From the Department of Medical Biochemistry and Molecular Biology, University of Turku, FIN-20520 Turku, Finland
Received for publication, October 4, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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H2O2 is an
unavoidable cytotoxic by-product of aerobic life. Dpr, a recently
discovered member of the Dps protein family, provides a means for
catalase-negative bacteria to tolerate H2O2. Potentially, Dpr could bind free intracellular iron and thus inhibit the Fenton chemistry-catalyzed formation of toxic hydroxyl radicals (H2O2 + Fe2+ Aerobic metabolism is essential for many living organisms for the
production of energy. However, partially reduced forms of oxygen
superoxide anion radical (O Dpr (Dps-like peroxide resistance
protein) is a recently discovered aerotolerance and
H2O2 resistance factor of Streptococcus mutans (11). Although Dpr is widely conserved in other
Gram-positive bacteria, including the important human pathogens
Streptococcus pyogenes and Streptococcus
pneumoniae (11), relatively little is known of the molecular
mechanisms of its action. The primary amino acid sequence shares
similarity with Escherichia coli Dps (DNA-binding
protein from starved cells) (12, 13), a prototype for a large group of similar oligomeric proteins (14), which is
abundantly expressed in starved cells and involved in DNA protection by
DNA-Dps biocrystal formation (15, 16). Studies on Dps family members
also indicate that the protective function of Dpr against H2O2 might be mediated by
H2O2 degradation due to a catalase-like activity (14) or by chelation of free intracellular iron (17).
It is known that toxicity of H2O2
is relatively weak (3), although it easily diffuses across biological
membranes (18, 19) and oxidize thiols (3). However, if reduced
transition metal ions, especially iron, are present,
H2O2 is nonenzymatically cleaved into highly
toxic hydroxyl radicals by Fenton chemistry (H2O2 + Fe2+ Streptococcus suis is an important pig pathogen that causes
severe infections such as sepsis and meningitis, and it
occasionally causes life-threatening disease also in humans
(30). We have previously identified in S. suis a
galactose-specific adhesion activity (31-33). One of the proteins
identified as a candidate adhesin displaying binding activity to
glycoproteins had a 64% primary amino acid sequence identity with
S. mutans Dpr.1
S. suis seemed an ideal model organism to study the in
vivo function of Dpr in H2O2 resistance
because it not only lacks catalase but also lacks NADH peroxidase (34).
The results of the present study demonstrate that the putative
ferroxidase center of Dpr is involved in iron incorporation and that
the H2O2 resistance mediated by Dpr depends on
its iron incorporation activity in vivo.
Bacterial Strains, Plasmids, and Primers
The bacterial strains, plasmids, and primers used
in this study are listed in Tables I and
II. S. suis was cultured
aerobically or under 6% CO2 at 37 °C with or without
agitation in Todd Hewitt Broth medium (DIFCO) supplemented with
0.5% (w/v) yeast extract (Biokar Diagnostics)
(THY).2 E. coli
was grown aerobically at 37 °C with agitation in Luria-Bertani medium. When needed, media were solidified by 1.5% agar. All bacteria were stored at ·OH + -OH + Fe3+). We explored the in
vivo function of Dpr in the catalase- and NADH
peroxidase-negative pig and human pathogen Streptococcus suis. We show that: (i) a Dpr allelic exchange knockout mutant was hypersensitive (~106-fold) to
H2O2, (ii) Dpr incorporated iron in
vivo, (iii) a putative ferroxidase center was present in Dpr,
(iv) single amino acid substitutions D74A or E78A to the putative
ferroxidase center abolished the in vivo iron
incorporation, and (v) the H2O2 hypersensitive phenotype was complemented by wild-type Dpr or by a membrane-permeating iron chelator, but not by the site-mutated forms of Dpr. These results demonstrate that the putative ferroxidase center of Dpr is functionally active in iron incorporation and that the
H2O2 resistance is mediated by Dpr in
vivo by its iron binding activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
·OH + -OH + Fe3+) (20, 21). Dps family members share
a conserved amino acid motif, similar to mammalian ferritin
L-subunit iron nucleation center, in the N-terminal halves
of the proteins (14, 22-24). Because several members, including the
streptococcal Dpr (11, 25), are known to bind iron (17, 22, 26, 27),
the conserved motif is believed to serve a functional role. It has been
suggested that the motif catalyzes iron oxidation by its putative
ferroxidase activity and also directs the formation of an iron core
into the inner cavity of the oligomer (17, 22, 28, 29). However, there
is no direct experimental evidence for any of the Dps family members to
support the functionality of the putative ferroxidase center in iron
incorporation in vivo. Furthermore, the biological significance of iron incorporation is not well established.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C in growth media containing 15% (v/v)
glycerol. Antibiotics (Sigma) were used at the following concentrations unless otherwise indicated: (i) E. coli, 100 µg/ml
ampicillin, 30 µg/ml kanamycin, 100 µg/ml spectinomycin, and 30 µg/ml chloramphenicol; and (ii) S. suis, 20 µg/ml
ampicillin, 500 µg/ml kanamycin, 1000 µg/ml spectinomycin, and 10 µg/ml chloramphenicol.
