Ferritin-like Proteins Research Articles

The two Dps of Edwardsiella tarda are involved in resistance against oxidative stress and host infection

DNA-binding protein from starved cells (Dps) is a member of ferritin-like proteins that exhibit properties of nonspecific DNA binding and iron oxidation and storage. Although studies of Dps from many bacterial species have been reported, no investigations on Dps from fish pathogens have been documented. In this study, we examined the biological function of two Dps proteins, Dps1 and Dps2, from Edwardsiella tarda, an important fish bacterial pathogen that can also infect humans. Dps1 and Dps2 are, respectively, 163- and 174-residue in length and each contains the conserved ferroxidase center of Dps. Expression of dps1 and dps2 was growth phase-dependent and reached high levels in stationary phase. Purified recombinant Dps1 and Dps2 were able to mediate iron oxidation by H 2O 2 and bind DNA. Compared to the wild type strain, (i) the dps1 mutant (TXDps1) and the dps2 mutant (TXDps2) were unaffected in growth, while the dps2 mutant with interfered dps1 expression (TXDps2RI) exhibited a prolonged lag phase; (ii) TXDps1, TXDps2, and especially TXDps2RI were significantly reduced in H 2O 2 and UV tolerance and impaired in the capacity to invade into host tissues and replicate in head kidney macrophages; (iii) TXDps1, TXDps2, and TXDps2RI induced stronger macrophage respiratory burst activity and thus were defective in the ability to block the bactericidal response of macrophages. Taken together, these results indicate that Dps1 and Dps2 are functional analogues that possess ferroxidase activity and DNA binding capacity and are required for optimum oxidative stress resistance and full bacterial virulence.

Antibody targeting the ferritin-like protein controls Listeria infection.

The acquisition of iron during the infection process is essential for the growth of pathogenic microorganisms (S. C. Andrews, Adv. Microb. Physiol. 40:281-351, 1998; H. M. Baker, B. F. Anderson, and E. N. Baker, Proc. Natl. Acad. Sci. U. S. A. 100:3579-3583, 2003). Since the solubility of iron is low and it is toxic at low concentrations, following uptake, iron is stored in subcellular microenvironments in the iron storage protein ferritin (C. Cheers and M. Ho, J. Reticuloendothel. Soc. 34:299-309, 1983). Here, we show that ferritin-like proteins (Frl) are highly conserved in the genus Listeria and demonstrate that these proteins are present in both the cytoplasm and cell wall fractions of these bacteria. Even though Frl is expressed under different growth conditions, transcriptional mapping revealed that its regulation is complex. When bacteria are grown in brain heart infusion medium, extracellular expression involves both sigma A (SigA)- and sigma B (SigB)-dependent promoters; however, during intracellular growth, initiation of transcription is additionally SigB dependent. The expression of Frl is greatly enhanced in bacteria grown in the presence of blood, and a mutant strain lacking the frl gene was defective for growth in this medium. Using the monoclonal antibody (MAb) specific for Frl, we demonstrate that administration of anti-Frl MAb prior to infection confers antilisterial resistance in vivo, evidenced in reduced bacterial load and increased survival rates, thereby demonstrating the in vivo significance of upregulated cell surface-associated Frl expression. In vitro studies revealed that the antilisterial resistance is due to increased listerial phagocytosis.

Structural basis of the zinc- and terbium-mediated inhibition of ferroxidase activity in Dps ferritin-like proteins

Streptococcus suis Dpr is an iron-binding protein involved in oxidative stress resistance. It belongs to the bacterial Dps protein family whose members form dodecameric assemblies. Previous studies have shown that zinc and terbium inhibit iron incorporation in Listeria innocua Dps protein. In order to gain structural insights into the inhibitory effect of zinc and terbium, the crystal structures of Streptococcus suis Dpr complexes with these ions were determined at 1.8 A and 2.1 A, respectively. Both ions were found to bind at the ferroxidase center and in the same location as iron. In addition, a novel zinc-binding site formed by His40 and His44 was identified. Both His residues were found to be present within all known Streptococcus suis Dpr variants and in Streptococcus pneumoniae, Streptococcus gordonii, and Streptococcus sanguinis Dpr proteins. Amino acid sequence alignment of Dpr with other Dps family members revealed that His44 is highly conserved, in contrast to His40. The inhibitory effect of zinc and terbium on iron oxidation in Dpr was studied in vitro, and it was found that both ions at concentrations >0.2 mM almost completely abolish iron binding. These results provide a structural basis for the inhibitory effect of zinc and terbium in the Dps family of proteins, and suggest a potential role of the Dps proteins in zinc detoxification mechanisms involving the second zinc-binding site.

