Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy

Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe²⁺ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.     

The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage: x-ray crystal structure,iron binding,and hydroxyl-radical scavenging properties

Agrobacterium tumefaciens Dps (DNA-binding proteins from starved cells), encoded by the dps gene located on the circular chromosome of this plant pathogen, was cloned, and its structural and functional properties were determined in vitro. In Escherichia coli Dps, the family prototype, the DNA binding properties are thought to be associated with the presence of the lysine-containing N-terminal tail that extends from the protein surface into the solvent. The x-ray crystal structure of A. tumefaciens Dps shows that the positively charged N-terminal tail, which is 11 amino acids shorter than in the E. coli protein, is blocked onto the protein surface. This feature accounts for the lack of interaction with DNA. The intersubunit ferroxidase center characteristic of Dps proteins is conserved and confers to the A. tumefaciens protein a ferritin-like activity that manifests itself in the capacity to oxidize and incorporate iron in the internal cavity and to release it after reduction. In turn, sequestration of Fe(II) correlates with the capacity of A. tumefaciens Dps to reduce the production of hydroxyl radicals from H2O2 through Fenton chemistry. These data demonstrate conclusively that DNA protection from oxidative damage in vitro does not require formation of a Dps-DNA complex. In vivo, the hydroxyl radical scavenging activity of A. tumefaciens Dps may be envisaged to act in concert with catalase A to counteract the toxic effect of H2O2, the major component of the plant defense system when challenged by the bacterium.     

Overexpression and characterization of an iron storage and DNA-binding Dps protein from Trichodesmium erythraeum

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 N(2)-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 Dps(T. erythraeurm) protein (Dps(tery)) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps(tery), and we found that Dps(tery), like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps(tery) binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps(tery) indicated that it has an iron core that resembles that of horse spleen ferritin.     

Antioxidant Dps protein from the thermophilic cyanobacterium Thermosynechococcus elongatus

DNA-binding proteins from starved cells (Dps proteins) protect bacteria primarily from oxidative damage. They are composed of 12 identical subunits assembled with 23-symmetry to form a compact cage-like structure known to be stable at temperatures > 70 degrees C and over a wide pH range. Thermosynechococcus elongatus Dps thermostability is increased dramatically relative to mesophilic Dps proteins. Hydrophobic interactions at the dimeric and trimeric interfaces called Dps-like are replaced by salt bridges and hydrogen bonds, a common strategy in thermophiles. Moreover, the buried surface area at the least-extended Dps-like interface is significantly increased. A peculiarity of T. elongatus Dps is the presence of a chloride ion coordinated with threefold symmetry-related arginine residues lining the opening of the Dps-like pore toward the internal cavity. T. elongatus Dps conserves the unusual intersubunit ferroxidase centre that allows the Dps protein family to oxidize Fe(II) with hydrogen peroxide, thereby inhibiting free radical production via Fenton chemistry. This catalytic property is of special importance in T. elongatus (which lacks the catalase gene) in the protection of DNA and photosystems I and II from hydrogen peroxide-mediated oxidative damage.     

Helicobacter hepaticus Dps protein plays an important role in protecting DNA from oxidative damage

The ferritin-like DNA-binding protein from starved cells (Dps) family proteins are present in a number of pathogenic bacteria. Dps in the enterohepatic pathogen, Helicobacter hepaticus is characterized and a H. hepaticus dps mutant was generated by insertional mutagenesis. While the wild type H. hepaticus cells were able to survive in an atmosphere containing up to 6.0% O₂, the dps mutant failed to grow in 3.0% O₂, and it was also more sensitive to oxidative reagents like H₂O₂, cumene hydroperoxide and t-butyl hydroperoxide. Upon air exposure, the dps^-  cells had more damaged DNA than the wild type; they became coccoid or lysed and they contained ∼6-fold higher amount of 8-oxoguanine (8-oxoG) DNA lesions than wild type cells. Purified H. hepaticus Dps was shown to be able to bind both iron and DNA. The iron-loaded form of Dps protein had much greater DNA binding ability than the native Dps or the iron-free Dps.     

Structural studies on the second Mycobacterium smegmatis Dps: invariant and variable features of structure, assembly and function

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.     

Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.

Reactive oxygen species can damage most cellular components, but DNA appears to be the most sensitive target of these agents. Here we present the first evidence of DNA protection against the toxic and mutagenic effects of oxidative damage in metabolically active cells: direct protection of DNA by Dps, an inducible nonspecific DNA-binding protein from Escherichia coli. We demonstrate that in a recA-deficient strain, expression of Dps from an inducible promoter prior to hydrogen peroxide challenge increases survival and reduces the number of chromosomal single-strand breaks. dps mutants exhibit increased levels of the G x C-->T x A mutations characteristic of oxidative damage after treatment with hydrogen peroxide. In addition, expression of Dps from the inducible plasmid reduces the frequency of spontaneous G x C-->T x A and A x T-->T x A mutations and can partially suppress the mutator phenotype of mutM (fpg) and mutY alleles. In a purified in vitro system, Dps reduces the number of DNA single-strand breaks and Fpg-sensitive sites introduced by hydrogen peroxide treatment, indicating that the protection observed in vivo is a direct effect of DNA binding by Dps. The widespread conservation of Dps homologs among prokaryotes suggests that this may be a general strategy for coping with oxidative stress.     

