Regulation of the Intracellular Free Iron Pool by Dpr Provides Oxygen Tolerance to Streptococcus mutans
ABSTRACT
Dpr is an iron-binding protein required for oxygen tolerance in Streptococcus mutans. We previously proposed that Dpr could confer oxygen tolerance to the bacterium by sequestering intracellular free iron ions that catalyze generation of highly toxic radicals (Y. Yamamoto, M. Higuchi, L. B. Poole, and Y. Kamio, J. Bacteriol. 182:3740-3747, 2000; Y. Yamamoto, L. B. Poole, R. R. Hantgan, and Y. Kamio, J. Bacteriol. 184:2931-2939, 2002). Here, we examined the intracellular free iron status of wild-type (WT) and dpr mutant strains of S. mutans, before and after exposure to air, by using electron spin resonance spectrometry. Under anaerobic conditions, free iron ion concentrations of WT and dpr strains were 225.9 ± 2.6 and 333.0 ± 61.3 μM, respectively. Exposure of WT cells to air for 1 h induced Dpr expression and reduced intracellular free iron ion concentrations to 22.5 ± 5.3 μM; under these conditions, dpr mutant cells maintained intracellular iron concentration at 230.3 ± 28.8 μM. A decrease in cell viability and genomic DNA degradation was observed in the dpr mutant exposed to air. These data indicate that regulation of the intracellular free iron pool by Dpr is required for oxygen tolerance in S. mutans.
Iron is an essential and beneficial nutrient for most organisms but has toxic properties in the presence of oxygen (4, 15, 31). Iron ions stimulate the generation of highly reactive and toxic oxygen species such as hydroxyl radicals (11, 15). In vitro experiments have shown that Fe(II) catalyzes nonenzymatic hydroxyl radical formation from hydrogen peroxide via the Fenton reaction, whereas hydrogen peroxide remained intact in the absence of iron ions at physiological pH (11). Intracellular iron source for the Fenton reaction is considered to be a free iron pool in cells (15, 17). Tight regulation of iron metabolism, especially the intracellular free iron pool, is therefore regarded as a determining factor for survival of an organism in air (11, 15, 17, 31).
Methods to investigate the intracellular free iron pool in intact cells were recently developed, and the presence of several factors affecting the free iron status was reported in both prokaryotes and eukaryotes (17, 19). It has been reported that, in Escherichia coli and Saccharomyces cerevisiae, accumulation of intracellular superoxide, owing to a superoxide dismutase deficiency, increases the level of free iron pool by releasing iron ions from proteins containing iron-sulfur clusters (19, 29). In an E. coli fur mutant, aberrant regulation of iron uptake was associated with an increase in the level of free iron (19). In mammalian cells, repression of ferritin H subunit expression increased the level of intracellular free iron (17). In all reported cases, an increase in the free iron pool correlated with an increase in oxidative stress (17, 19, 29, 31).
Streptococcus mutans, a principal causative agent of human dental caries, cannot synthesize heme and lacks both a respiratory chain and catalase, which are required for elimination of hydrogen peroxide in most aerobic organisms. However, S. mutans grows under aerobic conditions and induces several antioxidant proteins when cells are exposed to air (12, 13, 24, 27, 35-37). We previously identified dpr (for dps-like peroxide resistance) as a potential peroxide resistance gene from S. mutans. Studies of a series of dpr-deficient strains led us to conclude that dpr plays a vital role in aerobic survival of S. mutans (36). Our further studies on the purified dpr gene product showed that Dpr forms ferritin-like spherical dodecamers and binds up to 480 iron atoms per complex (37). Primary amino acid sequence homologies indicate that Dpr is a member of the Dps (for DNA-binding protein from starved cells) (3) protein family (36). Dps is a nonspecific DNA-binding protein that is induced by oxidative or nutrient stress in E. coli (3). Stable Dps-DNA complex formation is believed to protect DNA from hydrogen peroxide action (3, 21, 33). However, in the case of S. mutans, Dpr could not bind DNA (37). We therefore proposed another mode of cell protection from oxidative stress by Dpr, based on its sequestration of intracellular iron ions. We demonstrated in vitro that Dpr prevents iron-dependent hydroxyl radical formation (36). At almost the same time, Zhao et al. reported iron-binding and iron-detoxifying properties of E. coli Dps (38). It was also reported that some Dps family proteins having iron binding, but not DNA-binding ability, were involved in oxidative stress resistance (8, 16, 28). The crystal structure of Dps family proteins, including Streptococcus suis Dpr homologue, revealed a ferritin-like structure of the proteins, indicating that this class of proteins could incorporate iron ions as ferritin does (8, 10, 14, 18, 38). Taken together with our data on Dpr properties, it was suggested that Dps family proteins could affect the cellular free iron ion status, thereby conferring oxygen tolerance. In the present study, we measured the intracellular free iron pool of wild-type (WT) and dpr strains of S. mutans and clarified the role of Dpr in regulating the intracellular free iron pool and on bacterial survival in air.