Structural bioinformatics analysis of enzymes involved in the biosynthesis pathway of the hypermodified nucleoside ms2io6A37 in tRNA

TRNAs from all organisms contain posttranscriptionally modified nucleosides, which are derived from the four canonical nucleosides. In most tRNAs that read codons beginning with U, adenosine in the position 37 adjacent to the 3' position of the anticodon is modified to N(6)-(Delta(2)-isopentenyl) adenosine (i(6)A). In many bacteria, such as Escherichia coli, this residue is typically hypermodified to N(6)-isopentenyl-2-thiomethyladenosine (ms(2)i(6)A). In a few bacteria, such as Salmonella typhimurium, ms(2)i(6)A can be further hydroxylated to N(6)-(cis-4-hydroxyisopentenyl)-2-thiomethyladenosine (ms(2)io(6)A). Although the enzymes that introduce the respective modifications (prenyltransferase MiaA, methylthiotransferase MiaB, and hydroxylase MiaE) have been identified, their structures remain unknown and sequence-function relationships remain obscure. We carried out sequence analysis and structure prediction of MiaA, MiaB, and MiaE, using the protein fold-recognition approach. Three-dimensional models of all three proteins were then built using a new modeling protocol designed to overcome uncertainties in the alignments and divergence between the templates. For MiaA and MiaB, the catalytic core was built based on the templates from the P-loop NTPase and Radical-SAM superfamilies, respectively. For MiaB, we have also modeled the C-terminal TRAM domain and the newly predicted N-terminal flavodoxin-fold domain. For MiaE, we confidently predict that it shares the three-dimensional fold with the ferritin-like four-helix bundle proteins and that it has a similar active site and mechanism of action to diiron carboxylate enzymes, in particular, methane monooxygenase (E.C.1.14.13.25) that catalyses the biological hydroxylation of alkanes. Our models provide the first structural platform for enzymes involved in the biosynthesis of i(6)A, ms(2)i(6)A, and ms(2)io(6)A, explain the data available from the literature and will help to design further experiments and interpret their results.

ThedpsGene of Symbiotic “CandidatusLegionella jeonii” inAmoeba proteusResponds to Hydrogen Peroxide and Phagocytosis

To survive in host cells, intracellular pathogens or symbiotic bacteria require protective mechanisms to overcome the oxidative stress generated by phagocytic activities of the host. By genomic library tagging, we cloned a dps (stands for DNA-binding protein from starved cells) gene of the symbiotic "Candidatus Legionella jeonii" organism (called the X bacterium) (dps(X)) that grows in Amoeba proteus. The gene encodes a 17-kDa protein (pI 5.19) with 91% homology to Dps and DNA-binding ferritin-like proteins of other organisms. The cloned gene complemented the dps mutant of Escherichia coli and conferred resistance to hydrogen peroxide. Dps(X) proteins purified from E. coli transformed with the dps(X) gene were in oligomeric form, formed a complex with pBlueskript SKII DNA, and protected the DNA from DNase I digestion and H(2)O(2)-mediated damage. The expression of the dps(X) gene in "Candidatus Legionella jeonii" was enhanced when the host amoeba was treated with 2 mM H(2)O(2) and by phagocytic activities of the host cell. These results suggested that the Dps protein has a function protective of the bacterial DNA and that its gene expression responds to oxidative stress generated by phagocytic activities of the host cell. With regard to the fact that invasion of Legionella sp. into respiratory phagocytic cells causes pneumonia in mammals, further characterization of dps(X) expression in the Legionella sp. that multiplies in a protozoan host in the natural environment may provide valuable information toward understanding the protective mechanisms of intracellular pathogens.