Strains and plasmids
Oligonucleotide primers
DNA Techniques
Genomic DNA of S. suis was isolated as
described previously (38). Standard protocols were used for PCR, DNA
modification, cloning, E. coli transformation, and Southern
and colony hybridization as described by Sambrook and Russel (39).
DNA-modifying enzymes were purchased from Promega and Fermentas, and
Vent DNA polymerase was purchased from New England Biolabs. DNA
molecular mass markers were from Promega. Plasmids amplified in
E. coli DH5 were isolated using QIAprep Spin Miniprep Kit
(Qiagen) as described by the manufacturer. DNA fragments were purified
from agarose gels using QIAquick Gel Extraction Kit or from PCR and
other enzymatic reactions using QIAquick PCR Purification Kit as
described by the manufacturer (Qiagen). DNA sequences were determined
by ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit
(PerkinElmer Life Sciences) with AmpliTaq DNA Polymerase FS (Roche
Molecular Biochemicals). Sequencing primers were purchased from
Interactiva Biotechnologie GmbH. For Southern, Northern, and colony
hybridization, radioactive DNA probes were labeled with
[
-32P]CTP (Amersham Biosciences) using Prime-a-Gene
Labeling System (Promega) according to the instructions of the
manufacturer. Templates for labeling were generated by PCR using
primers DPR-5'-IN and DPR-3'-IN specific for dpr and 16S-5'
and 16S-3' specific for 16S rRNA genes and gel-isolated before
labeling. The hybridized membranes were analyzed with Fujifilm
BA-2500 Phosphor Imaging Plate System (Fuji Photo Film Co.) according
to the instructions of the manufacturer.
Cloning and Sequence Analysis of the dpr Locus
Southern hybridization was used to identify a 6.5-kb
ClaI-EcoRI genomic fragment that seemed to
contain the entire dpr from S. suis serotype 2 strain 628 (data not shown). To clone the 6.5-kb fragment, we ligated
gel-isolated, ClaI- and EcoRI-digested DNA fragments ranging from 6 to 7 kb into
EcoRI-NarI-digested pBR322, electrotransformed
the ligation mixture into E. coli DH5 cells, and screened
the transformants by colony hybridization. One hybridizing clone, designated pDPR6500, was isolated and sequenced. Sequencing data
were assembled, and the consensus sequence was edited using the MAC
DNAsis software (Hitachi). The web-based program ORF Finder (www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to predict the coding
regions. The BLAST software package at the National Center for
Biotechnology Information (www.ncbi.nlm.nih.gov) was used to search for
protein sequences homologous to the deduced amino acid sequences.
Promoter sequence features were searched using WWW Signal Scan
(bimas.dcrt.nih.gov/molbio/signal).
Electrotransformation of S. suis
Overnight S. suis cultures were diluted 100-fold into
50 ml of fresh THY supplemented with 30 mM glycin. Cultures
were incubated at 37 °C with slight agitation to
A600 of ~0.2. Cells were harvested by
centrifugation (2000 × g, 20 min, 4 °C) and washed
twice with 10 ml of ice-cold 0.5 M sucrose (2000 × g, 20 min, 4 °C) and once with 10 ml of ice-cold 0.5 M sucrose supplemented with 15% (v/v) glycerol (2000 × g, 20 min, 4 °C). The cells were resuspended in 50 µl of ice-cold 0.5 M sucrose supplemented with 15%
glycerol (v/v) and were either used directly or stored at 70 °C.
Electrotransformations were done using the Gene Pulser II
Electroporation System (Bio-Rad). 1 µg of suicide vector or 100 ng of
shuttle vector was mixed with 50 µl of the electrocompetent cells on
ice. The mixtures were transferred into prechilled sterile Gene Pulser
cuvettes (interelectrode distance, 0.1 cm; Bio-Rad) and pulsed with a
setting of 15 microfarads, 1.8 kV, and 200 ohms. After the electric
pulse, the cells were diluted in 1 ml of THY supplemented with 0.3 M sucrose and incubated for 2 h at 37 °C under 6%
CO2. The cells were then plated on THY agar containing the
appropriate antibiotics. The cells routinely yielded ~106
transformants/µg shuttle vector.
