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PubMed1988

Duplication insertion of drug resistance determinants in the radioresistant bacterium Deinococcus radiodurans.

Escherichia coli drug resistance plasmids were introduced into Deinococcus radiodurans by cloning D. radiodurans DNA into the plasmids prior to transformation. The plasmids were integrated into the chromosome of the transformants and flanked by a direct repeat of the cloned D. radiodurans segment. The plasmid and one copy of the flanking chromosomal segment constituted a unit ("amplification unit") which was found repeated in tandem at the site of chromosomal integration. Up to 50 copies of the amplification unit were present per chromosome, accounting for approximately 10% of the genomic DNA. Circular forms of the amplification unit were also present in D. radiodurans transformants. These circles were introduced into E. coli, where they replicated as plasmids. The drug resistance determinants which have been introduced into D. radiodurans in this fashion are cat (from Tn9) and aphA (from Tn903). Transformation of D. radiodurans to drug resistance was efficient when the donor DNA was from D. radiodurans or E. coli, but was greatly reduced when the donor DNA was linearized with restriction enzymes prior to transformation. In the course of the study, a plasmid, pS16, was discovered in D. radiodurans R1, establishing that all Deinococcus strains so far examined contain plasmids.

Cloning, Molecular↗
PubMed1995

Interchromosomal recombination in the extremely radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans and other members of the genus Deinococcus are remarkable for their extreme resistance to ionizing radiation and many other agents that damage DNA. We have recently shown that recombinational processes participate in interplasmidic repair following in vivo irradiation. We now present direct studies on interchromosomal recombination among chromosomes irradiated in vivo during stationary phase (four chromosomes per cell). Following an exposure to 1.75 Mrad (the dose required to achieve a survival of 37%, which degrades the cells' four chromosomes into about 500 fragments), we determined that there may be as many as 175 crossovers per chromosome (700 crossovers per nucleoid) undergoing repair. In addition, these studies suggest that many of the crossovers occurring during repair are nonreciprocal.

Alleles↗
PubMed1995

Glucosyl diglyceride lipid structures in Deinococcus radiodurans.

The structures of two lipids from the radiation-resistant bacterium Deinococcus radiodurans are reported here: 1,2-diacyl-3-alpha-glucopyranosyl-glycerol and 3-O-[6'-O-(1",2"-diacyl- 3"-phosphoglycerol)-alpha-glucopyranosyl]-1,2-diacylglycerol. These lipids are strikingly different from previously characterized polar lipids from this organism, in that they are not unique to the genus Deinococcus and indeed have counterparts in both gram-negative and gram-positive bacteria. Moreover, as examples of glucose-containing lipids, they further illustrate the diversity of carbohydrate-containing lipids in D. radiodurans, from which lipids containing galactose and N-acetylglucosamine have already been structurally characterized.

Carbon Isotopes↗
PubMed2002

RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization.

The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E. coli RecA (RecA(Ec)) proteins are close functional homologues. RecA(Dr) forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA(Ec). The RecA(Dr) protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA(Dr) protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA(Dr) protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA(Dr) protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA(Dr) protein binds much better to duplex DNA than the RecA(Ec) protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.

Adenosine Triphosphate↗
PubMed2002

Firm but slippery attachment of Deinococcus geothermalis.

Bacterial biofilms impair the operation of many industrial processes. Deinococcus geothermalis is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria. It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known. High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of D. geothermalis E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium. Extensive slime matrix was absent from the D. geothermalis E50051 biofilms. AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed. We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible. In spite of this, AFM imaging of living D. geothermalis E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells. The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface. Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water. Biofilms were evaluated for their strength of attachment. D. geothermalis E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of Burkholderia cepacia F28L1 or the well-characterized biofilm former Staphylococcus epidermidis O-47. Deinococcus radiodurans strain DSM 20539(T) also formed tenacious biofilms. This paper shows that D. geothermalis has firm but laterally slippery attachment not reported before for a nonmotile species.

Bacterial Adhesion↗
PubMedDate not supplied

Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans.