Structure of the DPS-like protein from Sulfolobus solfataricus reveals a bacterioferritin-like dimetal binding site within a DPS-like dodecameric assembly.

The superfamily of ferritin-like proteins has recently expanded to include a phylogenetically distinct class of proteins termed DPS-like (DPSL) proteins. Despite their distinct genetic signatures, members of this subclass share considerable similarity to previously recognized DPS proteins. Like DPS, these proteins are expressed in response to oxidative stress, form dodecameric cage-like particles, preferentially utilize H(2)O(2) in the controlled oxidation of Fe(2+), and possess a short N-terminal extension implicated in stabilizing cellular DNA. Given these extensive similarities, the functional properties responsible for the preservation of the DPSL signature in the genomes of diverse prokaryotes have been unclear. Here, we describe the crystal structure of a DPSL protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Although the overall fold of the polypeptide chain and the oligomeric state of this protein are indistinguishable from those of authentic DPS proteins, several important differences are observed. First, rather than a ferroxidase site at the subunit interface, as is observed in all other DPS proteins, the ferroxidase site in SsDPSL is buried within the four-helix bundle, similar to bacterioferritin. Second, the structure reveals a channel leading from the exterior surface of SsDPSL to the bacterioferritin-like dimetal binding site, possibly allowing divalent cations and/or H(2)O(2) to access the active site. Third, a pair of cysteine residues unique to DPSL proteins is found adjacent to the dimetal binding site juxtaposed between the exterior surface of the protein and the active site channel. The cysteine residues in this thioferritin motif may play a redox active role, possibly serving to recycle iron at the ferroxidase center.

Design of Functional Ferritin-Like Proteins with Hydrophobic Cavities

Ferritin four-helix bundle subunits self-assemble to create a stable multimer with a large central hydrophilic cavity where metal ions bind. To explore the versatility of this reaction vessel, computational design was used to generate cavities with increasingly apolar surface areas inside a dodecameric ferritin-like protein, Dps. Cavity mutants, in which as many as 120 surface accessible hydrophilic residues were replaced with hydrophobic amino acids, were shown to still assemble properly using size-exclusion chromatography and dynamic light scattering measurements. Wild-type Dps exhibited highly cooperative subunit folding and assembly, which was monitored by changes in Trp fluorescence and UV circular dichroism. The hydrophobic cavity mutants showed distinctly less cooperative unfolding behavior, with one mutant forming a partially assembled intermediate upon guanidine denaturation. Although the stability of Dps to such denaturation decreased with increasing apolar surface area, all proteins exhibited high melting temperatures, T(m) = 74-90 degrees C. Despite the large number of mutations, near-native ability to mineralize iron was maintained. This work illustrates the versatility of the ferritin scaffold for engineering large protein cavities with novel properties.

Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus

Oxidative stress is a universal phenomenon experienced by organisms in all domains of life. Proteins like those in the ferritin-like di-iron carboxylate superfamily have evolved to manage this stress. Here we describe the cloning, isolation, and characterization of a Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus ( PfDps-like). Phylogenetic analysis, primary structure alignments and higher order structural predictions all suggest that the P. furiosus protein is related to proteins within the broad superfamily of ferritin-like di-iron carboxylate proteins. The recombinant PfDps protein self-assembles into a 12 subunit quaternary structure with an outer shell diameter of ∼10 nm and an interior diameter of ∼5 nm. Dps proteins functionally manage the toxicity of oxidative stress by sequestering intracellular ferrous iron and using it to reduce H 2O 2 in a two electron process to form water. The iron is converted to a benign form as Fe(III) within the protein cage. This Dps-mediated reduction of hydrogen peroxide, coupled with the protein’s capacity to sequester iron, contributes to its service as a multifunctional antioxidant.

