Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Deinococcus”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 145 records · Page 8Linked to original sources
PubMed2006

Engineering of Deinococcus radiodurans R1 for bioprecipitation of uranium from dilute nuclear waste.

Genetic engineering of radiation-resistant organisms to recover radionuclides/heavy metals from radioactive wastes is an attractive proposition. We have constructed a Deinococcus radiodurans strain harboring phoN, a gene encoding a nonspecific acid phosphatase, obtained from a local isolate of Salmonella enterica serovar Typhi. The recombinant strain expressed an approximately 27-kDa active PhoN protein and efficiently precipitated over 90% of the uranium from a 0.8 mM uranyl nitrate solution in 6 h. The engineered strain retained uranium bioprecipitation ability even after exposure to 6 kGy of 60Co gamma rays. The PhoN-expressing D. radiodurans offers an effective and eco-friendly in situ approach to biorecovery of uranium from dilute nuclear waste.

Acid Phosphatase↗
PubMed2002

Involvement of two putative alternative sigma factors in stress response of the radioresistant bacterium Deinococcus radiodurans.

Two genes bearing similarity to alternative sigma factors were identified in the Deinococcus radiodurans genome sequence and designated sig1 and sig2. These genes were cloned and inactivated, and both were found to be important for survival during heat and ethanol stress, although the sig1 mutants displayed a more severe phenotype than the sig2 mutants. Reporter gene fusions to the groESL and dnaKJ operons transformed into these mutant backgrounds indicated that sig1 is required for the heat shock induction of groESL and dnaKJ, whereas sig2 mutants show a more moderate defect in dnaKJ induction and are not impaired for groESL induction. Essentiality tests suggested that neither sig1 nor sig2 is essential under all conditions. Sequence comparisons demonstrated that the sig1 gene product is classed distinctly with extracytoplasmic function (ECF) sigma factors, whereas Sig2 appears to be a more divergent sigma factor ortholog. These results suggest that sig1 encodes the major ECF-derived heat shock sigma factor in D. radiodurans and that it plays a central role in the positive regulation of heat shock genes. sig2, in contrast, appears to play a more minor role in heat shock protection and may serve to modulate the expression of some heat protective genes.

Amino Acid Sequence↗
PubMed2002

The IrrE protein of Deinococcus radiodurans R1 is a novel regulator of recA expression.

IRS24 is a DNA damage-sensitive strain of Deinococcus radiodurans strain 302 carrying a mutation in an uncharacterized locus designated irrE. Five overlapping cosmids capable of restoring ionizing radiation resistance to IRS24 were isolated from a genomic library. The ends of each cloned insert were sequenced, and these sequences were used to localize irrE to a 970-bp region on chromosome I of D. radiodurans R1. The irrE gene corresponds to coding sequence DR0167 in the R1 genome. The irrE gene encodes a 35,000-Da protein that has no similarity to any previously characterized peptide. The irrE locus of R1 was also inactivated by transposon mutagenesis, and this strain was sensitive to ionizing radiation, UV light, and mitomycin C. Preliminary findings indicate that IrrE is a novel regulatory protein that stimulates transcription of the recA gene following exposure to ionizing radiation.

Bacterial Proteins↗
PubMed2002

Protein splicing of the Deinococcus radiodurans strain R1 Snf2 intein.

Adjacent intein fragments fused to a Snf2/Rad54 helicase-related protein and Snf2/Rad54 helicase were reported for Deinococcus radiodurans R1, leading to the speculation that a frameshift was required for splicing or that trans splicing occurred. However, a type strain (ATCC 13939, RF18410) yielded a single protein that splices by the Ala1 protein splicing pathway, with splicing dependent on adjacent residues.

Cloning, Molecular↗
PubMed2003

Structure of a coenzyme A pyrophosphatase from Deinococcus radiodurans: a member of the Nudix family.

Gene Dr1184 from Deinococcus radiodurans codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA). Nudix enzymes with the same specificity have been found in yeast, humans, and mice. The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily. The structure of the complex of the enzyme with Mg(2+), its activating cation, reveals the position of the catalytic site. A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding. Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data.

Amino Acid Sequence↗
PubMed2005

Global transcriptional and proteomic analysis of the Sig1 heat shock regulon of Deinococcus radiodurans.

