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Applied and Environmental Microbiology, April 2006, p. 2918-2924, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2918-2924.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Overexpression and Characterization of an Iron Storage and DNA-Binding Dps Protein from Trichodesmium erythraeum

M. Castruita,1* M. Saito,3 P. C. Schottel,1 L. A. Elmegreen,1 S. Myneni,2 E. I. Stiefel,1 and F. M. M. Morel2

Department of Chemistry,1 Department of Geosciences, Princeton University, Princeton, New Jersey 08540,2 Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 025433

Received 8 November 2005/ Accepted 17 January 2006


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ABSTRACT
 
Although the role of iron in marine productivity has received a great deal of attention, no iron storage protein has been isolated from a marine microorganism previously. We describe an Fe-binding protein belonging to the Dps family (DNA binding protein from starved cells) in the N2-fixing marine cyanobacterium Trichodesmium erythraeum. A dps gene encoding a protein with significant levels of identity to members of the Dps family was identified in the genome of T. erythraeum. This gene codes for a putative DpsT. erythraeurm protein (Dpstery) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dpstery, and we found that Dpstery, like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dpstery binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dpstery indicated that it has an iron core that resembles that of horse spleen ferritin.


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INTRODUCTION
 
Iron is used in numerous metabolic reactions and is essential in most living organisms. However, the low solubility of iron in oxygenated waters limits its bioavailability to aquatic organisms. This is the case in the surface waters of the ocean, where Fe is found primarily as particulate oxyhydroxides and Fe input from the atmosphere and deep seawater can be limited (8, 24). Recent studies have shown that low availability of Fe to phytoplankton directly limits primary production in some regions of the ocean (5, 14, 15, 30). Other studies have suggested that Fe may limit nitrogen fixation by marine cyanobacteria in other oceanic regions and thus control primary production by limiting the input of fixed nitrogen (17, 34).

Trichodesmium spp. are diazotrophic marine cyanobacteria that play a key role in the tropical and subtropical regions of the oceans not only as major primary producers but also as suppliers of new nitrogen through N2 fixation (9). Trichodesmium is responsible for an estimated input of at least 65 Tg fixed N2 per year. Nitrogenase is a two-component metalloenzyme with MoFe and Fe cofactors. The MoFe and Fe proteins contain metalloclusters (Fe-S cores) that are required for nitrogenase activity (18, 26). Because of the high Fe content of the nitrogenase enzyme, the iron requirement of nitrogen fixers has been estimated to be 2.5 to 5.2 times higher than that of other phytoplankton (40). The mechanisms by which Trichodesmium acquires and utilizes Fe are thus of great interest. In this study we focused on the question of Fe storage, which is critical because of the episodic nature of Fe input into many tropical and subtropical regions of the oceans.

Free iron in cells is extremely toxic as it catalyzes the generation of reactive oxygen species, such as the hydroxyl radical ( · OH), which causes oxidative damage (Haber-Weiss/Fenton reactions). Thus, iron storage in cells is effected by ferritins, a superfamily of proteins that store iron in a readily available yet nontoxic form (4). Mammalian-type ferritins (i.e., ferritins proper), bacterioferritins, and Dps proteins (DNA binding proteins from starved cells) belong to this family of proteins. Ferritins and bacterioferritins are composed of 24 subunits that form a spherical protein shell with a hollow center in which Fe can be stored (up to 4,500 Fe atoms in ferritins and 2,000 Fe atoms in bacterioferritins) (4, 20). Bacterioferritins differ from mammalian ferritins in that they have up to 12 noncovalently bound heme groups (4, 39). The heme centers have bis-methionine ligation (12, 13). Dps proteins have not been as thoroughly characterized as ferritins or bacterioferritins yet. These proteins are composed of only 12 subunits and can accommodate correspondingly fewer Fe atoms (~500 atoms) (22). Iron is incorporated into the central cavity of ferritins by oxidation of Fe2+, followed by formation of a microcrystalline ferrihydrite-phosphate core. The conversion of Fe2+ to Fe3+ is catalyzed by a ferroxidase center that is found in the subunits in ferritins and bacterioferritins (4) and between subunits in Dps proteins (22, 35, 38). Studies with various organisms have confirmed that the role of all ferritins is to store Fe and prevent its toxicity (41). In addition, it has been found that Dps protects DNA against oxidative agents by cocrystallization of the nucleic acid with the protein (3, 31, 51). DNA binding appears not to be sequence specific since no DNA binding motifs have been identified in Dps proteins yet. The protection of DNA appears to occur through oxidization and sequestration of Fe2+ ions, avoiding Fenton chemistry (31, 51).

