the rate at which hydrogen
peroxide
was utilized. The results of this assay are shown in
Table
2. In the wild-type strain, KatA activity
was almost completely
absent under iron-replete conditions. The
perR mutation substantially
increased KatA activity under
iron-replete conditions but particularly
under low-iron conditions,
where KatA activity was 10-fold higher
than that in the wild type.
Interestingly, KatA activity was still
2-fold iron repressed in the
perR mutant, and in the
fur mutant
KatA activity
was 4-fold lower than that in the wild type under
low-iron conditions
but higher than that in the wild type under
high-iron conditions. Such
an effect of the
fur mutation on iron-repressed
expression
of
katA suggests that Fur coregulates
katA
expression
with PerR. This hypothesis is supported by the observation
that
in the
perR fur double mutant KatA levels were almost
as high
as those in the
perR mutant, but regulation in
response to iron
levels is no longer evident (Table
2).
| |
DISCUSSION |
Reactive oxygen species cause damage to DNA, proteins, and
membranes. Bacteria have developed systems which include catalase, alkyl hydroperoxide reductase, and superoxide dismutase to clear these
reactive oxygen species. In the Enterobacteriaceae, most oxidative-stress proteins have been identified as being induced by
different kinds of oxidative stress and can be subdivided in two
separately regulated classes; (i) the O2
(superoxide) stress proteins and (ii) the peroxide stress proteins. The
O2 stress proteins include
manganese-containing superoxide dismutase (SodA) and endonuclease IV
(13). Catalase and alkyl hydroperoxide reductase are members
of the peroxide regulon (13).
Oxidative-stress defense proteins are expressed at a basal level under
normal nonstressed conditions, but all these systems need to be able to
be upregulated in conditions of increased oxidative stress. The
regulation of these systems has been extensively investigated in
E. coli, where there are two regulatory systems for
oxidative-stress genes. The SoxR-SoxS system regulates the superoxide
regulon (1), and mutations in soxR or
soxS fail to induce the members of their regulon
(13). OxyR regulates the OxyR regulon, which is part of the
peroxide stress regulon. The OxyR protein is a transcription factor
that senses oxidative stress through disulfide bond formation, and,
under conditions of oxidative stress, transcription of its regulon is
induced following a change in OxyR conformation due to this disulfide
bond formation (26, 27, 33). OxyR is considered a global
regulator, and H. influenzae oxyR mutants are unable to
respond to oxidative stress (18).
Homologs of the oxyR gene have been identified in many
gram-negative bacteria, but no oxyR homologs have been
identified in the genome sequence of Helicobacter pylori
(28), and we did not find a homolog in the C. jejuni genome (24). This indicates that in C. jejuni as well as H. pylori regulation of
oxidative-stress defense is organized differently. Three
oxidative-stress defense genes
superoxide dismutase (sodB)
(21, 22), catalase (katA) (15), and
alkyl hydroperoxide reductase (ahpC) (3)have
been identified in C. jejuni and the closely related species
C. coli. The transcription of the katA and
ahpC genes in C. jejuni is responsive to the iron
concentration in the growth medium (3). Iron-responsive regulation is usually mediated by the Fur protein, and putative Fur
binding sequences were identified upstream of sodB,
katA, and ahpC. However, it was demonstrated that
in a C. jejuni fur mutant expression of katA and
ahpC was still iron responsive, unlike the expression of
iron-uptake systems (31). These findings led us to search
the C. jejuni genome sequence (24) for the presence of an ORF encoding a second Fur homolog. We found this second
fur homolog (named perR) and have now
demonstrated using insertional mutagenesis that the product of
perR is responsible for regulating expression of the
katA and ahpC genes. Expression of both KatA and
AhpC was very high in the perR mutants, such that these
proteins were now also detected in the periplasmic fraction. The
increase in KatA and AhpC expression is higher than that observed under
conditions of iron limitation, indicating that even under iron
limitation transcription from the promoters of katA and
ahpC is still not maximal. Thus, there are likely to be
other environmental stimuli regulating expression of these oxidative-stress defense genes. This agrees with the observation that
in C. coli KatA expression is induced under conditions of oxidative stress (19). As yet it is unknown whether other
stimuli are important in oxidative-stress responses in
Campylobacter.