Isolation of Total RNA and Northern Hybridization
For extraction of total RNA, overnight S. suis
cultures were diluted 100-fold into fresh THY and grown at 37 °C
with vigorous shaking. At various levels of turbidity, 1-ml aliquots
were taken from the cultures. The cells were harvested by
centrifugation (15,000 × g, 2 min, 4 °C),
immediately snap-frozen in liquid N2, and stored at
70 °C. The cells were later thawed, and total RNA was extracted
using the RNeasy Mini kit (Qiagen) according to instructions of the
manufacturer. 20 µg of RNA was electrophoresed on a 1% agarose gel
containing 2.2 M formaldehyde. The RNA Ladder (New England
Biolabs) served as a molecular mass marker. After electrophoresis, the
RNA was transferred to a Hybond-N+ membrane (Amersham Biosciences).
Hybridization and washing conditions were performed as described by
Sambrook and Russel (39).
Preparation of Cellular Protein Extracts of S. suis
Overnight S. suis cultures were diluted 100-fold into fresh THY and grown at 37 °C with vigorous shaking. At various levels of turbidity, cells were harvested by centrifugation (2000 × g, 20 min, 4 °C), washed with Pi/NaCl (10 mM sodium phosphate buffer and 0.15 M NaCl (pH 7.4)) (2000 × g, 20 min, 4 °C), and resuspended in 750 µl of Pi/NaCl. The cells were disrupted by sonication five times (20 s each time), with a chilling interval of 1 min between the sonications. EDTA-free Protease Inhibitor Mixture Tablets (Roche Molecular Biochemicals) were added to the sonicates according to instructions of the manufacturer. After unbroken cells and cell debris were removed by centrifugation (15,000 × g, 30 min, 4 °C), the cleared lysate was collected and stored at 4 °C. Protein concentrations were determined at least in triplicate using the Bio-Rad Protein Assay, based on the Bradford dye binding procedure (40), using bovine serum albumin (BSA) (Sigma) as a standard.
Preparation of Polyclonal Antibodies Specific for Dpr
Expression and purification of recombinant Dpr have been described previously (41). Two rabbits were immunized subcutaneously with 1.0 ml of a mixture of the recombinant Dpr (100 µg/ml) and Freund's complete adjuvant (1:1, v/v). Booster injections with the same protein mixture in Freund's incomplete adjuvant were given at 28 and 56 days, and sera were collected 14 days after each immunization. The sera collected 14 days after the last booster had the highest activity against Dpr and were used for subsequent work.
Western Analysis of Dpr Expression
For Western blotting, the proteins were resolved under denaturing conditions in a 12% polyacrylamide gel or under nondenaturing conditions in a 4-15% gradient Tris-HCl Ready Gel (Bio-Rad) using SDS-PAGE Low Range (Bio-Rad) or equine spleen type I ferritin (Sigma) and BSA as the molecular mass markers, respectively. Proteins were subsequently transferred to a Protran Nitrocellulose Transfer Membrane (Schleicher & Schell) using the 2117 Multiphor II Electrophoresis Unit (LKB Bromma). The membranes were saturated with 3% (w/v) BSA and 0.1% (v/v) Tween 20 in Pi/NaCl at room temperature for 1 h. The anti-Dpr polyclonal antibodies were diluted 1:10,000 in 1% (w/v) BSA and 0.05% (v/v) Tween 20 in Pi/NaCl, and the membranes were incubated at room temperature for 30 min. After washing with Pi/NaCl, the membranes were incubated at room temperature for 30 min in 1% BSA (w/v) and 0.05% (v/v) Tween 20 in Pi/NaCl containing a 1:10,000 dilution of peroxidase-conjugated goat anti-rabbit immunoglobulins (DAKO). After washing with Pi/NaCl, the ECL chemiluminescence detection kit (Amersham Biosciences) was used to detect the binding according to instructions of the manufacturer.
Inactivation of dpr in S. suis
We generated a genetically stable deletion mutant
(D282dpr) of the dpr by adopting the double
cross-over method.
Construction of the Suicide Vector pDPR2-- A 2.8-kb fragment around the dpr was amplified by PCR using primers TOT-5' and TOT-3'. The resulting PCR product was digested with BclI and KpnI, and the resulting 2.6-kb fragment was cloned into the KpnI-BamHI site of pID700 to generate pDPR1. The dpr was deleted from the pDPR1 by PCR with primers DEL-5' and DEL-3' and replaced by a spectinomycin resistance gene (spc) devoid of terminator generated by PCR with primers SPCS and SPCA using pKUN19-Spc as the template. The resulting plasmid, pDPR2, was analyzed by restriction enzyme digestions and PCR to contain the spc in the same direction of transcription as the dpr.
Generation of the Mutant-- Because strain 628 has been poorly transformable in our hands, we used D282 cells of same serotype and with identical dpr sequence as strain 628, and colonies with the single cross-over genotype (ChlR, SpcR) were selected. One ChlR and SpcR colony was chosen, and the double cross-over genotype (SpcR) was selected as follows. Single cross-over mutant was first grown overnight with spectinomycin (1000 µg/ml), allowing the vector to excise out of the genome, leaving the spc in the place of the dpr. Resulting double cross-over mutants were enriched by using the bacteriostatic activity of chloramphenicol (10 µg/ml) and at the same time killing the dividing bacteria (still having the vector insertion) by using ampicillin (20 µg/ml). The enriched bacteria were plated on THY with spectinomycin, and the double cross-over genotype (SpcR) was verified by replica plating. The replacement of dpr by spc was further verified by PCR with primers DPR-5'-OUT and DPR-3'-OUT annealing upstream and downstream of dpr, respectively, and by Western analysis.