Sensitization of D-glucuronic acid transport system of E. coli to protein group reagents in presence of substrate or absence of energy source

N-ethylmaleimide and 1-fluoro-2,4-dinitrobenzene inactivate D-glucuronic acid transport in $\text{E. coli}$ K12. The inactivation is highly enhanced by the two substrates of the transport system, D-glucuronate and D-galacturonate, or by inhibitors of respiratory energy production. The significance of these results is discussed in the framework of a model of a mobile carrier which can exist in two or more distinct conformational states.     

Structure, function and regulation of the DNA-binding protein Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review

Dps, the DNA‐binding protein from starved cells, is capable of providing protection to cells during exposure to severe environmental assaults; including oxidative stress and nutritional deprivation. The structure and function of Dps have been the subject of numerous studies and have been examined in several bacteria that possess Dps or a structural/functional homologue of the protein. Additionally, the involvement of Dps in stress resistance has been researched extensively as well. The ability of Dps to provide multifaceted protection is based on three intrinsic properties of the protein: DNA binding, iron sequestration, and its ferroxidase activity. These properties also make Dps extremely important in iron and hydrogen peroxide detoxification and acid resistance as well. Regulation of Dps expression in E. coli is complex and partially dependent on the physiological state of the cell. Furthermore, it is proposed that Dps itself plays a role in gene regulation during starvation, ultimately making the cell more resistant to cytotoxic assaults by controlling the expression of genes necessary for (or deleterious to) stress resistance. The current review focuses on the aforementioned properties of Dps in E. coli, its prototypic organism. The consequences of elucidating the protective mechanisms of this protein are far‐reaching, as Dps homologues have been identified in over 1000 distantly related bacteria and Archaea. Moreover, the prevalence of Dps and Dps‐like proteins in bacteria suggests that protection involving DNA and iron sequestration is crucial and widespread in prokaryotes.     

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.     

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

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 Dps_{T. erythraeurm} protein (Dps_tery) with 69% primary amino acid sequence similarity to Synechococcus DpsA. We expressed and purified Dps_tery, and we found that Dps_tery, like other Dps proteins, is able to bind Fe and DNA and protect DNA from degradation by DNase. We also found that Dps_tery binds phosphate, like other ferritin family proteins. Fe K near-edge X-ray absorption of Dps_tery indicated that it has an iron core that resembles that of horse spleen ferritin.     

Dps proteins prevent Fenton-mediated oxidative damage by trapping hydroxyl radicals within the protein shell

Dps (DNA-binding proteins from starved cells) proteins belong to a widespread bacterial family of proteins expressed under nutritional and oxidative stress conditions. In particular, Dps proteins protect DNA against Fenton-mediated oxidative stress, as they catalyze iron oxidation by hydrogen peroxide at highly conserved ferroxidase centers and thus reduce significantly hydroxyl radical production. This work investigates the possible generation of intraprotein radicals during the ferroxidation reaction by Escherichia coli and Listeria innocua Dps, two representative members of the family. Stopped-flow analyses show that the conserved tryptophan and tyrosine residues located near the metal binding/oxidation center are in a radical form after iron oxidation by hydrogen peroxide. DNA protection assays indicate that the presence of both residues is necessary to limit release of hydroxyl radicals in solution and the consequent oxidative damage to DNA. In general terms, the demonstration that conserved protein residues act as a trap that dissipates free electrons generated during the oxidative process brings out a novel role for the Dps protein cage.     

Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity.

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid. We expressed and purified 12 species of the DNA-binding protein, i.e. CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td(-) phenotype A). The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays. Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities. Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA. From the apparent dissociation constant (K(d)) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nM (HU binding to the non-curved DNA) to 250 nM (Hfq binding to the non-curved DNA), under the assay conditions employed.     

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation.     

Dps proteins, an efficient detoxification and DNA protection machinery in the bacterial response to oxidative stress

The proteins belonging to the Dps (DNA-binding proteins from starved cells) family play an important role within the bacterial defence system against oxidative stress. They act on Fe(II) and hydrogen peroxide that are potentially toxic in the presence of air. Fe(II) forms spontaneously insoluble Fe(III) and reacts with molecular oxygen or its reduced forms to yield the highly damaging hydroxyl radicals. All Dps proteins have the distinctive capacity to annul the toxic combination of iron and hydrogen peroxide as they use the latter compound to oxidise Fe(II). In addition to this intrinsic DNA protection capacity, several members of the family, including the archetypical Escherichia coli Dps, protect DNA physically by shielding it in large Dps-DNA complexes. The structural and functional characteristics that endow Dps proteins with the chemical and physical protection mechanism are presented and discussed also in the framework of the varied situations that may be encountered in different bacterial species.      