Despite the current interest in the use of iron by autotrophs and particularly by nitrogen fixers in the oceans, no protein belonging to the ferritin family has been isolated or characterized from a marine microorganism previously. Here we describe the identification, isolation, and characterization of a Dps protein in Trichodesmium erythraeum.


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METHODS AND MATERIALS
 
Bacterial strains.
The DH5 strain of Escherichia coli was used for cloning, and the BL21(DE3)pLysS strain (Novagen) was used for protein production. Genomic DNA from T. erythraeum IMS101 was used for PCR amplification (kindly provided by Eric Webb, Woods Hole Oceanographic Institution). For DNA damage assays, pUC19 (Sigma-Aldrich) from E. coli was used.

Cloning of the dps gene.
The T. erythraeum IMS101 dps gene was amplified by PCR with oligonucleotide primers 5'-GAAATAAATCATATGTCTAACGC-3' (upper primer) and 5'-CATTTTTCTAGATCTTGCAGG-3' (lower primer). The upper primer was designed to recognize restriction endonuclease NdeI, and the lower primer was designed to recognize BglII. The resulting PCR product was cloned into the pCR 2.1 vectors (Invitrogen) by the TA cloning method and was subcloned into pRSET A (Invitrogen) by utilizing the NdeI and BglII restriction sites, which resulted in pRSET-dps. DNA sequencing reactions were performed by using the plasmid template pRSET-dps with ABI Prism BigDye 3.1 sequencing kits (Applied Biosystems) and an ABI PRISM 3100 genetic analyzer (Applied Biosystems). For protein production the recombinant plasmid pRSET-dps was transferred to the BL21(DE3)/pLysS host, which is inducible with isopropyl-ß-D-thiogalactopyranoside (IPTG).

Overexpression and purification of Dps.
E. coli BL21(DE3)pLysS containing pRSET-dps was grown to an optical density at 600 nm of 0.7 in 1 liter of Luria-Bertani broth containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol. IPTG (0.4 mM) was added to the cultures, and this was followed by incubation for 2 h for production of recombinant protein. The cells were collected by centrifugation at 4,000 x g for 15 min and resuspended in 250 ml of 20 mM Tris-HCl (pH 8.0). The cells were disrupted by using a sonicator (Branson Sonifier) at 50% power for 30 s (repeated three times) and then heat shocked at 70°C for 10 min. The supernatant was separated by centrifugation at 10,000 x g for 15 min and dialyzed overnight in 50 mM Tris-HCl (pH 7.9)-50 mM NaCl. Final purification of the protein was performed by using a Source 30-Q anion-exchange column and a Superdex-30 size exclusion column. To concentrate the purified protein, a Centriprep YM-50 MW centrifugal filter was used. The purity of the overexpressed Dps protein was checked by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), followed by staining with Coomassie brilliant blue R250. The protein concentration was determined by the method of Lowry et al. (29).

Preparation of 55Fe-loaded Dps.
To obtain radiolabeled cultures, E. coli cells harboring the pRSET-dps and pRSET vectors were grown in LB medium containing 55FeCl3 and purified as described above. The native PAGE gel containing the Dps protein was then exposed to a phosphor screen and scanned with a STORM 860 Phosphorimager (Molecular Dynamics) to detect 55Fe.

Gel retardation assays.
Dpstery was mixed with pUC19 at DNA/protein molar ratios 1:500, 1:800, and 1:103 in 50 mM Tris-HCl (pH 7.9) and incubated for 30 min at 30°C. The complexes obtained were resolved by 1% agarose gel electrophoresis in 0.5% Tris-borate-EDTA (TBE) buffer and were detected by ethidium bromide staining. To determine the kd, 0.15 nM pUC19 was titrated with Dpstery. The solutions were then incubated for 30 min at 30°C, and the products were resolved by 1% agarose gel electrophoresis in 0.5% TBE buffer and detected by ethidium bromide staining. DNA was quantified by using the IMAGE J program, and the method used for determination of kd was based on the method of Carey (1, 10).

