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.     

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.     

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.     

Evidence for glycosylation on a DNA-binding protein of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

Background  

All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column.     

Growth regulation in irradiance limited marine Synechococcus sp. WH 7803

Examinations of the macromolecular components of the protein synthesizing system (RNA, DNA and protein) have been made in the marine cyanobacterium, Synechococcus sp. WH 7803. Slowly growing, irradiance limited cells have less RNA and lower rates of RNA synthesis than do those growing at rapid rates. RNA content and synthesis increase in conjunction with division rate. Protein content is variable. Protein synthesis increases up to a plateau at division rates less the maximum observed. The results imply that there is extra protein synthetic capacity produced at high, irradiance limited growth rates. Synechococcus sp. WH 7803 responds to an increase in irradiance through a rapid shiftup in macromolecular synthesis. RNA, protein and DNA increase in a sequential fashion which precedes the onset of cell division. After decreases in irradiance, protein synthesis is maintained despite reductions in RNA. This suggests that there is some degree of physiological buffering which occurs in this species. These studies indicate that, as in more extensively studied procaryotic models, the protein synthesizing system plays a central role in the global mechanisms regulating growth in Synechococcus sp. WH 7803.Abbreviations PSS protein synthesizing system - HMW high molecular weight - LMW low molecular weight - TCA trichloroacetic acid     

Structure and Mechanism of Iron Translocation by a Dps Protein from Microbacterium arborescens

Dps (DNA protection during starvation) enzymes are a major class of dodecameric proteins that bacteria use to detoxify their cytosol through the uptake of reactive iron species. In the stationary growth phase of bacteria, Dps enzymes are primarily used to protect DNA by biocrystallization. To characterize the wild type Dps protein from Microbacterium arborescens that displays additional catalytic functions (amide hydrolysis and synthesis), we determined the crystal structure to a resolution of 2.05 Å at low iron content. The structure shows a single iron at the ferroxidase center coordinated by an oxo atom, one water molecule, and three ligating residues. An iron-enriched protein structure was obtained at 2 Å and shows the stepwise uptake of two hexahydrated iron atoms moving along channels at the 3-fold axis before a restriction site inside the channels requires removal of the hydration sphere. Supporting biochemical data provide insight into the regulation of this acylamino acid hydrolase. Moreover, the peroxidase activity of the protein was determined. The influence of iron and siderophores on the expression of acylamino acid hydrolase was monitored during several stages of cell growth. Altogether our data provide an interesting view of an unusual Dps-like enzyme evolutionarily located apart from the large Dps sequence clusters.     

A novel nucleic acid-binding protein in the cyanobacterium Synechococcus sp. PCC6301: a soluble 33-kDa polypeptide with high sequence similarity to ribosomal protein S1

Cyanobacteria are prokaryotes that carry out plant-type photosynthesis and contain several eukaryotic-type RNA-binding proteins. Using a single-stranded DNA column, a 33-kDa protein was isolated and characterized from Synechococcus sp. PCC6301. This protein of 293 amino acids is similar in overall structure to the ribosomal protein S1 found in the same species, and contains three repeated units that are highly similar to the S1 motif originally found in the ribosomal protein S1 of Escherichia coli. However, the 33-kDa protein was found not to be associated with ribosomes and its nucleic acid binding specificity is distinct from that of the ribosomal protein S1. As this protein has high affinity for both single- and double-stranded DNA, as well as for poly(G) and poly(A), we tentatively named it nucleic acid-binding protein 1 (Nbp1). Received: 8 October 1999 / Accepted: 24 January 2000     

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.     

Expression and mutagenesis of the dpsA gene of Synechococcus sp. PCC7942, encoding a DNA-binding protein involved in oxidative stress protection

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.     