The iron-binding protein Dps confers hydrogen peroxide stress resistance to Campylobacter jejuni

We identified and characterized the iron-binding protein Dps from Campylobacter jejuni. Electron microscopic analysis of this protein revealed a spherical structure of 8.5 nm in diameter, with an electron-dense core similar to those of other proteins of the Dps (DNA-binding protein from starved cells) family. Cloning and sequencing of the Dps-encoding gene (dps) revealed that a 450-bp open reading frame (ORF) encoded a protein of 150 amino acids with a calculated molecular mass of 17,332 Da. Amino acid sequence comparison indicated a high similarity between C. jejuni Dps and other Dps family proteins. In C. jejuni Dps, there are iron-binding motifs, as reported in other Dps family proteins. C. jejuni Dps bound up to 40 atoms of iron per monomer, whereas it did not appear to bind DNA. An isogenic dps-deficient mutant was more vulnerable to hydrogen peroxide than its parental strain, as judged by growth inhibition tests. The iron chelator Desferal restored the resistance of the Dps-deficient mutant to hydrogen peroxide, suggesting that this iron-binding protein prevented generation of hydroxyl radicals via the Fenton reaction. Dps was constitutively expressed during both exponential and stationary phase, and no induction was observed when the cells were exposed to H(2)O(2) or grown under iron-supplemented or iron-restricted conditions. On the basis of these data, we propose that this iron-binding protein in C. jejuni plays an important role in protection against hydrogen peroxide stress by sequestering intracellular free iron and is expressed constitutively to cope with the harmful effect of hydrogen peroxide stress on this microaerophilic organism without delay.     

Spatial organization of Dps and DNA–Dps complexes

DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA–Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA–Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA–Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA–Dps complexes. Angle analysis has demonstrated that in DNA–Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet.     

The neutrophil-activating Dps protein of Helicobacter pylori, HP-NAP, adopts a mechanism different from Escherichia coli Dps to bind and condense DNA

The Helicobacter pylori neutrophil-activating protein (HP-NAP), a member of the Dps family, is a fundamental virulence factor involved in H.pylori-associated disease. Dps proteins protect bacterial DNA from oxidizing radicals generated by the Fenton reaction and also from various other damaging agents. DNA protection has a chemical component based on the highly conserved ferroxidase activity of Dps proteins, and a physical one based on the capacity of those Dps proteins that contain a positively charged N-terminus to bind and condense DNA. HP-NAP does not possess a positively charged N-terminus but, unlike the other members of the family, is characterized by a positively charged protein surface. To establish whether this distinctive property could be exploited to bind DNA, gel shift, fluorescence quenching and atomic force microscopy (AFM) experiments were performed over the pH range 6.5–8.5. HP-NAP does not self-aggregate in contrast to Escherichia coli Dps, but is able to bind and even condense DNA at slightly acid pH values. The DNA condensation capacity acts in concert with the ferritin-like activity and could be used to advantage by H.pylori to survive during host-infection and other stress challenges. A model for DNA binding/condensation is proposed that accounts for all the experimental observations.     

Comparison of the kinetics of iron release from a marine (Trichodesmium erythraeum) Dps protein and mammalian ferritin in the presence and absence of ligands

The ferritin superfamily of iron storage proteins includes ferritin proper and Dps (DNA binding protein from starved cells) along with bacterioferritin. We examined the release of Fe from the Dps of Trichodesmium erythraeum (Dps(tery)) and compared it to the release of Fe from horse spleen ferritin (HoSF) under various conditions. Both desferrioxamine B (DFB), a Fe(III) chelator, and ascorbic acid were able to mobilize Fe from Dps(tery) at rates comparable to those observed for HoSF. The initial Fe release rate from both proteins increased linearly with the concentration of DFB, suggesting that the chelator binds to Fe in the protein. A small but significant rate obtained by extrapolation to zero concentration of DFB implies that Dps(tery) and HoSF might release Fe(III) spontaneously. A similar result was observed for HoSF in the presence of sulfoxine. In a different experiment, Fe(III) was transferred from holoferritin to apotransferrin across a dialysis membrane in the absence of chelator or reducing agent. The apparent spontaneous release of Fe from HoSF and Dps(tery) brings forth the hypothesis that the Fe core in Fe storage proteins might be continuously dissolving and re-precipitating in vivo, thus maintaining it in a highly reactive and bioavailable form.