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George S. Bullerjahn - One of the best experts on this subject based on the ideXlab platform.

  • Expression and mutagenesis of the DpsA gene of Synechococcus sp. PCC7942, encoding a DNA-binding Protein involved in oxidative stress protection
    FEMS Microbiology Letters, 2006
    Co-Authors: Kshitij Dwivedi, Aparna Sen, George S. Bullerjahn
    Abstract:

    The Dps family of Proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long-term nutrient limitation. Some members of the Dps Protein family have been shown to form abundant, large (∼150 kD) hexameric complexes that bind chromosomal DNA with little sequence specificity. Previous work from this lab has demonstrated that the Dps Proteins are divergent members of the bacterioferritin/bacterioferritin superfamily, and that the Synechococcus sp. PCC7942 Dps homolog, named DpsA, is a DNA-binding hemoProtein having heme-dependent catalytic activity. We speculated that this Protein may yield a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggested that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Current work has examined the expression of DpsA both under nutrient stress and during the growth phase; whereas DpsA mRNA is detectable in the exponential phase, transition to stationary phase yields a 20-fold increase in steady-state mRNA levels. Mapping the promoter region identifies a TAGAAT −10 sequence likely recognized by a cyanobacterial RpoS homolog. Lastly, site-directed mutants lacking DpsA function exhibit a severe phenotype impaired under all conditions yielding photooxidative stress; these include high light and treatment with paraquat. This supports our contention that the DpsA Protein serves an important protective function in an obligate photoautotroph.

  • Immunocytochemical localization of the stress-induced DpsA Protein in the cyanobacterium Synechococcus sp. strain PCC 7942
    Journal of basic microbiology, 2002
    Co-Authors: Kathryn A. Durham, George S. Bullerjahn
    Abstract:

    Proteins of the Dps family are divergent ferritins that have been shown to bind DNA with high affinity during periods of nutrient and oxidative stress. Such binding protects the chromosome from peroxide attack. Surprisingly, we show by immunocytochemistry that the cyanobacterial Dps homolog, DpsA, localizes preferentially to the thylakoid membrane in Synechococcus sp. strain PCC7942. We propose that two DpsA pools are functioning in this species--an insoluble fraction bound to the chromosome, and a soluble fraction acting as a ferritin involved metal homeostasis of the photosynthetic apparatus. This model is presented in light of recent work on the E. coli Dps Protein showing that DNA binding is regulated by the metal-binding capacity of the Dps complex (Frenkiel-Krispin et al. 2001). Additionally, the pattern of DpsA localization in cells as they progress through the growth curve suggests that the DpsA complex may be involved in metal ion transport across the cell envelope.

  • The DpsA Protein of Synechococcus sp. Strain PCC7942 Is a DNA-binding HemoProtein LINKAGE OF THE Dps AND BACTERIOFERRITIN Protein FAMILIES
    Journal of Biological Chemistry, 1995
    Co-Authors: Maria Marjorette O. Peña, George S. Bullerjahn
    Abstract:

    Abstract The Dps family of Proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long term nutrient limitation. Some members of the Dps Protein family have been shown to form large (150-kDa), hexameric complexes that bind chromosomal DNA with little sequence specificity. In this paper we report the nucleotide sequence of the DpsA gene from Synechococcus sp. PCC7942 encoding a cyanobacterial Dps homolog. The deduced amino acid sequence of the Synechococcus sp. DpsA Protein revealed that a carboxyl-terminal domain of the Protein was >60% homologous to the COOH-terminal half of bacterioferritin. Other known Dps family members lack such high similarity to the bacterioferritins. Purification and spectroscopic analysis of the Synechococcus sp. DpsA Protein complex revealed that the complex contains heme and has a weak catalase activity in vitro. Activity staining of nondenaturing polyacrylamide gels showed that the Protein complex comigrated with both the heme and the catalase activity, and O evolution measurements yielded a maximal specific activity of 1.7 μmol of HO consumed/μg of Protein min. We speculate that the Protein may have a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggest that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Last, the evolutionary link between the Dps Protein family and the bacterioferritins is discussed.

  • Purification and characterization of a Synechococcus sp. strain PCC 7942 polypeptide structurally similar to the stress-induced Dps/PexB Protein of Escherichia coli
    Archives of Microbiology, 1995
    Co-Authors: Maria Marjorette O. Peña, William Burkhart, George S. Bullerjahn
    Abstract:

    A stable DNA/Protein complex having an apparent molecular mass of approximately 150kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps Protein of Escherichia coli ; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB . The 150-kDa complex dissociated into a 22-kDa Protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the Protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like Protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like Protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.