The sig1 gene, predicted to encode an extracytoplasmic function-type heat shock sigma factor of Deinococcus radiodurans, has been shown to play a central role in the positive regulation of the heat shock operons groESL and dnaKJ. To determine if Sig1 is required for the regulation of additional heat shock genes, we monitored the global transcriptional and proteomic profiles of a D. radiodurans R1 sig1 mutant and wild-type cells in response to elevated temperature stress. Thirty-one gene products were identified that showed heat shock induction in the wild type but not in the sig1 mutant. Quantitative real-time PCR experiments verified the transcriptional requirement of Sig1 for the heat shock induction of the mRNA of five of these genes-dnaK, groES, DR1314, pspA, and hsp20. hsp20 appears to encode a new member of the small heat shock protein superfamily, DR1314 is predicted to encode a hypothetical protein with no recognizable orthologs, and pspA is predicted to encode a protein involved in maintenance of membrane integrity. Deletion mutation analysis demonstrated the importance in heat shock protection of hsp20 and DR1314. The promoters of dnaKJE, groESL, DR1314, pspA, and hsp20 were mapped and, combined with computer-based pattern searches of the upstream regions of the 26 other Sig1 regulon members, these results suggested that Sig1 might recognize both sigma70-type and sigma(W)-type promoter consensus sequences. These results expand the D. radiodurans Sig1 heat shock regulon to include 31 potential new members, including not only factors with cytoplasmic functions, such as groES and dnaK, but also those with extracytoplasmic functions, like pspA.

Bacterial Proteins↗
PubMed2005

Characterization of pathways dependent on the uvsE, uvrA1, or uvrA2 gene product for UV resistance in Deinococcus radiodurans.

The genome of a radiation-resistant bacterium, Deinococcus radiodurans, contains one uvsE gene and two uvrA genes, uvrA1 and uvrA2. Using a series of mutants lacking these genes, we determined the biological significance of these components to UV resistance. The UV damage endonuclease (UvsE)-dependent excision repair (UVER) pathway and UvrA1-dependent pathway show some redundancy in their function to counteract the lethal effects of UV. Loss of these pathways does not cause increased sensitivity to UV mutagenesis, suggesting either that these pathways play no function in inducing mutations or that there are mechanisms to prevent mutation other than these excision repair pathways. UVER efficiently removes both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) from genomic DNA. In contrast, the UvrA1 pathway does not significantly contribute to the repair of CPDs but eliminates 6-4PPs. Inactivation of uvrA2 does not result in a deleterious effect on survival, mutagenesis, or the repair kinetics of CPDs and 6-4PPs, indicating a minor role in resistance to UV. Loss of uvsE, uvrA1, and uvrA2 reduces but does not completely abolish the ability to eliminate CPDs and 6-4PPs from genomic DNA. The result indicates the existence of a system that removes UV damage yet to be identified.

Bacterial Proteins↗
PubMed2005

Fine structure of the Deinococcus radiodurans nucleoid revealed by cryoelectron microscopy of vitreous sections.

Transmission electron microscopy revealed that the nucleoid of the extremely radioresistant bacteria Deinococcus radiodurans may adopt an unusual ring shape. This led to the hypothesis that the tight toroidal package of the D. radiodurans genome might contribute to radioresistance by preventing diffusion of ends of double-stranded DNA breaks. The molecular arrangement of DNA in the nucleoid, which must be determined to test this hypothesis, is not discernible by conventional methods of electron microscopy. We have applied cryoelectron microscopy of vitreous sections and found that the DNA arrangement in D. radiodurans differs from toroidal spooling. Diffuse coralline nucleoids of exponentially growing D. radiodurans do not reveal any particular molecular order. Electron-dense granules are generally observed in the centers of nucleoids. In stationary-phase cells, the nucleoid segregates from cytoplasm and DNA filaments show locally parallel arrangements, with increasing aspects of cholesteric liquid crystalline phase upon prolonged starvation. The relevance of the observed nucleoid organization to the radiation resistance of D. radiodurans is discussed.

Cryoelectron Microscopy↗
PubMed2004

Characterization of a nudix hydrolase from Deinococcus radiodurans with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates.

BACKGROUND: Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism. RESULTS: The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli. Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxy)nucleoside 5'-diphosphates (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP). Km for dGDP was 110 microM and kcat was 0.18 s-1 under optimal assay conditions (pH 9.4, 7.5 mM Mg2+). 8-Hydroxy-2'-deoxyguanosine 5'-diphosphate (8-OH-dGDP) was also a substrate with a Km of 170 microM and kcat of 0.13 s-1. Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP. Limited pyrophosphatase activity was also observed with NADH and some (di)adenosine polyphosphates but no other substrates. Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase. Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene. CONCLUSION: Nucleotide substrates for nudix hydrolases conform to the structure NDP-X, where X can be one of several moieties. Thus, a preference for (d)NDPs themselves is most unusual. The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the function of DR09075 may be to assist in the recycling of nucleotides under the very different metabolic requirements of stationary phase. Thus, if DR0975 does contribute to radiation resistance, this contribution may be indirect.