An archaeal antioxidant: Characterization of a Dps-like protein from Sulfolobus solfataricus

Evolution of an oxygenic atmosphere required primordial life to accommodate the toxicity associated with reactive oxygen species. We have characterized an archaeal antioxidant from the hyperthermophilic acidophile Sulfolobus solfataricus. The amino acid sequence of this approximately 22-kDa protein shares little sequence similarity with proteins with known function. However, the protein shares high sequence similarity with hypothetical proteins in other archaeal and bacterial genomes. Nine of these hypothetical proteins form a monophyletic cluster within the broad superfamily of ferritin-like diiron-carboxylate proteins. Higher order structural predictions and image reconstructions indicate that the S. solfataricus protein is structurally related to a class of DNA-binding protein from starved cells (Dps). The recombinant protein self assembles into a hollow dodecameric protein cage having tetrahedral symmetry (SsDps). The outer shell diameter is approximately 10 nm, and the interior diameter is approximately 5 nm. Dps proteins have been shown to protect nucleic acids by physically shielding DNA against oxidative damage and by consuming constituents involved in Fenton chemistry. In vitro, the assembled archaeal protein efficiently uses H2O2 to oxidize Fe(II) to Fe(III) and stores the oxide as a mineral core on the interior surface of the protein cage. The ssdps gene is up-regulated in S. solfataricus cultures grown in iron-depleted media and upon H2O2 stress, but is not induced by other stresses. SsDps-mediated reduction of hydrogen peroxide and possible DNA-binding capabilities of this archaeal Dps protein are mechanisms by which S. solfataricus mitigates oxidative damage.

Dps/Dpr ferritin‐like protein: insights into the mechanism of iron incorporation and evidence for a central role in cellular iron homeostasis in Streptococcus suis

The Dps family members constitute a distinct group of multimeric and ferritin-like iron binding proteins (up to 500 iron atoms/12-mer) that are widespread in eubacteria and archaea and implicated in oxidative stress resistance and virulence. Despite the wealth of structural knowledge, the mechanism of iron incorporation has remained elusive. Here, we provide evidence on Dpr of the swine and human pathogen Streptococcus suis that: (i) iron incorporation proceeds by Fe(II) binding, Fe(II) oxidation and subsequent storage as Fe(III); (ii) Fe(II) atoms enter the 12-mer cavity through four hydrophilic pores; and (iii) Fe(II) atoms are oxidized inside the 12-mer cavity at 12 identical inter-subunit sites, which are structurally different but functionally equivalent to the ferroxidase centres of classical ferritins. We also provide evidence, by deleting and ectopically overexpressing Dpr, that Dpr affects cellular iron homeostasis. The key residues responsible for iron incorporation in S. suis Dpr are well conserved throughout the Dps family. A model for the iron incorporation mechanism of the Dps/Dpr ferritin-like protein is proposed.

The Dps-like protein Fri of Listeria monocytogenes promotes stress tolerance and intracellular multiplication in macrophage-like cells

Members of the ferritin-like Dps protein family are found in a number of bacterial species, where they demonstrate the potential to bind iron, and have been implicated in tolerance to oxidative stress. In this study of the food-borne pathogen Listeria monocytogenes, the fri gene encoding a Dps homologue was deleted, and, compared to wild-type cells, it was found that the resulting mutant was less resistant to hydrogen peroxide, and demonstrated reduced survival following long-term (7-11 days) incubation in laboratory media. In view of this, it is shown that fri gene expression is controlled by the hydrogen peroxide regulator PerR, as well as the general stress sigma factor sigmaB. When fri mutant cells were transferred to iron-limiting conditions, growth was retarded relative to wild-type cells, indicating that Fri may be required for iron storage. This notion is supported by the observation that the L. monocytogenes genome appears not to encode other ferritin-like proteins. Given the role of Fri in resistance to oxidative stress, and growth under iron-limiting conditions, the ability of the fri mutant to infect mice was examined. When injected by the intraperitoneal route, the fri mutant demonstrated a reduced capacity to proliferate in the organs of infected mice relative to the wild-type, whereas when the bacteria were supplied intravenously this effect was mitigated. In addition, the mutant was impaired in its ability to survive and grow in J774.A1 mouse macrophage cells. Thus, the data suggest that Fri contributes to the ability of L. monocytogenes to survive in environments where oxidative stress and low iron availability may impede bacterial proliferation.