  • purification and characterization of a synechococcus sp strain pcc 7942 polypeptide structurally similar to the stress induced Dps pexb Protein of escherichia coli
    Archives of Microbiology, 1995
    Co-Authors: Maria Marjorette O. Peña, William Burkhart, George S. Bullerjahn
    Abstract:

    A stable DNA/Protein complex having an apparent molecular mass of approximately 150kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps Protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa Protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the Protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like Protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like Protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.

Emilia Chiancone - One of the best experts on this subject based on the ideXlab platform.

Ferric C. Fang - One of the best experts on this subject based on the ideXlab platform.

  • The role of ferritins in the physiology of Salmonella enterica sv. Typhimurium: a unique role for ferritin B in iron-sulphur cluster repair and virulence.
    Molecular microbiology, 2007
    Co-Authors: Jyoti Velayudhan, Margaret Castor, Anthony R. Richardson, Kara L. Main-hester, Ferric C. Fang
    Abstract:

    Ferritins are ubiquitous iron (Fe) storage Proteins that play a fundamental role in cellular Fe homeostasis. The enteric pathogen Salmonella enterica serovar Typhimurium possesses four ferritins: bacterioferritin, ferritin A, ferritin B and Dps. The haem-containing bacterioferritin (Bfr) accounts for the majority of stored Fe, followed by ferritin A (FtnA). Inactivation of bfr elevates the intracellular free Fe concentration and enhances susceptibility to H2O2 stress. The DNA-binding Dps Protein provides protection from oxidative damage without affecting the steady-state intracellular free Fe concentration. FtnB appears to be particularly important for the repair of oxidatively damaged Fe-sulphur clusters of aconitase and, in contrast to Bfr and FtnA, is required for Salmonella virulence in mice. Moreover, ftnB and Dps are repressed by the Fe-responsive regulator Fur and induced under conditions of Fe limitation, whereas bfr and ftnA are maximally expressed when Fe is abundant. The absence of a conserved ferroxidase domain and the potentiation of oxidative stress by FtnB in some strains lacking Dps suggest that FtnB serves as a facile cellular reservoir of Fe2+.

  • The Ferritin-Like Dps Protein Is Required for Salmonella enterica Serovar Typhimurium Oxidative Stress Resistance and Virulence
    Infection and immunity, 2004
    Co-Authors: Thomas A. Halsey, Andrés Vázquez-torres, Daniel J. Gravdahl, Ferric C. Fang, Stephen J. Libby
    Abstract:

    Resistance to phagocyte-derived reactive oxygen species is essential for Salmonella enterica serovar Typhimurium pathogenesis. Salmonella can enhance its resistance to oxidants through the induction of specific genetic pathways controlled by SoxRS, OxyR, sigma(S), sigma(E), SlyA, and RecA. These regulons can be found in a wide variety of pathogenic and environmental bacteria, suggesting that evolutionarily conserved mechanisms defend against oxidative stress both endogenously generated by aerobic respiration and exogenously produced by host phagocytic cells. Dps, a ferritin-like Protein found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress by sequestering iron and limiting Fenton-catalyzed oxyradical formation. In Escherichia coli and some other bacterial species, Dps has been shown to accumulate during stationary phase in a sigma(S)-dependent fashion, bind nonspecifically to DNA, and form a crystalline structure that compacts and protects chromatin from oxidative damage. In the present study, we provide evidence that Dps protects Salmonella from iron-dependent killing by hydrogen peroxide, promotes Salmonella survival in murine macrophages, and enhances Salmonella virulence. Reduced numbers of Dps mutant bacteria in the livers and spleens of infected mice are consistent with a role of Dps in protecting Salmonella from oxidative stress encountered during infection.

Maria Marjorette O. Peña - One of the best experts on this subject based on the ideXlab platform.