Ectopic Expression of Dpr in D282dpr
The dpr mutation was complemented by introducing
wild-type dpr into D282dpr in an E. coli/Streptococcus sp. shuttle vector pLZ12-Km/Spc.
pLZ12-Km/Spc was generated by ligating the spc generated by
PCR with primers SPCS and SPCB using pKUN19-Spc as the template to
NcoI/EcoRI-digested pLZ12-Km. Two
dpr-containing DNA fragments, L4A and P3, were amplified by
PCR using primers 5'-DCOMP and 3'-COMP or 5'-UPCOMP and 3'-COMP,
respectively. These DNA fragments were digested with KpnI
and ligated into KpnI-digested and dephosphorylated pLZ12-Km/Spc to generate the plasmid constructs pLZ12-L4A and pLZ12-P3,
respectively. In these constructs, dpr transcription is
under the control of spc promoter or the possible promoter activity of dpr upstream sequence. The constructs were
sequenced to ensure that no mistakes were introduced into
dpr during the amplification and introduced into
D282
dpr, and complementation genotype (SpcR,
KanR) was selected on agar plates. The expression of Dpr
was verified by Western analysis.
Site-directed PCR Mutagenesis
Codons encoding Asp-74 (GAT) and Glu-78 (GAG) were independently
mutated into GCA (Ala) in the pLZ12-L4A, resulting in plasmids pLZ12-L4A-74 and pLZ12-L4A-78, respectively. Briefly, pLZ12-L4A was
linearized by PCR using the mutagenic oligonucleotides (DPR-AspA and
DPR-NAT-3'A for D74A and DPR-GluB and DPR-NAT-3'B for E78A) as follows:
40 s at 94 °C, 30 s at 65 °C, and 6 min at 72 °C for 15 cycles. The products from tubes containing varying amounts of
Mg2+ were pooled, purified, and digested with
DpnI to reduce the number of parental molecules. The
linearized PCR products were treated with T4 polynucleotide kinase,
gel-isolated, religated, and transformed to E. coli DH5.
All mutations were confirmed by DNA sequencing. The resulting
constructs were introduced into D282
dpr, and
complementation genotype (SpcR, KanR) was
selected on agar plates. Expression of Dpr was verified by Western analysis.
Radiometric Determination of Iron Content of Dpr
Overnight S. suis cultures were diluted 100-fold into 25 ml of fresh THY containing 55FeCl3 (663.3 MBq/mg; PerkinElmer Life Sciences) as 0.35 MBq/ml and grown to early-stationary phase at 37 °C with vigorous shaking. The bacteria were harvested by centrifugation (2000 × g, 20 min, 4 °C), washed once with Pi/NaCl (2000 × g, 20 min, 4 °C), and resuspended in 750 µl of Pi/NaCl. Cellular protein extracts were prepared as described. For determination of iron content of Dpr, 50 µg of cellular protein extracts was resolved under nondenaturing conditions in a 4-15% gradient Tris-HCl Ready Gel (Bio-Rad). After the run, one gel was stained with Coomassie Blue to show the pattern of protein bands for equal loading in each lane, and another gel was placed between two plastic sheets and analyzed for 55Fe-containing proteins using Fujifilm BA-2500 Phosphor Imaging Plate System (Fuji Photo Film Co.) according to instructions of the manufacturer.
H2O2 Sensitivity Assays
Overnight S. suis cultures were diluted 100-fold into
fresh THY and grown to early-stationary phase at 37 °C with vigorous shaking. At this point, 1-ml aliquots were taken from the cultures and
exposed to H2O2 in concentrations of 0, 1.0, 2.5, and 5.0 mM. Cells were left in contact with
H2O2 for 2 h at 37 °C without agitation. Immediately after the incubation, cells were diluted in
Pi/NaCl and plated for viability counts onto THY agar.
Colonies were counted after overnight incubation under 6%
CO2 at 37 °C. In the iron chelator complementation
analysis of the H2O2 hypersensitivity of
D282dpr, 1-ml aliquots of early-stationary phase cultures were first incubated with deferoxamine mesylate (DFOM) (Sigma) or
diethylene triamine pentaacetic acid (DTPA) (Sigma) at concentrations of 0, 10, 50, and 100 µM for 30 min at 37 °C without
agitation. The cells were then incubated with or without 5.0 mM H2O2 for 2 h at 37 °C
without agitation. Immediately after the incubation, cells were diluted
in Pi/NaCl and plated for viability counts onto THY agar.