MATERIALS AND METHODS
Strains, media, and growth conditions.
S. mutans GS-5 (WT strain) and DES (dpr-deficient mutant) (36) were used in the present study. Cells were prepared for analysis by electron spin resonance (ESR) spectrometry as follows. A 10-ml preculture of S. mutans, prepared in Todd-Hewitt broth (THB; Difco Laboratories, Detroit, Mich.) under anaerobic conditions (in an anaerobic glove box [Hirasawa Works, Tokyo, Japan] in an atmosphere of 80% nitrogen, 10% carbon dioxide, and 10% hydrogen), was added to a 300-ml culture in the same medium. The culture was incubated at 37°C for 3.5 h (A660 = ∼0.8) under anaerobic conditions. At this time, part of the culture (100 ml) was removed and used as the zero time sample. The remaining culture (200 ml) was centrifuged at 7,800 × g for 5 min, resuspended in the same volume of fresh THB medium, transferred to 500-ml flasks, and then incubated at 37°C with shaking (120 cycles/min). After 30 min of incubation, 100 ml of the culture was removed as the 30-min sample. The rest of the culture was incubated for another 30 min and used as the 60-min sample.
ESR spectrometry sample preparation.
Portions (100 ml) of the cultures described above were centrifuged at 7,800 × g for 5 min. Pellets were resuspended in 5 ml of THB medium with or without 20 mM deferoxamine (Sigma) and then incubated at 37°C with shaking (170 cycles/min) for 10 min under aerobic conditions. Cells were collected by centrifugation at 7,800 × g for 5 min, washed with ice-cold 20 mM Tris-HCl buffer at pH 7.0, and resuspended in 0.3 ml of the same buffer containing 10% (vol/vol) glycerol. An aliquot of each sample was taken to measure the optical density at 660 nm. Then, 200 μl of each cell suspension was transferred to a quartz ESR tube, immediately frozen, and stored at −80°C until ESR measurements were carried out.
ESR spectrometry.
ESR spectra were recorded on an RE-3X ESR spectrometer (JEOL, Ltd., Tokyo, Japan). Samples were maintained at −196°C by using a finger Dewar vessel filled with liquid nitrogen. Experimental conditions used for low-temperature Fe(III) electron paramagnetic resonance (EPR) were as follows: center field, 250 mT; sweep width, 150 mT (250 mT for wider sweep); frequency, 9.21 GHz; microwave power, 5 mW; modulation amplitude, 1 mT; modulation frequency, 100 kHz; receiver gain, 1×100; sweep time, 4 min; and time constant, 0.03 s. The g value was calculated by using the standard formula g = hv/βH, where h is Planck's constant, v is the frequency, β is the Bohr magneton, and H is the external magnetic field at resonance.
Calculation of intracellular free iron concentration.
The double-integrated intensities of the g = 4.3 signal of each sample were converted to intracellular free iron ion concentrations as follows. The amount of deferoxamine-Fe(III) in the ESR sample was quantified by using the EPR signals of deferoxamine-Fe(III) of known concentrations. First, 1 ml of cell suspension (optical density at 600 nm of 1.0) was calculated to contain 0.58 μl of intracellular water volume, based on (i) the reported internal water content in S. mutans cells of 1.6 μl per mg (dry weight) (25) and (ii) the fact that 1 ml of cell suspension (A660 = 1.0) contained 0.365 ± 0.034 mg (dry weight). We used this value, along with the ESR signal from an external Fe(III) standard and the optical density of the ESR sample, to quantify intracellular free iron concentrations.
Measurement of total iron.
S. mutans cells were collected by centrifugation at 7,800 × g for 10 min. Cells were washed once with phosphate-buffered saline (pH 7.0) and twice with Milli-Q water (Millipore Corp., Tokyo, Japan). Washed cells were resuspended in 1 ml of Milli-Q water and then transferred to a Teflon container. Water was removed from cells by incubation at 90°C for 20 h, and the bacterial dry weight was measured. Next, 2 ml of concentrated nitric acid (Ultratrace analysis grade; Wako Pure Chemical Industries, Osaka, Japan) and 0.2 ml of concentrated perchloric acid (Ultrapure AA-100; Tama Chemicals, Kanagawa, Japan) were added to about 100 mg of dried bacterial cells in a Teflon container, and the cells were dissolved into liquid by microwave treatment as described previously (23). After the cells were dissolved, the containers were heated on a hot plate at 160°C to near dryness and then dissolved in 5 ml of 5% nitric acid solution for analysis by atomic absorption spectrometry with an atomic absorption spectrometer (170-30; Hitachi, Tokyo, Japan). The iron content and bacterial cell dry weight of samples, coupled with the reported internal water content in S. mutans cells of 1.6 μl per mg (dry weight) (25), allowed us to quantify the total iron concentration in the cell.