Bacterial Proteins↗
PubMed2005

Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of Haloarcula marismortui and Deinococcus radiodurans.

The number and position of the pseudouridines of Haloarcula marismortui and Deinococcus radiodurans large subunit RNA have been determined by a combination of total nucleoside analysis by HPLC-mass spectrometry and pseudouridine sequencing by the reverse transcriptase method and by LC/MS/MS. Three pseudouridines were found in H. marismortui, located at positions 1956, 1958, and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586, respectively. The three pseudouridines are all in locations found in other organisms. Previous reports of a larger number of pseudouridines in this organism were incorrect. Three pseudouridines and one 3-methyl pseudouridine (m3Psi) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m3Psi), 1900, and 2584, the m3Psi site being determined by a novel application of mass spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917, and 2605, which are also pseudouridines in E. coli (1915 is m3Psi). The pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans is conserved in eubacteria and a chloroplast but is not found in archaea or eukaryotes, whereas pseudouridine 2621 in H. marismortui is more conserved in eukaryotes and is not found in eubacteria. All the pseudoridines are near, but not exactly at, nucleotides directly involved in various aspects of ribosome function. In addition, two D. radiodurans Psi synthases responsible for the four Psi were identified.

Base Sequence↗
PubMed2006

Characterization of monofunctional catalase KatA from radioresistant bacterium Deinococcus radiodurans.

Catalase plays a key role in protecting cells against toxic reactive oxygen species. Here we report on the cloning, purification and characterization of a catalase (KatA, DR1998) from the extremely radioresistant bacterium Deinococcus radiodurans. The size of purified D. radiodurans KatA monomer was 65 kDa while gel filtration revealed that the size of the enzyme was 240 kDa, suggesting that KatA formed a homotetramer in solution. Purified KatA displayed a final specific activity of 68,800 U/mg of protein. The catalase activity of KatA was inhibited by sodium azide, sodium cyanide and 3-amino-1,2,4-triazole. The absorption spectrum of KatA exhibited a Soret band at 408 nm. The position of the spectral peak remained unchanged following reduction of KatA with dithionite. No peroxidase activity was found for KatA. These results demonstrate that D. radiodurans KatA is a typical monofunctional heme-containing catalase. The stability of KatA with respect to H2O2 stress was superior to that of commercially available Aspergillus niger and bovine liver catalases. The relative abundance of KatA in cells in addition to the H2O2 resistance property may play a role in the survival strategy of D. radiodurans against oxidative damage.

Amino Acid Sequence↗
PubMed2003

Characterization and distribution of IS8301 in the radioresistant bacterium Deinococcus radiodurans.

The insertion sequence element IS8301 isolated from the radiation resistant bacterium Deinococcus radiodurans strain KD8301 was characterized. IS8301 is comprised of 1,736-bp, lacks terminal inverted repeats and does not duplicate target DNA upon its insertion. The amino acid sequence homology of two open reading frames encoded in IS8301 indicates that this insertion sequence element belongs to the IS200/IS605 group. There were seven loci completely identical with the IS8301 sequence in the published D. radiodurans R(1) genome sequence. The genome distribution profiles of IS8301 in strain KD8301 as well as in the three different laboratory isolates (KR(1), MR(1), and R(1)) of wild-type D. radiodurans were investigated using genomic hybridization analysis. At least 21 strong hybridization signals were detected in strain KD8301 while only one hybridization signal was detected in strain KR(1), the parent strain of KD8301. In strain MR1, a different wild-type isolate, six strong hybridization signals were detected. In spite of the identification of seven copies of IS8301 in the published D. radiodurans R(1) genome sequence, only one hybridization signal was detected in strain R(1) purchased from American Type Culture Collection. Using inverse PCR and sequencing analyses, total 13 different insertion loci of IS8301 in the D. radiodurans genome were identified. Sequence comparison of the flanking region of insertion sites indicated that the sequence 5'-TTGAT-3' preceded the left end of IS8301 in all cases.

Amino Acid Sequence↗
PubMed2003

Cloning of structural gene of Deinococcus radiodurans UV-endonuclease beta.