In vitro DNA damage assay.
DNA-Dps complexes were used to assess the ability of the overexpressed protein to protect the DNA from digestion by DNase I. pUC19 and DNA-Dps complexes obtained from incubation of pUC19 and Dpstery were incubated with 1 U of DNase I for 5 min at room temperature, and the reaction was stopped with 50 mM EDTA. The resulting products were then resolved on a 1% agarose gel with 0.5% TBE buffer and stained with ethidium bromide.

In vitro loading of Fe and phosphate.
The Dps protein was loaded with iron in the presence of O2 by using the method of Ishikawa et al. (23). To a 100-µg/ml Dps solution in 20 mM Tris-HCl (pH 7.0)-150 mM NaCl-0.1 mM EDTA, 500 µM freshly prepared iron(II) ammonium sulfate hexahydrate was added, and the preparation was left at room temperature for 3 h. After incubation, the protein-iron solution was dialyzed against 20 mM Tris-HCl (pH 7.0)-150 mM NaCl-0.1 mM EDTA. To test hydrogen peroxide as an oxidizing agent, a 150-µg/ml Dps solution in 20 mM Tris-HCl (pH 7.0)-150 mM NaCl-0.1 mM EDTA under N2 was added to 250 µM iron(II) ammonium sulfate hexahydrate. Hydrogen peroxide (125 µM) was immediately added, and the solution was left at room temperature for 3 h and dialyzed as described above.

The protein was loaded with both phosphate and iron by using the method described by Aitkin-Rogers et al. for bacterioferritin (2). Solutions containing 1 mM and 5 mM phosphate were prepared using potassium phosphate (monobasic) in 20 mM Tris-HCl (pH 7.0)-150 mM NaCl-0.1 mM EDTA, and to both solutions 300 µg of Dps was added and incubated for 5 min. Then 500 µM iron(II) was added, and the solutions were incubated for 3 h.

After the protein solutions were dialyzed, they were washed with a solution containing 50 mM EDTA and 100 mM sodium oxalate (pH 7) to remove unbound iron. This was done by adding 500 µl of the EDTA-oxalate solution to concentrated protein, followed by 10 min of incubation. The EDTA-oxalate solution was removed by centrifugation at 3,313 x g for 30 min in a Centricon 10 tube. Then 1 ml of 50 mM Tris-HCl (pH 7.9) buffer was added and centrifuged for an additional 30 min to remove any remaining EDTA-oxalate solution.

Fe and phosphate analysis.
The iron concentration was determined chemically by the method of Stookey, modified for protein use (43). Protein (10 µl) was digested with 1 M HCl (50 µl) for 1 h and neutralized with 1 M NaOH (50 µl). After neutralization, 500 µl of 2 M sodium dithionite was added, and the preparation was incubated for 30 min. Finally, 250 µl of 0.004 M ferrozine and 250 µl of 0.5 M sodium acetate buffer were added, and the solution was left overnight. The absorbance at 562 nm of the samples was determined with a UV-visible spectrophotometer. Phosphate was measured using the malachite green method, as described by Linge and Oldham and modified for protein use (28). Protein (20 µl) was digested with 50 µl of 1 M HCl for 1 h and neutralized with 50 µl of 1 M NaOH. Then 1 ml of the malachite green reagent was added, and the preparation was incubated at room temperature for 30 min. The absorbance at 625 nm was determined with a UV-visible spectrophotometer.

Fe K-edge X-ray absorption.
Fe K near-edge X-ray absorption (XANES) spectra were collected at beamline 11-2 at the Stanford Synchrotron Radiation Laboratory, Stanford, CA, using an Si (220) monochromator crystal. The incident photon flux was measured using an ion chamber filled with N2. The XANES spectra of all samples and standards were collected in fluorescence mode using a 30-element Ge detector. The standards were also examined in transmission mode to minimize saturation effects in the fluorescence yield spectra. All spectra were normalized to the incident photon flux, and their energies were calibrated with respect to the pre-edge peaks in goethite with the high-energy pre-edge transition set to 7,101.1 eV. The slits were set to 6 x 1 mm to obtain high-resolution XANES spectra.