Colonies were counted after overnight incubation under 6%
CO2 at 37 °C.
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RESULTS |
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General Features of S. suis dpr Locus--
We have previously
identified in S. suis a galactose-specific adhesion activity
(31-33) and purified a candidate adhesin (42). Recently, the
corresponding gene was cloned, based on peptide sequence data of the
purified protein and by genome walking.1 The gene turned
out to encode a protein with 64% identity and 82% similarity to
S. mutans Dpr (NCBI Protein Database accession number
BAA96472) at the level of primary amino acid sequence. In the
present study, we identified by Southern hybridization and subsequently
cloned and sequenced a 6.5-kb ClaI-EcoRI fragment containing the entire dpr. As shown in Fig.
1A, we identified eight
possible ORFs in addition to dpr, some of which were in the
same direction of transcription. This indicated that dpr
might be transcribed in a multicistronic mRNA. To study this
possibility, we first analyzed the upstream region of dpr
for sequence features commonly associated with bacterial promoters. As
shown in Fig. 2, we found a possible
-factor
10 recognition sequence TATAAT and a
35 recognition
sequence TTGCAA between ORFI and dpr. A putative
Shine-Dalgarno box was found a few nucleotides upstream of the
initiation codon. Northern hybridization demonstrated that dpr is transcribed monocistronically with an approximate
mRNA size of 550 bp (Fig. 1B). Thus, there seems to be a
functional promoter between ORFI and dpr possibly including
the sequence features found in this study (Fig. 2). However, further
work including primer extension analysis is needed to characterize the
putative dpr promoter.
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Inactivation and Complementation of dpr in S. suis--
The
sequence information of the 6.5-kb ClaI-EcoRI
fragment allowed us to construct a suicide plasmid pDPR2 and
subsequently generate a dpr knockout mutant
(D282dpr) using allelic replacement (Fig.
3A). PCR analysis with primers
DPR-5'-OUT and DPR-3'-OUT annealing upstream and downstream of
dpr, respectively, confirmed the replacement of
dpr by spc by homologous recombination (Fig. 3B). This was further verified by Western analysis (Fig.
3C). The dpr mutation was complemented by
introducing wild-type dpr into D282
dpr in an
E. coli/Streptococcus sp. shuttle vector
pLZ12-Km/Spc (Fig. 2). In pLZ12-L4A, dpr was inserted into
spc containing only its putative Shine-Dalgarno box, leading
to a Dpr expression level comparable with that of the wild-type strain
D282 at the early-stationary phase (Fig. 3C). In pLZ12-P3,
dpr was inserted into spc with its own putative
promoter and Shine-Dalgarno box, leading to overexpression of Dpr at
the early-stationary phase (Fig. 3C). The overexpression might have resulted from better stability of pLZ12-P3 or from inclusion
of the putative dpr promoter. In any case, the
complementation strains constructed allowed us to analyze the function
of Dpr with two cellular levels of the protein. Both of the constructed plasmids pLZ12-L4A and pLZ12-P3 induced a constant Dpr expression level
in early-stationary phase as analyzed by Western blotting of several
independent protein extracts.
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Role of Dpr in Aerotolerance of S. suis--
Dpr is an important
aerotolerance factor for S. mutans (11). To study whether
Dpr is involved in aerotolerance of S. suis, we analyzed the
growth of our strains in solid and liquid THY incubated under normal or
6% CO2 atmosphere. At the same time, we quantified
dpr mRNA and Dpr levels at different time points of the
growth phase. We first grew D282 and D282dpr to
early-stationary phase in THY at 37 °C under 6% CO2.
The cultures were then diluted 100-fold into fresh THY and incubated at
37 °C with vigorous shaking, which causes an extensive aeration of
the culture medium. The growth was monitored by measuring
A600 (Fig.
4A). As shown in Fig.
4B, Dpr expression was transcriptionally induced just after the culture entered the actively growing state. The transcriptional activity sharply decreased when the bacterial population reached the
stationary phase. The protein levels, on the other hand, reached the
maximum in the actively growing state and remained relatively unchanged
at the stationary phase (Fig. 4C). Thus, wild-type D282 cells clearly expressed Dpr during the growth period assayed. Yet, as
shown in Fig. 4A, there was no significant growth
retardation of D282
dpr as compared with D282. As analyzed
by plating of early-stationary phase cultures, there were no
significant differences between the colony forming abilities of
D282
dpr and D282, regardless of whether the THY plates
were incubated aerobically or under 6% CO2 (data
not shown). Taken together, the results indicate that Dpr is not an
essential aerotolerance factor for S. suis.