The IdiA protein of Synechococcus sp. PCC 7942 functions in protecting the acceptor side of Photosystem II under oxidative stress

Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from Q_A, gave a decreased O₂ evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Crystal structures of Streptococcus pyogenes Dpr reveal a dodecameric iron-binding protein with a ferroxidase site

DNA-binding protein from starved cells (Dps)-like proteins are key factors involved in oxidative stress protection in bacteria. They bind and oxidize iron, thus preventing the formation of harmful reactive oxygen species that can damage biomolecules, particularly DNA. Dps-like proteins are composed of 12 identical subunits assembled in a spherical structure with a hollow central cavity. The iron oxidation occurs at 12 intersubunit sites located at dimer interfaces. Streptococcus pyogenes Dps-like peroxide resistance protein (Dpr) has been previously found to protect the catalase-lacking S. pyogenes bacterium from oxidative stress. We have determined the crystal structure of S. pyogenes Dpr, the second Dpr structure from a streptococcal bacterium, in iron-free and iron-bound forms at 2.0- and 1.93-Å resolution, respectively. The iron binds to well-conserved sites at dimer interfaces and is coordinated directly to Asp77 and Glu81 from one monomer, His50 from a twofold symmetry-related monomer, a glycerol molecule, and a water molecule. Upon iron binding, Asp77 and Glu81 change conformation. Site-directed mutagenesis of active-site residues His50, His62, Asp66, Asp77, and Glu81 to Ala revealed a dramatic decrease in iron incorporation. A short helix at the N-terminal was found in a different position compared with other Dps-like proteins. Two types of pores were identified in the dodecamer. Although the N-terminal pore was found to be similar to that of other Dps-like proteins, the C-terminal pore was found to be blocked by bulky Tyr residues instead of small residues present in other Dps-like proteins.     

Structural Analysis of Photosystem II: Comparative Study of Cyanobacterial and Higher Plant Photosystem II Complexes

Oxygen evolving photosystem II (PSII-OEC) complexes and PSII core complexes were isolated from spinach and the thermophilic cyanobacteriumSynechococcussp. OD24 and characterized by gel electrophoresis, immunoblotting, and absorbance spectroscopy. The mass of the core complexes was determined by scanning transmission electron microscopy (STEM) and found to be 281 ± 65 kDa for spinach and 313 ± 52 kDa forSynechococcussp. OD24. The mass of the spinach PSII-OEC complex was 327 ± 64 kDa. Digital images of negatively stained PSII-OEC and PSII core complexes were recorded by STEM and analyzed by single particle averaging. All monomeric complexes showed similar morphologies and were of comparable length (14 nm) and width (10 nm). The averages revealed a pseudo-twofold symmetry axis, which is a prominent structural element of the monomeric form. Difference maps between the averaged projections of the oxygen evolving complexes and the core complexes from both species indicated where the 33-kDa extrinsic manganese stabilizing protein is bound. A symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex, is proposed.     

Nucleotide sequence and homology comparison of two genes of the sulfate transport operon from the cyanobacterium Synechocystis sp. PCC 6803

The genome of Synechocystis sp. PCC 6803 contains an operon with homology to the sulfate permease of other prokaryotes. We used antibodies raised against cytoplasmic membrane protein to find three genes with strong homology to sbpA, orf81 and cysT genes of the cyanobacterium Synechococcus sp. PCC 7942, Escherichia coli, Salmonella typhymurium and Marchantia polymorpha. It is likely that the permease genes are expressed and the proteins are inserted into the cytoplasmic membrane.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

Characterization of the photosystem II 32 kDa protein in Synechococcus PCC7942

A rapidly labeled photosynthetic membrane protein was identified in the cyanobacterium Synechococcus PCC7942 R2 as the 32 kDA protein that is involved in electron transport and quinone binding in the photosystem II complex. Partial proteolysis of the membrane-bound protein indicates that the internal architecture and the topology of the Synechococcus 32 kDa protein resembles the analogous protein of higher plants. In addition to the R2 wild-type strain, we characterized three psbA-inactivated Synechococcus strains, in which two of the three endogenous psbA genes were inactivated. In all strains, a 32 kDa protein cross-reacts with an antiserum that was raised against a higher-plant 32 kDa protein and displays in vivo light-dependent turnover. In Synechococcus, the herbicide DCMU inhibits the 32 kDa protein turnover at similar concentration ranges as in higher plants; however, a fraction of the molecules always displays a DCMU-insensitive degradation.     