Nucleotide sequence accession number.
The nucleotide sequence encoding Dpstery has been deposited in the EMBL nucleotide sequence database under accession number AJ875220. The same sequence (contig 414 gene 1836) has been annotated in the Trichodesmium genome as a Dps-like protein gene (http://jgi.doe.gov).


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RESULTS AND DISCUSSION
 
Identification of iron storage proteins.
We searched the previously published genome of T. erythraeum IM101 (http://www.jgi.doe.gov) and identified two genes, designated genes A and B, that encode amino acid sequences homologous to known bacterioferritin sequences (Fig. 1). Gene A exhibits sequence similarity with bacterioferritin genes from Azotobacter vinelandii (13%), Pseudomonas putida (17%), Synechocystis sp. strain PCC 6803 (14%), and Desulfovibrio desulfuricans (18%). Analysis of an alignment of the sequence encoded by Trichodesmium gene A with bacterioferritin sequences revealed conservation of the amino acid residues that act as ligands to the ferroxidase center (5). Amino acids Glu-18, Glu-50, His-54, Glu-94, Glu-127, and His-130, which form the ferroxidase center in A. vinelandii, are conserved in the gene A product as Glu-23, Glu-55, His-59, Glu-100, Glu-132, and His-135 (5). However, the gene A product lacks the heme-methionine ligand that is conserved in all bacterioferritins, which indicates that it is likely a bacterial ferritin instead.


Figure 1
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  		FIG. 1. Sequence alignment for bacterioferritins and a homologous protein in T. erythraeum (encoded by gene A). The residues involved in metal binding are indicated by stars, and the methionine heme ligand is underlined. The bacterioferritin sequences of A. vinelandii (BFR_Azotobacter), P. putida (BFR_Pseudomonas), Synechocystis sp. strain PCC 6803 (BFR_Synechocystis), and D. desulfuricans (BFR_Desulfuricans) were used to identify the homologous protein in Trichodesmium (BFR_Trichodesmium). Residues identical in all sequences are shown with a black background, and conserved residues are indicated by a gray background.

Gene B codes for a sequence consisting of 180 amino acids (molecular mass, ~20.23 kDa) with sequence identity to known bacterioferritin sequences of Synechocystis (19%), P. putida (19%), D. desulfuricans (15%), and A. vinelandii (13%). Although identified with bacterioferritin sequences, the protein encoded by this gene does not contain all the amino acids residues involved in the di-iron ferroxidase coordination site in bacterioferritins. A comparison of the gene B product with known Dps proteins revealed high levels of similarity with members of the Dps family (Fig. 2). Gene B thus codes for a putative Dps protein, which we designated Dpstery; this protein exhibits 69% primary amino acid sequence identity with Synechococcus DpsA, 32% primary amino acid sequence identity with E. coli Dps, and 30% primary amino acid sequence identity with Listeria innocua Flp.


Figure 2
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  		FIG. 2. Alignment of the Dpstery sequence with known and putative Dps sequences. Residues identical in all the sequences are indicated by a black background, and conserved residues are indicated by a gray background. Amino acid residues that are red are the residues known to be involved in metal binding in Listeria ferritin (Listeria_flp), and these residues are aligned with homologous positions in Dpstery (Tricho_Dps) Crocosphaera watsonii putative Dps (Crocosphaera), Synechococcus sp. strain PCC 7942 DpsA (Synp7_DspA), and E. coli Dps (Ecoli_Dps).

Sequence alignment of Dpstery and other proteins revealed conservation of the amino acid motifs that are thought to be involved in the formation of the intersubunit dinuclear ferritin-like ferroxidase center in Dps proteins (Fig. 2). In the crystal structure of the Flp protein from L. innocua, a member of the Dps family, 12 iron atoms have been observed occupying the putative ferroxidase centers. The amino acids involved in the coordination of iron are His-31, His-43, Asp-47, Asp-58, and Glu-62 (22). Equivalent amino acid residues were found in Dpstery (His-53, His-65, Glu-70, Glu-81, and Glu-84). Recent work in vivo has shown that the amino acids involved with the putative ferroxidase center are crucial for the incorporation of iron. Site-directed mutagenesis of the negatively charged amino acids, Asp-74 and Glu-78, with Ala prevented iron incorporation by a Dps homologue in Streptococcus suis (37).