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FIG. 5.
Model illustrating Fur homolog-mediated negative
regulation of oxidative-stress genes and iron transport systems in
Campylobacter. The dotted arrow indicates a possible
influence of Fur on oxidative-stress gene regulation involving either a
direct interaction with oxidative-stress gene promoters or an indirect
effect via transport systems on intracellular iron concentrations.
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The protein profiles showed only two proteins whose expression was
affected by the perR mutation, and these proteins were identified as KatA and AhpC. In addition, unlike the fur
mutant (31), the C. jejuni perR mutant showed
growth characteristics similar to those of its parental strain,
indicating that in C. jejuni PerR may not be a global
regulator. In contrast, Fur regulates at least 13 proteins in C. jejuni (Fig. 3) (31) and also affects the expression of
KatA and possibly AhpC (Fig. 3 and Table 2). The apparently small PerR
regulon might explain why the perR fur double mutant was
still viable. We also tested the influence of the perR and
fur mutations on SodB activity. Although SOD activity was
strongly induced under high-iron conditions, neither the
perR or fur mutation influenced SOD enzyme
activity (data not shown). However, given that SodB is an
iron-containing enzyme, under low-iron conditions sodB
may be expressed but is not functional due to the iron limitation.
The perR system was first described for B. subtilis, where expression of KatA and AhpC is regulated by metal
ions and oxidative stress (9). The PerR regulator was
recently shown to be one of three Fur homologs (7), one
other being the iron uptake regulator (designated Fur), and the third
being the zinc uptake regulator (Zur) (14). Multiple Fur
homologs have been described for several other gram-positive organisms
and also for P. aeruginosa (32) and E. coli (20). The second E. coli Fur homolog
regulates zinc uptake and has been designated Zur (20). The
function of the second Fur homolog of P. aeruginosa is still
unknown (32). We have now demonstrated the function of a
second Fur homolog in C. jejuni, where it can be regarded as
a functional analog for OxyR (Fig. 5). It is noteworthy that there is
no second Fur or OxyR homolog in H. pylori, and thus, it
remains unclear whether and how H. pylori regulates
katA and ahpC expression.
Regulation of gene expression in C. jejuni is relatively
poorly understood. This report is the first description of peroxide stress regulation by a second Fur homolog in a gram-negative bacterium. We also describe the first C. jejuni double regulatory
mutant and show that both PerR and Fur have an effect on catalase
expression, demonstrating regulatory cross-talk in C. jejuni. Future work will focus on the mechanisms of PerR
regulation of peroxide stress resistance and further characterization
of the perR and perR fur mutants using
two-dimensional gel electrophoresis.
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ACKNOWLEDGMENTS |
This study was supported by the Wellcome Trust and a Royal
Society University Research Fellowship to J. M. Ketley. M.-L.A. Baillon was supported by a studentship from the Ministry of
Agriculture, Fisheries and Food.
We thank Kathryn Lilley for N-terminal amino acid sequencing and Gina
Manning, Andrey Karlyshev, and Diane Newell for helpful discussions.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Genetics, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom. Phone: 44-116-2523434. Fax: 44-116-2523378. E-mail: ket{at}le.ac.uk.
Present address: Departments of Medical Microbiology & Gastroenterology, Faculty of Medicine, Vrije Universiteit Amsterdam, 1081 BT Amsterdam, The Netherlands.
Present address: Waltham Centre for Pet Nutrition,
Waltham-on-the-Wolds, Melton Mowbray LE14 4RT, United Kingdom.
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Abstract
Introduction