Free radical scavenging effects of the cellular protein extracts from two strains of Deinococcus radiodurans and Escherichia coli against O2-, H2O2 and *OH were investigated by chemiluminescence (CL) methods. The cellular protein extracts of D. radiodurans R1 and KD8301 showed higher scavenging effects on O2- than that of E. coli. D. radiodurans R1 and KD8301 also strongly scavenged H2O2 with an EC50 (50% effective concentration) of 0.12 and 0.2 mg/mL, respectively, compared to that of E. coli (EC50 = 3.56 mg/mL). The two strains of D. radiodurans were effective in scavenging *OH generated by the Fenton reaction, with EC50 of 0.059 and 0.1 mg/mL, respectively, compared to that of E. coli (EC50 > 1 mg/mL). Results from the chemiluminescence assay of *OH-induced DNA damage and the plasmid pUC18 DNA double-strand break (DSB) model in vitro showed that D. radiodurans had remarkably inhibitory effect on the *OH-induced oxidative damage of DNA. The scavenging effects of D. radiodurans on reactive oxygen species (ROS) played an important role in the response to oxidation stress and preventing against DNA oxidative damage, and may be attributed to intracellular scavenging proteins, including superoxide dismutase (SOD) and catalase.

Catalase↗
PubMed2004

Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate.

We report the one- and two-dimensional (1-D and 2-D) capillary electrophoresis separation of Deinococcus radiodurans protein homogenate. Proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), which reacts with lysine residues and creates a highly fluorescent product. Detection is by laser-induced fluorescence. 1-D capillary sieving electrophoresis (CSE) produces over 150,000 plates and micellar electrokinetic capillary chromatography (MEKC) produces over 900,000 plates for components in a D. radiodurans protein homogenate. In a 2-D separation, proteins are first separated by CSE. Fractions are repetitively transferred to a second capillary for further separation based on MEKC. The 2-D separation has a approximately 550 spot capacity. Over 150 components are partially resolved from the homogenate. Resolution is limited in the first dimension by diffusion of proteins during the long separation period and in the second dimension by the combination of a long fraction-transfer time and short separation period.

Automation↗
PubMed2005

Proteomic analysis of Deinococcus radiodurans recovering from gamma-irradiation.

In order to reveal the mechanisms of the extreme radioresistance and DNA repair in Deinococcus radiodurans, we examined proteome changes in a wild-type strain following gamma-irradiation using two-dimensional polyacrylamide gel electrophoresis and Silver-staining. The expression levels of 26 protein spots showed significant changes under radiation stress. Of these spots, 21 were identified with peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry after tryptic in-gel digestion. These proteins exhibited various cellular functions, including (i) translation; (ii) transcription; (iii) signal transduction; (iv) post-translational modification, protein turnover, chaperones; (v) carbohydrate transport and metabolism; (vi) energy production and conversion; (vii) nucleotide transport and metabolism; (viii) inorganic ion transport and metabolism; (ix) DNA replication, recombination and repair; and (x) yet unknown. Most of the proteins have not previously been reported to be relevant to radioresistance.

Bacterial Proteins↗
PubMed2004

Solution structure of hypothetical Nudix hydrolase DR0079 from extremely radiation-resistant Deinococcus radiodurans bacterium.

Using nuclear magnetic resonance (NMR) based methods, including residual dipolar coupling restraints, we have determined the solution structure of the hypothetical Deinococcus radiodurans Nudix protein DR0079 (171 residues, MW = 19.3 kDa). The protein contains eight beta-strands and three alpha-helices organized into three subdomains: an N-terminal beta-sheet (1-34), a central Nudix core (35-140), and a C-terminal helix-turn-helix (141-171). The Nudix core and the C-terminal helix-turn-helix form the fundamental fold common to the Nudix family, a large mixed beta-sheet sandwiched between alpha-helices. The residues that compose the signature Nudix sequence, GX5EX7REUXEEXGU (where U = I, L, or V and X = any amino acid), are contained in a turn-helix-turn motif on the face of the mixed beta-sheet. Chemical shift mapping experiments suggest that DR0079 binds Mg2+. Experiments designed to determine the biological function of the protein indicate that it is not a type I isopentenyl-diphosphate delta-isomerase and that it does not bind alpha,beta-methyleneadenosine 5'-triphosphate (AMPCPP) or guanosine 5'-[beta,gamma-imido]triphosphate (GMPPNP). In this article, the structure of DR0079 is compared to other known Nudix protein structures, a potential substrate-binding surface is proposed, and its possible biological function is discussed.

Amino Acid Motifs↗
PubMed2006

The aspartate transcarbamoylase-dihydroorotase complex in Deinococcus radiophilus has an active dihydroorotase.

Aspartate transcarbamoylase (ATCase) and dihydroorotase (DHOase) catalyse the first two steps unique to pyrimidine synthesis. In many bacteria they form non-covalently bonded complexes. There are two types of DHOase, type I and type II which share a common ancestry. Type I is the more ancient form and is present in the complexes. In recently evolved bacteria the DHOase is defective and its function has been replaced by a type II DHOase which is separate from the complex. Deinococcus radiophilus diverges early on the phylogenetic tree and so might be expected to have an active type I DHOase. Purification of the 500 kDa ATCase-DHOase complex, by conventional techniques, showed it to possess an active DHOase.