Amplification and cloning of Dpstery.
After overexpression of the putative Dpstery in E. coli, cell extracts were subjected to SDS-PAGE analysis. Figure 3 shows the presence of the expected 20-kDa band after induction of E. coli harboring pRSET-dps with IPTG. Examination of an 8% native PAGE gel containing the recombinant protein purified by using a ferritin purification protocol revealed a protein whose molecular mass is less than that of horse spleen ferritin (~450 kDa) but more than that of the bovines serum albumin dimer (~132 kDa) (Fig. 4). This result indicates that the native recombinant protein is not composed of 24 subunits and likely consists of 12 subunits (~240 kDa). On the basis of the amino acid sequence, we calculated a molecular mass of 20.23 kDa for one subunit and a molecular mass of ~242.76 kDa for the native protein.


Figure 3
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  		FIG. 3. Production of recombinant protein after induction of E. coli harboring pRSET-dps. A 12% SDS-PAGE gel was used to determine the protein production by two independent E. coli cultures before (lanes 1a and 2a) and after (lanes 1b and 2b) the addition of 0.4 mM IPTG. Lane 3 contained a protein marker. After induction a band at a molecular mass of ~20 kDa was present (lanes 1b and 2b).


Figure 4
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  		FIG. 4. Nondenaturing gel electrophoresis of Dpstery (lane 2), horse spleen ferritin (lane 1), and bovine serum albumin as a monomer and dimer (lane 3) on a 10% native PAGE gel.

Fe binding.
The iron binding ability of Dps proteins (or Dps homologs) has been confirmed with proteins isolated from E. coli, L. innocua, Mycobacterium smegmatis, Campylobacter jejuni, and Streptococcus mutans (19, 23, 42, 52, 54). Thus, we investigated the iron binding ability of Dpstery. E. coli cells with the pRSET (but not Dps) vector were grown in the same medium as a negative control. Upon exposure of the native PAGE gel to a phosphorimager, we observed the radiolabel iron in Dpstery, as expected. No 55Fe was observed in any of the protein bands from the control cells, demonstrating that the result obtained for Dpstery was not due to the presence of Dps from E. coli or to nonspecific iron binding (Fig. 5). The intense 55Fe band seen in the phosphorimage reflected the high iron binding capacity of Dpstery.


Figure 5
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  		FIG. 5. Iron binding ability of Dpstery. Protein extracts of cells grown in the presence of 55FeCl3 were resolved on a 10% native PAGE gel, stained with Coomassie blue (A), and then exposed to a phosphorimager to detect radiolabeled iron (B). The radiolabeled iron was detected in the cells that overexpressed the Dpstery protein (lane 2) and not in cells that lacked the pRSET-dps vector (lane 1).

The iron storage capacity of Dpstery was quantified by incubating the protein with ferrous ammonium sulfate in the presence of O2 or H2O2. We found 260 ± 20 Fe atoms/protein molecule in the presence of O2 and 270 ± 10 Fe atoms/protein molecule in the presence of H2O2. Because the maximum incorporation of Fe by Dps proteins has been reported to be 500 atoms per molecule, a solution of Dpstery that contained 260 Fe atoms per molecule was reincubated with a ferrous ammonium sulfate solution (7, 53). The additional incubation did not increase the incorporation of Fe by the protein (260 ± 40 Fe atoms/protein molecule). In E. coli Dps, H2O2 has been shown to be a more effective oxidant of Fe(II) than O2, but in Dpstery the maximum iron capacity remained the same regardless of the oxidant used (21). We do not know if the lower iron capacity that we observed in Dpstery than in Dps from E. coli corresponds to an intrinsic difference between the proteins or to a lack of optimization in our Fe loading protocol.