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Role of Dpr in H2O2 Resistance of S. suis--
Dpr is involved in H2O2 resistance
of S. mutans (11). Sensitivities of our S. suis
strains to H2O2 were tested by exposing early-stationary phase cultures to different concentrations of H2O2 and counting viable cells after plating
onto THY. Fig. 5 shows that over the
whole range of H2O2 concentrations used, there was only a slight loss of viability of the wild-type strain D282. In
contrast, D282dpr was highly sensitive toward
H2O2 with a ~106-fold reduced
viability after exposure to 5.0 mM
H2O2. To examine the possibility that the
H2O2 hypersensitivity of D282
dpr
was not due to its Dpr deficiency but rather was caused by polar
effects of the spc insertion, genetic complementation
analyses were done. These experiments were important because D282 was
used for mutant construction due to poor transformability of 628, which
served as the initial source of sequence information (Fig.
1A). Also, ORFV with a predicted gene product of 16.2 kDa
(Fig. 1A; Table III) was in
the complementary strand to dpr and was also deleted in D282
dpr. Fig. 5 shows that over the whole range of
H2O2 concentrations used, both of the
complementation strains were resistant to H2O2. This confirmed that Dpr was responsible for the detected
H2O2 resistance in S. suis and ruled
out the participation of ORFV and other downstream effects.
Overexpression of Dpr in D282P3 (Fig. 3C) did not
significantly increase the ability of bacteria to tolerate
H2O2 (Fig. 5).
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Iron Chelator Complementation of the H2O2
Hypersensitivity of D282dpr--
Free intracellular
Fe2+ nonezymatically cleaves H2O2
into hydroxyl radicals in Fenton chemistry fashion and is important for the actual toxicity of H2O2 (20, 21). We
analyzed how iron chelators with different membrane permeabilities
altered the H2O2 hypersensitivity of
D282
dpr. The hydrophilic iron chelator DFOM, a
siderophore produced by Streptomyces pilosus, has been used in several studies to interfere with the intracellular iron pool of
both prokaryotic and eukaryotic cells (43-46). DTPA, on the other
hand, has been used as an extracellular iron chelator (46). The effects
of these compounds on the H2O2 hypersensitivity
of D282
dpr were assayed by exposing early-stationary
phase cultures to 5.0 mM H2O2 after
preincubating the bacteria with different concentrations of the
chelators. As shown in Fig. 6 the
H2O2 hypersensitivity of D282
dpr
was complemented by a preincubation with 100 µM DFOM. In
contrast, DTPA did not complement the H2O2
hypersensitivity at any of the concentrations studied. The results
indicate that the intracellular but not the extracellular iron pool is
involved in H2O2 sensitivity of the bacteria
and that the absence of functional Dpr can be complemented by a
molecule capable of iron chelation.
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Site-directed Mutagenesis of the Putative Ferroxidase Center of
Dpr--
Amino acid sequence alignment of Dps family members with
known crystal structures revealed that S. suis Dpr contained
a putative ferroxidase center in its N-terminal half (Fig.
7). It has been suggested that this amino
acid motif is involved in iron incorporation in a fashion similar to
that of classical ferritins (22, 27, 28, 47), but there is no direct
experimental evidence to support this proposal in vivo. When
we analyzed cellular protein extracts from early-stationary phase
cultures grown in the presence of 55FeCl3, Dpr
had clearly incorporated iron (Fig. 8).
To investigate the possible relationship of the in vivo iron
incorporation activity with the putative ferroxidase center, we
utilized site-directed mutagenesis. The negatively charged residues
Asp-74 and Glu-78 of the putative ferroxidase center (Fig. 7) were
independently substituted with Ala. As shown in Fig. 8A, the
mutations had no apparent effects on the level of expression or
solubility of Dpr. The mutations also seemed to have no effects on the
oligomeric stability of Dpr as indicated by similar mobility to
wild-type Dpr in nondenaturing polyacrylamide gels (Fig.
8B). In contrast, the mutations independently caused a
complete inactivation of the iron incorporation activity in
vivo (Fig. 8C). Taken together, S. suis Dpr
was able to incorporate iron in vivo, and this process was
dependent on Asp-74 and Glu-78, the amino acids conserved in the
putative ferroxidase centers of several Dps family members (Fig. 7)
(14, 22, 27, 28, 47).
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H2O2 Sensitivities of D282dpr Expressing
Wild-type or Site-mutated Forms of Dpr--
The
H2O2 sensitivities of D282
dpr
expressing wild-type or site-mutated forms of Dpr were assayed by
exposing early-stationary phase cultures to different concentrations of
H2O2 and counting viable cells after plating
onto THY. As shown in Fig. 9 bacteria expressing wild-type Dpr were fully resistant to
H2O2. In contrast, bacteria expressing the
site-mutated and iron incorporation-negative forms of Dpr were
hypersensitive to H2O2 with a comparable
phenotype to dpr knockout. Thus, the detected
H2O2 resistance of S. suis was not
only critically dependent on Dpr but specifically on its iron
incorporation activity.