Monitoring survival, genomic DNA degradation, and expression of Dpr.
For viable cell determinations, culture dilutions were plated on solid THB medium supplemented with 500 U of bovine liver catalase (Sigma). After 48 h of incubation in an anaerobic box at 37°C, the CFU were counted. Genomic DNA of S. mutans was prepared as described previously (35), with some modifications. Cells were treated with both mutanolysin (200 U/ml; Sigma) and acromopeptidase (1,000 U/ml; Wako) for 15 min at 37°C in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA prior to lysis by sodium dodecyl sulfate. DNA samples (500 ng) were electrophoresed on a 1% Tris-acetate agarose gel and then visualized by ethidium bromide staining. For Western blot analyses, cell lysates were prepared as described previously (36) and separated by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis. Protein bands corresponding to Dpr were identified as described by using anti-Dpr antibody (37).
RESULTS AND DISCUSSION
Pool size of intracellular free iron in WT and dpr-deficient mutant of S. mutans.
The intracellular free iron pool was measured by ESR spectrometry essentially according to the method of Woodmansee and Imlay (34). Anaerobically grown S. mutans cells were treated with deferoxamine, a cell-permeable iron chelator, that converts “free iron” to the Fe(III) species. This species gives an intense ESR signal at g = 4.3 (34). Representative low-temperature Fe(III) ESR spectra of whole S. mutans cells are shown in Fig. 1. A strong signal at g = 4.3 was obtained for deferoxamine-treated S. mutans cells. The Fe(III) ESR signal at g = 4.3 is characteristic of ferric iron in a high spin complex (34). In contrast, the ESR spectrum of iron-loaded Dpr, which was prepared as previously described (37), gave a very broad ESR signal at a g value of ca. 2 but did not give an ESR signal at a g value of 4.3 (data not shown). Thus, the signal at g = 4.3, obtained for S. mutans cells treated with deferoxamine, is distinguishable from the signal corresponding to Dpr-bound iron.
We used ESR spectrometry to compare intracellular free iron concentrations in WT and dpr strains before and after exposure to air (Fig. 2). Under anaerobic conditions, similar intensities of signal at g = 4.3 were detected for both strains. Upon exposure to air, the intensity of signal obtained for WT cells decreased. In contrast, the intensity of signal for the dpr-deficient cells remained high (Fig. 2).
The double-integrated intensities of ESR signals were then converted to intracellular free iron ion concentrations (see Materials and Methods). Under anaerobic conditions, WT and dpr strains had intracellular free iron concentrations of 225.9 ± 2.6 and 333.0 ± 61.3 μM, respectively (Fig. 3A and B). After cell exposure to air for 1 h, the values of the WT strain dropped to 22.5 ± 5.3 μM; in contrast, values for the dpr mutant remained high, at 230.3 ± 28.8 μM (Fig. 3A and B). Total iron content in cells, determined by atomic absorption spectrometry, is also shown (Fig. 3A and B). In contrast to the rapid decrease of intracellular free iron in the WT strain, total iron content decreased by only 13% after 1 h of exposure to air, indicating that the decrease of free iron concentrations did not result from the change of total iron content but rather from the change of iron status inside the cell. Western blot analysis of Dpr expression under the different growth conditions showed that Dpr synthesis was induced by exposing cells to air (Fig. 3C). In WT cells, the decrease in intracellular free iron concentrations correlated with increased Dpr expression (Fig. 3). In order to examine whether Dpr synthesis is really responsible for attenuating intracellular free iron concentrations, chloramphenicol (50 μg/ml) was added to the WT culture prior to aerobic incubation. After 1 h of incubation in air, cells were analyzed by Western blotting and ESR spectrometry. Chloramphenicol treatment of WT cells prevented Dpr expression (data not shown) and resulted in higher intracellular free iron concentrations (468.5 ± 15.6 μM). These results strongly indicate that, in S. mutans, Dpr incorporates free iron ions in vivo and contributes to lowering the intracellular free iron ion concentration.