Aspartate Carbamoyltransferase↗
PubMed2006

Characterization of the Mn2+-stimulated (di)adenosine polyphosphate hydrolase encoded by the Deinococcus radiodurans DR2356 nudix gene.

The DR2356 nudix hydrolase gene from Deinococcus radiodurans has been cloned and the product expressed as an 18 kDa histidine-tagged protein. The enzyme hydrolysed adenosine and diadenosine polyphosphates, always generating ATP as one of the initial products. ATP and other (deoxy)nucleoside triphosphates were also substrates, yielding (d)NDP and Pi as products. The DR2356 protein was most active at pH 8.6-9.0 and showed a strong preference for Mn(2+) as activating cation. Mg(2+) ions at 15 mM supported only 5% of the activity achieved with 2 mM Mn(2+). K (m) and k (cat) values for diadenosine tetra-, penta- and hexaphosphates were 2.0, 2.4 and 1.1 microM and 11.4, 28.6 and 12.0 s(-1), respectively, while for GTP they were 20.3 microM and 1.8 s(-1), respectively. The K (m )for adenosine 5'-pentaphosphate was <1 microM. Expression analysis showed the DR2356 gene to be induced eight- to ninefold in stationary phase and in cells subjected to slow dehydration plus rehydration. Superoxide (but not peroxide) treatment and rapid dehydration caused a two-to threefold induction. The Mn-requirement and induction in stationary phase suggest that DR2356 may have a specific role in maintenance mode metabolism in stationary phase as Mn(2+) accumulates.

Acid Anhydride Hydrolases↗
PubMed2006

PprA: A protein implicated in radioresistance of Deinococcus radiodurans stimulates catalase activity in Escherichia coli.

PprA: a pleiotropic protein promoting DNA repair, role in radiation resistance of Deinococcus radiodurans was demonstrated. In this study, the effect of radiation and oxidative stress on transgenic Escherichia coli expressing pprA has been studied. The pprA gene from D. radiodurans KR1 was cloned and expressed in E. coli. Transgenic E. coli cells expressing PprA showed twofold to threefold higher tolerance to hydrogen peroxide as compared to control. The 2.8-fold in vivo stimulation of catalase activity largely contributed by KatE was observed as compared to nonrecombinant control. Furthermore, the purified PprA could stimulate the E. coli catalase activity by 1.7-fold in solution. The effect of PprA on catalase activity observed both in vivo and in vitro was reverted to normal levels in the presence of PprA antibodies. The results suggest that enhanced oxidative stress tolerance in E. coli expressing PprA was due to the PprA stimulation of catalase activity, perhaps through the interaction of these proteins.

Bacterial Proteins↗
PubMed2006

Development of a defined medium supporting rapid growth for Deinococcus radiodurans and analysis of metabolic capacities.

A morpholinepropanesulfonic acid (MOPS)-buffered rich defined medium (RDM) was optimized to support a reproducible 2.6-h doubling time at 35 degrees C for Deinococcus radiodurans R1 and used to gain insight into vitamin and carbon metabolism. D. radiodurans was shown to require biotin and niacin for growth in this medium. A glutamine-serine simple defined medium (SDM) was developed that supported a 4-h doubling time, and this medium was used to probe sulfur and methionine metabolism. Vitamin B(12) was shown to alleviate methionine auxotrophy, and under these conditions, sulfate was used as the sole sulfur source. Phenotypic characterization of a methionine synthase deletion mutant demonstrated that the B(12) alleviation of methionine auxotrophy was due to the necessity of the B(12)-dependent methionine synthase in methionine biosynthesis. Growth on ammonium as the sole nitrogen source in the presence of vitamin B(12) was demonstrated, but it was not possible to achieve reproducibly good growth in the absence of at least one amino acid as a nitrogen source. Growth on sulfate, cysteine, and methionine as sulfur sources demonstrated the function of a complete sulfur recycling pathway in this strain. These studies have demonstrated that rapid growth of D. radiodurans R1 can be achieved in a MOPS-based medium solely containing a carbon source, salts, four vitamins, and two amino acids.

Carbon↗
PubMed2002

Quantification of RecA protein in Deinococcus radiodurans reveals involvement of RecA, but not LexA, in its regulation.