The Physiological Role of Ferritin-Like Compounds in Bacteria

Iron, as the ferrous or ferric ion, is essential for the life processes of all eukaryotes and most prokaryotes; however, the element is toxic when in excess of that needed for cellular homeostasis. Ferrous ions can react with metabolically generated hydrogen peroxide to yield toxic hydroxyl radicals that in turn degrade lipids, DNA, and other cellular biomolecules. Mechanisms have evolved in living systems for iron detoxification and for the removal of excess ferrous ions from the cytosol. These detoxification mechanisms involve the oxidation of excess ferrous ions to the ferric state and storage of the ferric ions in ferritin-like proteins.There are at least three types of ferritin-like proteins in bacteria: bacterial ferritin, bacterioferritin, and dodecameric ferritin. These bacterial proteins are related to the ferritins found in eukaryotes. The structure and physical characteristics of the ferritin-like compounds have been elucidated in several bacteria. Unfortunately, the physiological roles of the bacterial ferritin-like compounds have been less thoroughly studied. A few studies conducted with mutants indicated that ferritin-like compounds can protect bacterial cells from iron overload, serve as an iron source when iron is limited, protect the bacterial cells against oxidative stress and/or protect DNA against enzymatic or oxidative attack. There is very little information available concerning the roles that ferritin-like compounds might play in the survival of bacteria in food, water, soil, or eukaryotic host environments.

The Small Heat Shock Protein Cage from Methanococcus jannaschii Is a Versatile Nanoscale Platform for Genetic and Chemical Modification

Nature has provided us with a range of reactive nanoscale platforms, in the form of protein cage architectures such as viral capsids and the cages of ferritin-like proteins. Protein cage architectures have clearly demarcated exterior, interior, and interface surfaces consisting of precisely located chemical functionalities. In the present work, we demonstrate that the small heat shock protein (MjHsp) cage from Methanococcus jannaschii is a new and versatile nanoscale platform whose exterior and interior surfaces are amenable to both genetic and chemical modification. Wild type and genetic mutants of the Hsp cage are shown to react with activated fluorescein molecules in a site specific manner. In addition, the 12 nm Hsp cage serves as a size constrained reaction vessel for the oxidative mineralization of iron, resulting in the formation of monodispersed 9 nm iron oxide nanoparticles. These results demonstrate the utility of the Hsp cage to serve as a nanoscale platform for the synthesis of both soft (orga...

The MrgA Multimeric Complex: a self-assembled nanostructure for inorganic synthesis

AbstractUtilization of protein cages in biomimetic chemistry allows for the deposition of inorganic materials within an organic construct, controlling the size and amount of material deposited within a constrained reaction environment. Previously, protein cages such as viral capsids and ferritins have been used in the constrained synthesis of inorganic materials. The MrgA protein displays a high sequence homology to both bacterial DNA-protecting proteins (Dps) found in many bacterial genera and Ferritin-like proteins (Flp), which have been shown to functionally sequester and store iron in a biologically available form. Here we demonstrate recombinant production, purification and characterization of the MrgA protein and provide evidence that this protein self-assembles to form a multimeric complex. This complex demonstrates characteristics similar to that of ferritin-like proteins such as resistance to iron toxicity, iron incorporation, and resistance to thermal and chemical denaturation. The ability to deposit iron within the putative internal cavity of this protein cage will allow the MrgA complex to be utilized as a spatially constrained reaction vessel for nanomaterial synthesis of other inorganic materials. From a materials science perspective, it will be interesting to see if these organic/inorganic hybrid materials can be harnessed for catalysis, nanomagnetics, and other applications.

Expression, purification and crystallization of Dpr, a ferritin-like protein from the Gram-positive meningitis-associated bacterium Streptococcus suis.

