Dps protein, a ferritin and bacterioferritin-like subfamily protein, has multifaceted roles in four major categories: 1) dodecameric assembly conformation structure for iron storage and homeostasis, 2) DNA protection roles from oxidative damage, 3) bacterial genome packaging as nucleoid protein, and 4) enzymatic activity (Andrews, 2010; Chiancone & Ceci, 2010; Zeth, 2012). It can oxidize iron to prevent oxidative free radicals or form a protein-DNA complex to protect DNA physically (Haikarainen & Papageorgiou, 2010). In addition, Dps can bind DNA without sequence specificity (Calhoun & Kwon, 2010). However, in Chip-seq analysis, the Dps protein of E. coli was reported to combine genomic DNA in a non-random manner (Antipov et al., 2017). DgDps1 (Dgeo_0281) and Dgeo_0257 also showed different DNA-binding specificity in the selected promoter and ORF regions (Fig. 2,4). The self-assembly characteristics and DNA-binding properties of Dps in solution are extensively agglomerated according to co-crystallization and general defense mechanism (Ceci et al., 2004). E. coli Dps has nucleoid clumping activity in Staphylococcus aureus resulting in H2O2 resistance and enzymatic activity (Ushijima et al., 2016). Dps has a ferroxidase activity that can catalyze oxidization of the ferrous irons (Fe2+) to the ferric state (Fe3+). In this process, H2O2 is reduced during oxidation of ferrous and Dps can detoxify H2O2 and protect DNA. Thus, in general, Dps is highly induced by direct treatment with H2O2 or relies on the growth phase in species specificity. Since E. coli Dps was first reported, various Dps has been reported in more than ten bacterial genera: Bacillus, Campylobacter, Lactobacillus, Helicobacter, Mycobacterium, Agrobacterium, Streptococcus, Pseudomonas, Vibrio together with Deinococcus, and several archaeal Dps in Solfolobus solfataricus, Pyrococcus furiosus, and Halobacterium salinarum (Tonello et al., 1999; Wiedenheft et al., 2005; Haikarainen & Papageorgiou, 2010; Ramsay et al., 2006; Huergo et al., 2013; Xia et al., 2017).
Dps expression in E. coli depends heavily on the growth phase (Azam & Ishihama, 1999; Calhoun & Kwon, 2010; Sato et al., 2013). During exponential growth, Dps is up-regulated by OxyR regulator, induced by hydrogen peroxide stress, and activates 𝜎70-RNA polymerase. When exposed to hydrogen peroxide during the stationary period, Dps is induced by RpoS, encoded by 𝜎s and expressed as a dominant protein. When cells are not in oxidative stress, Dps is down-regulated by nucleoid-associated proteins, Fis and H-NS, which prevent initiation of dps transcription by combining adjacent areas within the core dps promoter (Grainger et al., 2008; Calhoun & Kwon, 2010). However, the expression of dps was repressed by the OxyR containing one cysteine residue in the case of D. radiodurans (Chen et al., 2008).
VCO139 protein was strongly induced by exposure to hydrogen peroxide in a Gram-negative bacterium Vibrio cholerae (Xia et al., 2017). This protein encodes a Dps homologous protein and has ROS resistance. OxyR regulates Dps during the exponential phase and by RpoS during the stationary phase. This Dps protein can be expressed relatively without H2O2. However, Dps is dramatically induced in the exponential growth phase but less induced in the stationary growth phase under H2O2. The involvement of Dps in resistance to various environmental stresses has also been reported. For example, exposure of the Δdps strain to high iron levels during starvation resulted in a less viable Δdps strain compared with wild-type. These data suggest that Dps is vital for the survival of bacteria in starved cells due to its reaction to ROS and iron toxicity tolerance. As with V. cholerae, Dps is related to colonization but does not involve toxic gene expression (Xia et al., 2017).
Interestingly, Campylobacter jejuni Dps has unusually high-temperature tolerance and activates DNA binding by ferrous ions. However, this DNA interaction has been inhibited by NaCl and Mg2+ (Huergo et al., 2013; Sanchuki et al., 2015). In the hyperthermophilic Crenarchaeon S. solfataricus, Dps protein was directly induced by H2O2 treatment and ferrous ion depletion (Wiedenheft et al., 2005).
Dps protein forms a dimer or dodecamer structure and binds to DNA (Grove & Wilkinson, 2005). In DrDps1 of D. radiodurans, oligomeric states change depending on the growth phase and presence of DNA. This suggests that DrDps1 oligomeric forms and functional roles may adapt to environmental changes (Santos et al., 2015). On the other hand, the DrDps2 oligomeric form maintains a dodecameric structure without affecting the surrounding environment. DrDps2 is thought to be more selective about iron depending on the cell environment and involved in intracellular metal storage (Reon et al., 2012; Santos et al., 2015).