THE EFFECT OF PHOSPHATE STATUS ON THE KINETICS OF CYANOPHAGE INFECTION IN THE OCEANIC CYANOBACTERIUM SYNECHOCOCCUS SP. WH78031

Phycoerythrin-containing Synechococcus species are considered to be major primary producers in nutrient-limited gyres of subtropical and tropical oceanic provinces, and the cyanophages that infect them are thought to influence marine biogeochemical cycles. This study begins an examination of the effects of nutrient limitation on the dynamics of cyanophage/Synechococcus interactions in oligotrophic environments by analyzing the infection kinetics of cyanophage strain S-PM2 (Cyanomyoviridae isolated from coastal water off Plymouth, UK) propagated on Synechococcus sp. WH7803 grown in either phosphate-deplete or phosphate-replete conditions. When the growth of Synechococcus sp. WH7803 in phosphate-deplete medium was followed after infection with cyanophage, an 18-h delay in cell lysis was observed when compared to a phosphate-replete control. Synechococcus sp. WH7803 cultures grown at two different rates (in the same nutritional conditions) both lysed 24 h postinfection, ruling out growth rate itself as a factor in the delay of cell lysis. One-step growth kinetics of S-PM2 propagated on host Synechococcus sp. WH7803, grown in phosphate-deplete and-replete media, revealed an apparent 80% decrease in burst size in phosphate-deplete growth conditions, but phage adsorption kinetics ofS-PM2 under these conditions showed no differences. These results suggested that the cyanophages established lysogeny in response to phosphate-deplete growth of host cells. This suggestion was supported by comparison of the proportion of infected cells that lysed under phosphate-replete and-deplete conditions, which revealed that only 9.3% of phosphate-deplete infected cells lysed in contrast to 100% of infected phosphate-replete cells. Further studies with two independent cyanophage strains also revealed that only approximately 10% of infected phosphate-deplete host cells released progeny cyanophages. These data strongly support the concept that the phosphate status of the Synechococcus cell will have a profound effect on the eventual outcome of phage-host interactions and will therefore exert a similarly extensive effect on the dynamics of carbon flow in the marine environment.     

The cyanobacterium Synechococcus modulates Photosystem II function in response to excitation stress through D1 exchange

In this minireview we discuss effects of excitation stress on the molecular organization and function of PS II as induced by high light or low temperature in the cyanobacterium Synechococcus sp. PCC 7942. Synechococcus displays PS II plasticity by transiently replacing the constitutive D1 form (D1:1) with another form (D1:2) upon exposure to excitation stress. The cells thereby counteract photoinhibition by increasing D1 turn over and modulating PS II function. A comparison between the cyanobacterium Synechococcus and plants shows that in cyanobacteria, with their large phycobilisomes, resistance to photoinhibition is mainly through the dynamic properties (D1 turnover and quenching) of the reaction centre. In contrast, plants use antenna quenching in the light-harvesting complex as an important means to protect the reaction center from excessive excitation.Abbreviations D1 reaction center protein of Photosystem II - P₆₈₀ the reaction center of Photosystem II - Q_A the primary quinone acceptor of Photosystem II - Tyr_Z tyrosine electron donor to P₆₈₀     

Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp. PCC 6803

The gene products of sll0337 and slr0081 in Synechocystis sp. PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively. Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions. A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin. In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting. Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon. Although there are three assignments for putative alkaline phosphatase genes in the Synechocystis sp. PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation. This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcus sp. PCC 7942 [Ray, J.M., Bhaya, D., Block, M.A. and Grossman, A.R. (1991) J. Bact. 173: 4297–4309]. An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcus sp. PCC 7942.