In the dpr mutant, total iron amounts decreased more rapidly than in the WT strain after exposure of the cells to air (Fig. 3A and B). After 1 h of incubation of the dpr mutant in air, both total and free iron concentrations decreased by ca. 30%. This result implies that decreased iron uptake in the dpr mutant, due to cell death as described below, might reduce the intracellular free iron concentrations in the dpr mutant by reducing the total iron content.
Effects of high intracellular free iron concentrations on growth, survival, and DNA degradation of S. mutans.
In the presence of oxygen, excess amounts of intracellular free iron ions may catalyze the generation of reactive oxygen species that degrade cellular components and cause cell death (15, 31). We explored the effects of high intracellular free iron concentrations on growth and survival of the dpr mutant (Fig. 4A). Cell densities of both WT and dpr mutant strains slightly increased during the incubation period in air. However, the number of dpr mutant CFU decreased 100-fold after 1 h of exposure of the cells to air, whereas the number of WT strain CFU remained constant (Fig. 4A). A main target of oxygen-induced cellular damage is DNA (15, 31). The effect of aeration on genomic DNA extracted from WT and dpr mutant strains was examined by gel electrophoresis (Fig. 4B). Under anaerobic conditions, no significant differences in electrophoretic mobility were observed between genomic DNA from WT and dpr mutant strains. After exposure to air, however, marked degradation of DNA was observed in the dpr mutant extract (Fig. 4B). DNA integrity and cell survival of the dpr mutant were restored by the addition of catalase or deferoxamine, each of which removes a substrate for the Fenton reaction, to growth medium during cell exposure to air (Fig. 5). These results strongly indicate that iron-mediated generation of hydroxyl radicals via the Fenton reaction degraded cellular components such as DNA and caused cell death in the dpr mutant.
The present study provided the first direct evidence that Dps family proteins can affect the intracellular free iron pool. Pulliainen et al. recently identified a Dpr homologue in Streptococcus suis as an iron-binding protein and demonstrated, by introducing the site-mutated alleles of Dpr into an S. suis dpr mutant, that the iron-binding ability of the protein is required for hydrogen peroxide resistance (28). The result is in agreement with our present study with S. mutans. It is now evident that ferritin-like iron binding is a common feature of Dps family proteins (6, 8, 16, 28, 30, 32, 37, 38). It is therefore not surprising that Dps family proteins could potentially participate in the regulation and detoxification of intracellular free iron pool, as we described here. The presence of Dpr and Dps family proteins may prevent an iron-catalyzed Fenton reaction and, as a consequence, play an important role in oxygen tolerance, especially in catalase-deficient bacteria such as S. mutans and S. suis.
The addition of catalase to the medium restored the survival of the dpr mutant under air (Fig. 5), indicating the presence of hydrogen peroxide in the cells under this condition. Several lactic acid bacteria are known to accumulate hydrogen peroxide in the medium via the action of hydrogen peroxide-generating enzymes such as pyruvate oxidase and NADH oxidase (7, 22, 26). S. mutans lacks pyruvate oxidase but has hydrogen peroxide-forming NADH oxidase (Nox-1) (2, 13). Although Nox-1 functions as a component of bicomponents, peroxidase and the AhpC component (27), the expression of only Nox-1 (absence of AhpC) under conditions described previously (13) might allow the bacterium to produce hydrogen peroxide.
An interesting finding of the present study is that S. mutans cells contained significant amounts of intracellular iron (Fig. 3), particularly since lactic acid bacteria including streptococci are believed to require little or no iron for growth (5). The total iron contents in S. mutans (from 0.005 to 0.008% in dry weight) were some 2.5- to 4-fold less than that in E. coli grown in rich medium (1). Although the iron requirement in S. mutans reportedly depends on growth conditions (20), iron assimilation could facilitate metabolism, e.g., for amino acid biosynthesis utilizing the iron-containing protein aconitase (9) or potentially for activating iron-requiring ribonucleotide reductases identified in the genome sequence (2).
FIG. 1.
FIG. 2.
FIG. 3.
FIG. 4.
FIG. 5.
Acknowledgments
We are grateful to A. Gruss for critical reading of the manuscript.
This study was supported in part by grants-in-aid for scientific research 12876016 and 14656030 from the Japan Society for the Promotion of Science (JSPS), the Nagase Science and Technology Foundation, and the Noda Institute for Scientific Research Foundation. Y.Y. was the recipient of a predoctoral fellowship from JSPS.
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Published In
Journal of Bacteriology
Volume 186 • Number 18 • 15 September 2004
Pages: 5997 - 6002
PubMed: 15342568
Copyright
© 2004 American Society for Microbiology.
History
Received: 23 February 2004
Accepted: 22 March 2004
Published online: 15 September 2004
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