Incorporation of phosphate.
Although phosphate incorporation into the iron core of ferritins and bacterioferritins has been reported, until now the possibility that Dps proteins may contain phosphate has not been studied (46, 48). Purified Dpstery obtained from overexpression in E. coli was found to contain 10 ± 1 Pi molecules/protein molecule. When Dpstery was incubated with both ferrous ammonium sulfate and 1 mM or 5 mM potassium phosphate loading values of 66.4 ± 0.5 and 50 ± 4 Pi molecules/protein molecule were obtained, showing that the core has a Pi/Fe ratio of about 1:4. In horse spleen ferritin a Pi/Fe ratio of 1:8 has been reported, while in bacterioferritins Pi/Fe ratios between 1 and 2 have been observed (4, 46). Phosphate content is currently thought to affect the structure and size of the iron core in ferritins and bacterioferritins, and there is evidence that phosphate influences the availability of iron; however, the overall role of phosphate in the biochemistry of the protein is poorly understood (25, 45, 46). Nevertheless, the incorporation of phosphate into the iron core in Dpstery provides additional evidence for the ferritin-like properties of the core in Dps proteins.

DNA binding.
An unusual feature of Dps compared with other ferritin family proteins is that it binds DNA. This has been shown in E. coli, Porphyromonas gingivalis, Synechococcus, and M. smegmatis (3, 19, 36, 47). (Dps-like proteins composed of 12 subunits that do not appear to bind DNA, such as the protein found in L. innocua, may logically be classified as a separate type of ferritin.) To determine whether Dpstery binds DNA, we performed a gel mobility shift assay using the E. coli pUC19 plasmid as a template. Incubation of pUC19 DNA with Dpstery at 37°C for 30 min decreased the mobility of all the DNA bands on the agarose gel, and this effect was exaggerated at higher concentrations of protein (Fig. 6). Most strikingly, a large fraction of the DNA remained stationary and did not enter the agarose. For determination of the apparent dissociation constant (kd) of DNA with Dpstery, pUC19 (0.15 nM) was titrated with Dpstery, the products were resolved by agarose gel electrophoresis, and the bands were quantified using IMAGE J (1). The apparent kd of Dpstery calculated by measuring the protein concentration that resulted in 50% binding of the DNA was about 16 µM (Fig. 7), which is nearly 100-fold higher than the constant reported for the Dps protein from E. coli (kd, ~172 to 178 nM) (6, 10). Until more is known about the affinities of various Dps proteins for DNA, it is difficult to speculate on the meaning of this large apparent difference between the proteins from T. erythraeum and E. coli.


Figure 6
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  		FIG. 6. Binding of DNA by Dpstery. Dpstery was incubated with E. coli plasmid pUC19 for 30 min at 30°C in 50 mM Tris-HCl (pH 7.9)-50 mM NaCl. For supercoiled pUC19 DNA alone (lane 1), the different bands represent 100% coiled, 80% coiled, and nicked DNA. Lanes 2, 3, and 4 contained the Dpstery-DNA complex at DNA/protein molar ratios of 1:500, 1:800, and 1:103, respectively. Incubation of DNA with Dps led to the formation of a Dps-DNA complex that did not enter the agarose gel and remained in the well of the gel.


Figure 7
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  		FIG. 7. Dissociation constant of pUC19 for Dpstery. pUC19 (0.15 nM) was incubated with various concentration of Dpstery at 30°C for 30 min, and the products were resolved in a 1% agarose gel. Measurements were determined twice for each combination, and the averages were plotted. The standard deviation was less than 2%. The kd was determined by the method of Carey (10).

DNA protection.
Dps-DNA complexes have been shown to be extremely stable; in addition, DNA binding stabilizes the Dps structure (3). It has been shown that once a DNA-Dps complex is formed, the DNA is protected from attack by various nucleases, such as DNase I (19). To test the ability of Dpstery to protect DNA, we incubated pUC19 DNA with Dps for 30 min and then added DNase I. Nonincubated pUC19 DNA and pUC19 incubated with DNase I were used as controls. All samples were separated on a 1% agarose gel (Fig. 8). As Fig. 8, lanes 1 and 2, show, incubation of pUC19 DNA with DNase I resulted in complete degradation of the nucleic acid, and there were no visible bands on the gel. In contrast, DNA that was preincubated with Dpstery produced an intense band that remained in the loading well. Thus, the DNA-Dpstery complex appeared to be effectively protected from degradation by DNase I.