RecA protein is essential for the very high level of resistance of Deinococcus radiodurans to DNA damage induced by ionizing radiation or other DNA-damaging agents. Since the mechanism(s) involved in the control of recA expression and the extent of RecA induction following DNA damage in this species are still unclear, we have performed a genetic analysis of the recA locus and quantified the basal and induced levels of RecA protein in wild type, recA, and lexA mutants. We found that the two genes upstream of recA in the predicted cinA ligT recA operon appear to have no role in the regulation of recA expression or function, despite the fact that the reading frames in the operon overlap. By using a translational fusion of recA to a lacZ reporter gene, we showed that induction began with no delay following exposure to gamma-radiation or treatment with mitomycin, and continued at a constant rate until it reached a plateau. The induction efficiency increased linearly with inducer dose, levelling off at a concentration fourfold above the background. The basal concentration of RecA protein measured by Western blotting corresponded to approximately 11,000 monomers per cell, and the induced concentration to around 44,000 monomers per cell. These levels remained unchanged upon disruption of the lexA gene, indicating that LexA does not plays a role in recA regulation. However, inactivation of lexA caused cells to aggregate, suggesting that LexA may control the activity or expression of as yet undefined membrane functions. Cells bearing the recA670 mutation showed an elevated constitutive expression of recA in the absence of DNA damage. This phenotype did not result from the defect in DNA repair associated with the RecA670 protein, since the increased basal level of recA expression was also found in recA670/ recA(+) diploid cells that are proficient in DNA repair. These results suggest that RecA may be involved in regulating its own expression, possibly by stimulating proteolytic modification of other regulatory proteins.

Bacterial Proteins↗
PubMed2004

Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism Deinococcus radiodurans.

We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination. The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous RifR mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation. Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration--suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs. In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D. radiodurans, had no effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins were as resistant to gamma-rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage.

Amino Acid Sequence↗
PubMed2006

The crystal structure of Deinococcus radiodurans Dps protein (DR2263) reveals the presence of a novel metal centre in the N terminus.

The crystal structure of a DNA-binding protein from starved cells (Dps) (DR2263) from Deinococcus radiodurans was determined in two states: a native form, to 1.1-A resolution, and one soaked in an iron solution, to 1.6-A resolution. In comparison with other Dps proteins, DR2263 has an extended N-terminal extension, in both structures presented here, a novel metal binding site was identified in this N-terminal extension and was assigned to bound zinc. The zinc is tetrahedrally coordinated and the ligands, that belong to the N-terminal extension, are two histidines, one glutamate and one aspartate residue, which are unique to this protein within the Dps family. In the iron-soaked crystal structure, a total of three iron sites per monomer were found: one site corresponds to the ferroxidase centre with structural similarities to those found in other Dps family members; the two other sites are located on the two different threefold axes corresponding to small pores in the Dps sphere, which may possibly form the entrance and exit channels for iron storage.

Amino Acid Sequence↗
PubMed2004

Purification and some properties of superoxide dismutase from Deinococcus radiophilus, the UV-resistant bacterium.

The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg(-1) protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H2O2. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5+/-1 and 47.1+/-5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26 +/- 0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0-11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40 degrees C, but a linear reduction in activity above 50 degrees C. Inhibition of the purified SOD activity by beta-naphthoquinone-4-sulfonic acid, rho-diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(> D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.

Amino Acid Sequence↗
PubMed2005

Destruction of Deinococcus geothermalis biofilm by photocatalytic ALD and sol-gel TiO2 surfaces.

The aim of the present work was to explore possibilities of photocatalytic TiO2 coating for reducing biofilms on non-living surfaces. The model organism, Deinococcus geothermalis, known to initiate growth of durable, colored biofilms on machine surfaces in the paper industry, was allowed to form biofilms on stainless steel, glass and TiO2 film coated glass or titanium. Field emission electron microscopy revealed that the cells in the biofilm formed at 45 degrees C under vigorous shaking were connected to the surface by means of numerous adhesion threads of 0.1-0.3 microm in length. Adjacent cells were connected to one another by threads of 0.5-1 microm in length. An ultrastructural analysis gave no indication for the involvement of amorphous extracellular materials (e.g., slime) in the biofilm. When biofilms on photocatalytic TiO2 surfaces, submerged in water, were exposed to 20 W h m(-2) of 360 nm light, both kinds of adhesion threads were completely destroyed and the D. geothermalis cells were extensively removed (from >10(7) down to below 10(6) cells cm(-2)). TiO2 films prepared by the sol-gel technique were slightly more effective than those prepared by the ALD technique. Doping of the TiO2 with sulfur did not enhance its biofilm-destroying capacity. The results show that photocatalytic TiO2 surfaces have potential as a self-cleaning technology for warm water using industries.

Biofilms↗