  • The DpsA Protein of Synechococcus sp. Strain PCC7942 Is a DNA-binding HemoProtein LINKAGE OF THE Dps AND BACTERIOFERRITIN Protein FAMILIES
    Journal of Biological Chemistry, 1995
    Co-Authors: Maria Marjorette O. Peña, George S. Bullerjahn
    Abstract:

    Abstract The Dps family of Proteins are a diverse group of bacterial stress-inducible polypeptides that bind DNA and likely confer resistance to peroxide damage during periods of oxidative stress and long term nutrient limitation. Some members of the Dps Protein family have been shown to form large (150-kDa), hexameric complexes that bind chromosomal DNA with little sequence specificity. In this paper we report the nucleotide sequence of the DpsA gene from Synechococcus sp. PCC7942 encoding a cyanobacterial Dps homolog. The deduced amino acid sequence of the Synechococcus sp. DpsA Protein revealed that a carboxyl-terminal domain of the Protein was >60% homologous to the COOH-terminal half of bacterioferritin. Other known Dps family members lack such high similarity to the bacterioferritins. Purification and spectroscopic analysis of the Synechococcus sp. DpsA Protein complex revealed that the complex contains heme and has a weak catalase activity in vitro. Activity staining of nondenaturing polyacrylamide gels showed that the Protein complex comigrated with both the heme and the catalase activity, and O evolution measurements yielded a maximal specific activity of 1.7 μmol of HO consumed/μg of Protein min. We speculate that the Protein may have a peroxide-consuming mechanism located on the chromosomal DNA, and we also suggest that this activity may be a necessary feature to handle the endogenous oxidative stresses associated with oxygenic photosynthesis. Last, the evolutionary link between the Dps Protein family and the bacterioferritins is discussed.

  • Purification and characterization of a Synechococcus sp. strain PCC 7942 polypeptide structurally similar to the stress-induced Dps/PexB Protein of Escherichia coli
    Archives of Microbiology, 1995
    Co-Authors: Maria Marjorette O. Peña, William Burkhart, George S. Bullerjahn
    Abstract:

    A stable DNA/Protein complex having an apparent molecular mass of approximately 150kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps Protein of Escherichia coli ; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB . The 150-kDa complex dissociated into a 22-kDa Protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the Protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like Protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like Protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.

  • purification and characterization of a synechococcus sp strain pcc 7942 polypeptide structurally similar to the stress induced Dps pexb Protein of escherichia coli
    Archives of Microbiology, 1995
    Co-Authors: Maria Marjorette O. Peña, William Burkhart, George S. Bullerjahn
    Abstract:

    A stable DNA/Protein complex having an apparent molecular mass of approximately 150kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps Protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa Protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the Protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like Protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like Protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.

Dipankar Chatterji - One of the best experts on this subject based on the ideXlab platform.

  • flexible aspartates propel iron to the ferroxidation sites along pathways stabilized by a conserved arginine in Dps Proteins from mycobacterium smegmatis
    Metallomics, 2017
    Co-Authors: Sunanda Margrett Williams, Dipankar Chatterji
    Abstract:

    DNA-binding Proteins under starvation (Dps) are dodecameric nano-compartments for iron oxidation and storage in bacterial cells. These Proteins have roughly spherical structures with a hollow interior where iron is stored. Through mutational analysis of a conserved arginine residue in the second Dps Protein from Mycobacterium smegmatis, we have identified residues which stabilize the interfaces between the iron entry and ferroxidation sites. Also, we have used X-ray crystallography to determine the structures of co-crystals of iron and Dps in varying proportions and compare the changes in these ligand-bound forms with respect to the apo-Protein. The iron-loaded Proteins of low, medium and high iron-bound forms were found to exhibit aspartate residues with alternate conformations, some of which could be directly linked to the sites of ferroxidation and iron entry. We conclude that the increased flexibility of aspartates in the presence of iron facilitates its movement from the entry site to the ferroxidaton site, and the two active sites are stabilized by the interactions of a conserved arginine residue R73.

  • a histidine aspartate ionic lock gates the iron passage in miniferritins from mycobacterium smegmatis
    Journal of Biological Chemistry, 2014
    Co-Authors: Sunanda Margrett Williams, Dipankar Chatterji, Anu V Chandran, M S Vijayabaskar, Hemalatha Balaram, Saraswathi Vishveshwara, M Vijayan
    Abstract:

    Dps (DNA-binding Protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps Protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8–2.2 Å for the various mutants to compare structural alterations vis a vis the wild type Protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the Protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.

  • Estimation of Förster's distance between two ends of Dps Protein from mycobacteria: Distance heterogeneity as a function of oligomerization and DNA binding
    Biophysical chemistry, 2007
    Co-Authors: Rakhi Pait Chowdhury, Dipankar Chatterji
    Abstract:

    Dps Protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the Protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The Protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non- sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the Protein. We used frequency domain phase-modulation fluorescence spectroscopy and Forster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the Protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the Protein. Here, we report the Forster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA– Protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric Protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.