Figure 8
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  		FIG. 8. Dpstery-DNA complex protects DNA from DNase digestion. Lane 1 contained pUC19 plasmid DNA alone, and lane 2 contained pUC19 treated with 1 U of DNase I. The products obtained from incubation of DNA with Dpstery at DNA/protein molar ratios of 1:500 (lane 3), 1:800 (lane 4), and 1:103 (lane 5) were treated with DNase I. The DNA-Dpstery complexes remained intact after incubation with DNase and were visible in the wells of the 1% agarose gel.

Fe K near-edge X-ray absorption spectra.
Despite identification of Dps proteins in numerous microorganisms, little is known about the structure of the Fe core. Figure 9 shows the X-ray absorption spectra of horse spleen ferritin and Dpstery iron cores. The spectra of ferritin and Dpstery are nearly identical and have similar pre-edge features. The weak pre-edge observed at ~7,100 eV corresponds to the 1s -> 3d quadrupole-allowed, dipole-forbidden metal electronic transitions characteristic of octahedrally coordinated iron (49, 50). The energy and intensity of the 1s -> 3d transition are sensitive to the coordination environment and oxidation state of iron (49). Previous work has shown that the Fe K-edge 1s -> 3d pre-edge feature in the ferritin iron core observed at ~7,100 eV corresponds to high-spin ferric iron coordinated with six oxygen atoms (33). As the pre-edge feature of Dpstery is similar to that of horse spleen ferritin, we concluded that the iron core in Dpstery is composed of ferric iron that is octahedrally coordinated. Prior work has demonstrated that the chemistry of the Dps core from E. coli is similar to that of ferritin, and our XANES data also demonstrated that the core compositions of Dpstery and ferritin are similar (21).


Figure 9
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  		FIG. 9. Near-edge spectra of the iron K-edge of horse spleen ferritin (spectrum a) and Dpstery (spectrum b). The pre-edge feature of ferritin at ~7,100 eV is characteristic of ferric iron octahedrally coordinated to oxygen, and a similar pre-edge feature was observed for Dpstery.

Potential role of Dps proteins in the marine environment.
The Dpstery that we isolated is homologous to previously described Dps proteins and has a similar molecular mass. Dpstery also appears to have all the properties ascribed to such proteins; it binds iron, binds DNA, and protects DNA from degradation. Genomic analysis revealed that genes encoding Dps homologues are present in the genomes of Prochlorococcus sp. strain MIT 9313 and Crocosphaera watsonii, two of the few marine microorganisms that have been sequenced so far. All three of the functions that we demonstrated for Dpstery may be useful to photorophs that live in the surface ocean. Such organisms must survive in environments where Fe inputs are low or episodic and they are subjected to oxidative stress because of the presence of molecular oxygen and intense sunlight. These organisms may also need to protect their genetic material when they survive in some dormant form during low-nutrient periods or when they are advected out of the photic zone.

The problem of photoxidative damage may be particularly severe for Trichodesmium, which accumulates at the surface of the ocean during blooms. Along with carotenoids and other UV-absorbing compounds, the Dps protein identified in Trichodesmium may be part of an effective protective mechanism (11, 44). Mutants of Synechocystis, Synechococcus, and E. coli that lack the dps genes are extremely sensitive to photooxidative stress and peroxide (16, 27, 32). The protection provided by Dps against degradation by reactive oxygen species probably contributes to the survival of Trichodesmium, which must carry out oxygenic photosynthesis in the presence of intense sunlight.


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ACKNOWLEDGMENTS
 
This work was supported by the Center for Environmental Bioinorganic Chemistry (CHE 0221978).


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FOOTNOTES
 
* Corresponding author. Mailing address: Princeton University, 153A Guyot Hall, Princeton, NJ 08540. Phone: (609) 258-1052. Fax: (609) 258-5242. E-mail: mcastrui{at}princeton.edu. Back


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Applied and Environmental Microbiology, April 2006, p. 2918-2924, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2918-2924.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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