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DISCUSSION |
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In this paper, we provide for the first time direct in vivo evidence on how streptococcal Dpr mediates its protective function against H2O2. H2O2 resistance is a crucial property for streptococci because several species produce it as a part of their metabolism despite the lack of oxidative phosphorylation (4-7). H2O2 is also encountered as a part of host defenses (8), and under certain conditions, streptococci seem to utilize it as their own virulence factor (4, 9, 10). Thus, in addition to maintenance of normal cellular physiology, Dpr may also have a role in the pathogenesis of streptococcal infections. S. suis seemed an ideal model organism to study the in vivo function of Dpr because it not only lacks catalase, like other streptococci, but also lacks NADH peroxidase (34). This enzyme is capable, to some extent, of substituting for the absence of catalase (48, 49).
It is known that defects in the regulation of intracellular iron
homeostasis may lead to enhanced oxidative stress (46, 50, 51). Iron in
its reduced form nonenzymatically cleaves H2O2
into hydroxyl radicals, the most deleterious forms of reactive oxygen
intermediates, by Fenton chemistry (H2O2 + Fe2+ ·OH + -OH + Fe3+)
(20, 21, 52). Based on a primary amino acid sequence comparison, Dpr
has been reported to be a member of the Dps protein family (11), in
which one of the functional features is iron binding activity (17, 22,
26, 27). The family members seem to be able to oxidize Fe2+
and store it inside the oligomeric protein shell as Fe3+
(17, 29, 53, 54), resembling classical ferritins in this respect (23).
Indeed, Bozzi and co-workers (22) identified in L. innocua Flp, a member of the Dps family that is well characterized in vitro (53, 54), a homologous region to the iron
nucleation center of mammalian ferritin L-subunit (24).
They suggested that this region could carry out the initial steps in
iron core formation and also serve as a ferroxidase center. Although a
homologous region to this amino acid motif can be found in several Dps
family members (Fig. 7) (14), no direct experimental evidence is
available on its functionality in iron incorporation in
vivo. Furthermore, the biological significance of iron
incorporation is not well established. In the present study, a putative
ferroxidase center was identified in the N-terminal half of S. suis Dpr, and the structure-function relationship was studied by
site-directed mutagenesis.
The targets for the point mutations in the putative ferroxidase center were chosen by using the three-dimensional structure of L. innocua Flp (28). Flp shares 44% primary amino acid sequence identity with S. suis Dpr. In Flp crystals, 12 iron atoms have been directly observed occupying the putative ferroxidase centers, which are formed at the interfaces of two adjacent subunits (28). The amino acids protruding into the interface of Flp dodecamer and seemingly involved in the actual iron coordination (His-31, His-43, Asp-47, Asp-58, and Glu-62) (28) are all conserved and similarly spaced in S. suis Dpr as His-47, His-59, Asp-63, Asp-74, and Glu-78, respectively (Fig. 7). The involvement of these amino acids in forming a classical dinuclear ferritin-like ferroxidase center has been modeled for Flp (28). In the initial step, the first ferrous iron, guided into the interior of the dodecamer through hydrophilic channels, would bind to His-31 from the A helix and Asp-58 and Glu-62 from the B helix of an adjacent subunit (Fig. 7) (28). Based on the L. innocua Flp model, we decided to independently substitute two of the homologous negatively charged amino acids, Asp-74 and Glu-78, with Ala. The substitutions had no apparent effects on the expression, solubility, or oligomeric stability of the Dpr dodecamer. In contrast, Asp-74 and Glu-78 were crucial for the Dpr to incorporate iron in vivo. This result represents the first direct demonstration of the functional involvement of the putative ferroxidase center in iron incorporation in vivo for Dpr or any of the Dps family members.
The dpr knockout of S. suis had a
~106-fold reduced viability under exposure to 5.0 mM H2O2 as compared with the
wild-type strain. By analyzing the H2O2
sensitivities of dpr knockout expressing wild-type or
site-mutated forms of Dpr, we determined the role played by the iron
incorporation activity of Dpr in H2O2
resistance. Strikingly, bacteria expressing iron incorporation-negative
forms of Dpr were hypersensitive to H2O2 with a
phenotype comparable to that of dpr knockout. Thus, Dpr
seemed to protect the bacteria against H2O2 by
its iron incorporation activity. This was supported by the iron
chelator complementation analysis of the H2O2
hypersensitivity of D282dpr. DFOM, an iron chelator
interfering with the intracellular iron pool (43-46), was able to
rescue the viability defect of D282
dpr. Under the same
conditions, an extracellular iron chelator, DTPA (46), did not cause
any effect. Yamamoto et al. (25) recently reported data
indicating that S. mutans Dpr is capable of inhibiting the
action of Fenton chemistry in vitro. Thus, Dpr seems to
provide an indirect means for catalase-negative bacteria to tolerate
H2O2 by inhibiting the
Fe2+-catalyzed cleavage of H2O2
into more toxic reactive oxygen intermediates.