Ferritin-like proteins form a novel family of bacterial proteins with diverse functions, such as DNA binding, iron storage and cell activation. A common structural feature of these proteins is their ability to form spherical dodecamers. Dpr is a ferritin-like protein from the Gram-positive bacterium Streptococcus suis. Full-length and truncated Dpr were expressed and purified as 6xHis-tag fusion proteins. Crystals of truncated Dpr suitable for X-ray diffraction analysis were obtained after the removal of the N-terminal affinity tag by thrombin cleavage. A complete data set to 2.3 A resolution was collected using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 104.3, b = 137.6, c = 142.1 A and 12 molecules in the asymmetric unit.

Characterization of two genes encoding ferritin-like protein in Actinobacillus actinomycetemcomitans.

Two genes encoding ferritin-like protein, designated afnA and afnB, were identified in the upstream region of actX on the Actinobacillus actinomycetemcomitans chromosomal DNA. The actX has been reported to be a regulatory gene homologous to the Escherichia coli fnr, which controls the growth and virulence of A. actinomycetemcomitans under anaerobic conditions. The afnB located 340 bp-upstream from the actX, and the afnA located just 15 bp-upstream from afnB. The afnA and afnB encoded 161 and 165 amino acid residues, respectively, which were similar to ferritin-like proteins of other microorganisms. Western immunoblotting using rabbit antiserum against E. coli ferritin showed these two proteins, which are reactive with the serum with 19-kDa molecular masses, are produced from A. actinomycetemcomitans. The N-terminal amino acid sequences of the two proteins were consequent with those deduced from afnA and afnB. Northern hybridization revealed that the afnA and afnB constituted a bicistronic operon and the accumulation of afnA and afnB mRNA was upregulated under aerobic conditions. These findings suggested that the operon was regulated by the presence of oxygen. The two ferritin-like proteins may have important roles in the adaptation of A. actinomycetemcomitans to oxidative environmental changes.

The main cold shock protein of Listeria monocytogenes belongs to the family of ferritin-like proteins.

The transfer of the food-borne pathogen Listeria monocytogenes from 30 to 5 degrees C was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein has an isoelectric point at pH 5.1 and a molecular mass of about 18 kDa, as observed on two-dimensional gel electrophoresis (2-DE) pattern. Its N-terminal sequence, obtained from the 2-DE spot, shared a complete sequence identity with a Listeria innocua non-heme iron-binding ferritin. The purification of these ferritin-like proteins (Flp) revealed a native molecular mass of about 100-110 kDa which indicates a polypeptide composed of six 18 kDa-subunits. Northern analysis indicated the presence of a 0.8-kb monocistronic mRNA in exponential growing cells and an important increase inflp mRNA amount after a downshift but also an upshift in temperature.

The main cold shock protein of Listeria monocytogenes belongs to the family of ferritin-like proteins

The transfer of the food-borne pathogen Listeria monocytogenes from 30 to 5°C was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein has an isoelectric point at pH 5.1 and a molecular mass of about 18 kDa, as observed on two-dimensional gel electrophoresis (2-DE) pattern. Its N-terminal sequence, obtained from the 2-DE spot, shared a complete sequence identity with a Listeria innocua non-heme iron-binding ferritin. The purification of these ferritin-like proteins (Flp) revealed a native molecular mass of about 100–110 kDa which indicates a polypeptide composed of six 18 kDa-subunits. Northern analysis indicated the presence of a 0.8-kb monocistronic mRNA in exponential growing cells and an important increase in flp mRNA amount after a downshift but also an upshift in temperature.

Isolation, characterisation and expression of the bacterioferritin gene of Rhodobacter capsulatus

The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene ( bfr) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18 174 Da. The molecular mass of the purified protein was estimated to be 18 176.06 ± 0.80 Da by electrospray mass spectrometry. The bfr gene was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli. The amino acids which are involved in haem ligation, and those which provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conserved in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.

Isolation, characterisation and expression of the bacterioferritin gene ofRhodobacter capsulatus

The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene (bfr) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18,174 Da. The molecular mass of the purified protein was estimated to be 18,176. +/ 0.80 Da by electrospray mass spectrometry. The bfr was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli. The amino acids which are involved in haem ligation, and those provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conversed in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.