In this study, the protein form of Dgeo_0281, known as DgDps1, the homologous protein of DrDps1, was not identified in the dodecameric form. The maximum combining in gel filtration and EMSA test was the dimer structure. The DNA binding affinity was Kd 30 µM, half the maximum of the non-specific DNA sequence. Interestingly, the novel Dps protein (Dgeo_0257, called DgDps3) showed higher DNA-affinity than DgDps1 (Dgeo_0281) in just 10 µM. EMSA showed that the DgDps3-DNA complex was located near the upper pocket, unlike the DgDps1 found below. In the presence of metal ions together with DNA and protein, EMSA showed that those two DgDps proteins carried out the identical electrophoretic shift with DNA. Therefore, further analysis of protein structure conversion by metal ion and DNA-binding is needed.
Ferritin is a 24-mer protein with an inner diameter of 8 nm and stores 24,000 Fe atoms in vivo (Lawson et al., 1991; Harrison & Arosio, 1996; Yokoyama et al., 2012). However, the inner diameter of 5 nm Dps protein provides storage space for less than 500 iron atoms (Tonello et al., 1999; Ramsay et al., 2006). Thus, DNA-binding Dps proteins can store iron in a bioavailable form and protect cells against oxidative stress. The oxidative protection is achieved by binding Fe2+ ions and preventing the Fenton reaction-catalyzed formation of toxic hydroxyl radicals or binding DNA to block them from oxidative radicals (Imlay et al., 2013). Despite knowledge of the basic mechanisms of iron entry and oxidation, many essential questions regarding iron core formation and the iron release mechanism in response to cellular needs still need to be elucidated (Papinutto et al., 2002). Zeth et al. recently studied the metal position of three ions, Co2+, Zn2+, and La3+, and the translocation pathway in Listeria innocua Dps protein (Zeth et al., 2019). In addition, negatively charged residues inside the cavity cause dynamic change to Dps conformation. Thus, the Dps cage may be modified for different metal-holding specificities depending on the bioremediation of metal ions.
The ability of microorganisms to resist the toxic effects of metals is often related to their ability to transform metals into less soluble and less toxic chemical states with specific proteins (Brim et al., 2003). Dps of E. coli, involved in copper homeostasis, are good examples (Thieme & Grass, 2010). In the D. radiodurans experiment, Hg (II) and Hg (0) identified the ability to reduce toxicity in the cell (Brim et al., 2003). Fe (III), U (VI), Tc (VII), and Cr (VI) were also reduced under anaerobic and aerobic conditions (Fredrickson et al., 2000).
The original DR2263 (DrDps1) of D. radiodurans has a long N-terminal extension involved in DNA-binding and binding of several metals, such as iron and zinc (Romão et al., 2006; Nguyen & Grove, 2012; Ushijima et al., 2016). Due to their ability to act as protein cages for iron and various other metals, Dps-like proteins have recently become of considerable interest in nanotechnology. Furthermore, Dps can aggregate and remove metal ions harmful to bacteria and humans; therefore, the primary goal of our research is the industrial application of Dps. In addition to Fe (II) and Fe (III), Cr (VI), Dps is known to collect Hg (II), U (VI), and Cs (Brim et al., 2003). In this regard, we have established a vital research theme to ensure that Dgeo_0257 and Dgeo_0281 can react with these heavy and harmful metal ions. The various metal ions listed above and the interaction between the two proteins allowed us to compare the properties of Dgeo_0257 and Dgeo_0281. A better understanding of their functions and mechanisms can develop new biotechnology and nanotechnology (Haikarainen & Papageorgiou, 2010; Gerber et al., 2015).
We previously analyzed a transcriptomic analysis to define the functional role of putative Dps protein Dgeo_0257 using RNA-Seq technology (NCBI GEO accession number GSE151903; Lee et al., 2020). Tables S3 and S4 show a list of genes that have been over 3-fold up-regulated and 0.3 down-regulated. Interestingly, the 17 proteins/enzymes and ISDge5 transposases have also been up-regulated and 14 genes have been down-regulated. These results led us to believe that the putative Dps protein Dgeo_0257 was related to specific gene regulation together with DNA stabilization.
We conducted a study assuming that DgDps1 has existing Dps characteristics and DgDps3 has similar Dps characteristics lacking ferroxidase active center. Of particular interest, both DgDps proteins showed similar metal ion-sensing. However, a novel DgDps3 has higher DNA-binding affinity and more metal ion sensitivity than DgDps1. Two DgDps proteins have an intracellular responsibility for DNA protection, and detoxification of harmful iron depends on growth phases. Especially, DgDps1 as a dominant at early exponential growth phase does not induce at the stationary growth phase. When H2O2 treated stress was present in the DgDps3 disrupted mutant, DgDps1 was gradually induced on growth progress. However, DgDps3 was sensible to oxidative stress by H2O2 treatment, and when dominantly expressed DgDps1 was absent, substitutional induced at early exponential growth phase (Fig. 6). Therefore, the novel DgDps3 (Dgeo_0257) is proposed as one of the Dps DNA-binding proteins in D. geothermalis and will play a different role than DgDps1 (Dgeo_0281). Further investigation into DgDps3 is required to explain this protein´s function, structure, and network regulation through redox-sensing regulators in D. geothermalis.