To characterize its enzymic property we cloned and sequenced the gene of Deinococcus radiodurans encoding UV-endonuclease beta, an alternative enzyme to UvrABC repairing damaged DNA. Amino acid substitutions were found in UV-sensitive mutants. The putative amino acid sequence had some similarity with those of eukaryotic UV-endonucleases and with a sequence found in a protein data base of Bacillus subtilis.

Amino Acid Sequence↗
PubMed2004

Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1.

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3' ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.

Bacterial Proteins↗
PubMed2004

Heat shock and cold shock in Deinococcus radiodurans.

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42 degrees C maximized survival at the lethal temperature of 52 degrees C and a cold shock at 20 degrees C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I, and two proteins of unknown function.

Autoradiography↗
PubMed2006

Dynamics of Deinococcus radiodurans under controlled growth conditions.

Deinococcus radiodurans is a potent radiation resistant bacterium with immense potential in nuclear waste treatment. In this investigation, the translational and rotational dynamics of dilute suspensions of D. radiodurans cultured under controlled growth conditions was studied by the polarized and depolarized dynamic light-scattering (DLS) techniques. Additionally, confocal laser scanning microscopy was used for characterizing the cultured samples and also for identification of D. radiodurans dimer, tetramer, and multimer morphologies. The data obtained showed translational diffusion coefficients (DT) of 1.2 x 10(-9), 1.97 x 10(-9), and 2.12 x 10(-9) cm2 /s, corresponding to an average size of 3.61, 2.22, and 2.06 microm, respectively, for live multimer, tetramer, and dimer forms of D. radiodurans. Depolarized DLS experiments showed very slow rotational diffusion coefficients (DR) of 0.182/s for dimer and 0.098/s for tetramer morphologies. No measurable rotational diffusion was observed for multimer form. Polarized DLS measurements on live D. radiodurans confirmed that the bacterium is nonmotile in nature. The dynamics of the dead dimer and tetramer D. radiodurans were also studied using polarized and depolarized DLS experiments and compared with the dynamics of live species. The dead cells were slightly smaller in size when compared to the live cells. However, no additional information could be obtained for dead cells from the polarized and depolarized dynamic light-scattering studies.

Deinococcus↗
PubMed2006

Functional analysis of helicase and three tandem HRDC domains of RecQ in Deinococcus radiodurans.

RecQ is a highly conserved helicase necessary for maintaining genome stability in all organisms. Genome comparison showed that a homologue of RecQ in Deinococcus radiodurans designated as DR1289 is a member of RecQ family with unusual domain arrangement: a helicase domain, an RecQ C-terminal domain, and surprisingly three HRDC domain repeats, whose function, however, remains obscure currently. Using an insertion deletion, we discovered that the DRRecQ mutation causes an increase in gamma radiation, hydroxyurea and mitomycine C and UV sensitivity. Using the shuttle plasmid pRADK, we complemented various domains of the D. radiodurans RecQ (DRRecQ) to the mutant in vivo. Results suggested that both the helicase and helicase-and-RNase-D-C-terminal (HRDC) domains are essential for complementing several phenotypes. The complementation and biochemical function of DRRecQ variants with different domains truncated in vitro suggested that both the helicase and three HRDC domains are necessary for RecQ functions in D. radiodurans, while three HRDC domains have a synergistic effect on the whole function. Our finding leads to the hypothesis that the RecF recombination pathway is likely a primary path of double strand break repair in this well-known radioresistant organism.

Amino Acid Sequence↗
PubMed2003

Mechanism of thermotolerance induction by split-dose hyperthermia in Deinococcus radiodurans DNA repair deficient mutants.

We examined the phenomenon of thermotolerance induction in the radioresistant prokaryote, Deinococcus radiodurans, which was initially exposed to 30 min at 52 degrees C followed by various intervals up to 6 h at 30 degrees C in TGY medium and then re-exposed to 52 degrees C for various periods, i.e., split-dose hyperthermia. This thermotolerance induction was analyzed in DNA repair deficient mutants (strain 302, 251, UVS25, rec30 and KH840) and the wild-type strain MR1. The strain UVS25 is a double mutant for the mtcA and uvsD genes, and strain rec30 is a mutant for the deinococcal recA gene. The induction was suppressed to 1/10 and 1/25 in strains UVS25 and rec30 respectively, as compared with the maximum level in the wild-type strain MR1. However, the induction in strain 302 (mutant for the uvrA gene) was not suppressed. Therefore, we conclude that proteins synthesized during the interexposure interval, i.e., the products of the uvsD (UV endonuclease beta) and recA (RecA protein) genes contribute to the induction of thermotolerance in D. radiodurans.

Centrifugation, Density Gradient↗