Mechanisms other than iron incorporation have been suggested for Dps family members to explain the protection against H2O2, and synergy between them is possible (12, 14-16). A convincing body of evidence indicates that E. coli Dps protects DNA by a direct association, which leads to highly ordered DNA-Dps biocrystals (12, 15, 16, 55). However, Dps also contains a putative ferroxidase center in its N-terminal half, and a dual function in H2O2 resistance has been proposed. Dps could directly protect the DNA by biocrystal formation but could also inhibit the action of Fenton chemistry by its iron incorporation activity (17, 29, 47). Whether Dpr also binds and protects DNA remains contradictory. Yamamoto et al. (25) recently reported data indicating that S. mutans Dpr has no DNA binding activity. In our own studies using hydroxyl radical DNA footprinting assays, we have not detected any DNA binding activity for S. suis Dpr.3 Also, some other members of the Dps family seem to lack DNA binding activity (26, 27). The possibility that Dpr could differ from Dps in lacking DNA binding activity is not unexpected, considering that the primary amino acid sequence identity shared between S. suis Dpr and E. coli Dps is only 25%. It also seems unlikely that Dpr could have a catalase-like activity as reported for the DpsA of Synechococcus sp. (14). S. suis Dpr shares an extremely weak homology to DpsA (nondetectable in BLAST-P data base search) and does not seem to contain heme, which has been linked to the enzymatic activity of DpsA (14). Furthermore, Yamamoto et al. (25) have recently presented data indicating that S. mutans Dpr has no catalase-like activity. Indeed, the results of our present study indicate that it is the iron incorporation activity that is the molecular basis of Dpr-mediated H2O2 resistance.
The Dps family includes molecules from diverse taxonomic lineages (14) with striking similarities, even including a recent member in the archaeon Halobacterium salinarum (56). Proteins fold into four-helix bundles resembling the fold of mammalian ferritins and assemble into large hollow globular complexes (23, 27, 28, 47). Based on our present study, S. suis Dpr protected the bacteria against H2O2 by its iron incorporation activity. Whether this is true for the Dps family members in general is not known. However, the amino acid residues forming the putative ferroxidase centers are among the most conserved primary amino acid sequence features of the family (Fig. 7) (14). Also, all members have proven to be iron-binding proteins, if analyzed for this activity (11, 17, 22, 26, 27, 56). Thus, it is possible that in diverse bacterial species, Dps-related molecules might serve as functional ferritin-like molecules and protect bacteria against H2O2 as inhibitors of Fenton chemistry. However, it has become evident that, despite sharing the few highly conserved amino acid residues, the putative ferroxidase centers vary considerably (Fig. 7), which might lead to different functional properties. Indeed, Zhao et al. (17) have recently reported data on iron oxidation and incorporation properties of E. coli Dps that differ from those of L. innocua Flp in both the rates of Fe2+ binding and oxidation. Thus, it is obvious that more studies are needed to further elucidate the importance of iron incorporation activity for Dps family members in H2O2 resistance.
The functional divergence of the Dps family is an important area of
further investigation. The family members seem to be involved in
aerotolerance (11), H2O2 resistance (11, 12,
57), cold shock adaptation (58), starvation tolerance (12), iron
storage (22), neutrophil activation (59, 60), carbohydrate binding (61), and adhesion (62). The molecular determinants mediating these
activities are largely unknown. Additional in vivo studies together with in vitro work using recombinant site-mutated
forms of the molecules in combination with crystal structure
information (27, 28, 41, 47) are needed to reveal the molecular
mechanisms behind these activities and their relationships to each
other. Determining the structural basis of these biological activities may eventually contribute to the development of means to prevent and
treat diseases caused by pathogenic bacteria.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Drs. Hilde Smith and Michael Caparon for kindly providing bacterial strains and plasmids. Terttu Jompero, Jukka Karhu, and Tero Mustalahti are acknowledged for technical assistance.
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FOOTNOTES |
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* This study was supported by the grants from the Sigrid Jusélius Foundation and the Academy of Finland.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY154459.
To whom correspondence should be addressed: Dept. of Medical
Biochemistry and Molecular Biology, University of Turku,
Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Tel.: 358-2-333-7254;
Fax: 358-2-333-7229; E-mail: endonukleaasi@hotmail.com.
Published, JBC Papers in Press, December 24, 2002, DOI 10.1074/jbc.M210174200
1 K. Tikkanen, A. T. Pulliainen, V.-P. Korhonen, R. Segers, S. Haataja, J. Wahlfors, and J. Finne, submitted for publication.
3 A. T. Pulliainen and J. Finne, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: THY, Todd Hewitt Broth medium supplemented with 0.5% (w/v) yeast extract; ORF, open reading frame; BSA, bovine serum albumin; DFOM, deferoxamine mesylate; DTPA, diethylene triamine pentaacetic acid.
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