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1.
DNA Repair (Amst) ; 73: 144-154, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30527928

RESUMO

The Deinococcus radiodurans bacterium is one of the most radioresistant organisms known. It can repair hundreds of radiation-induced DNA double-strand breaks without loss of viability and reconstitute an intact genome through RecA-dependent and RecA-independent DNA repair pathways. Among the Deinococcus specific proteins required for radioresistance, the PprA protein was shown to play a major role for accurate chromosome segregation and cell division after completion of DNA repair. Here, we analyzed the cellular role of the deinococcal RecN protein belonging to the SMC family and, surprisingly, observed that the absence of the RecN protein suppressed the sensitivity of cells devoid of the PprA protein to γ- and UV-irradiation and to treatment with MMC or DNA gyrase inhibitors. This suppression was not observed when ΔpprA cells were devoid of SMC or SbcC, two other proteins belonging to the SMC family. The absence of RecN also alleviated the DNA segregation defects displayed by ΔpprA cells recovering from γ-irradiation. When exposed to 5 kGy γ-irradiation, ΔpprA, ΔrecN and ΔpprA ΔrecN cells repaired their DNA with a delay of about one hour, as compared to the wild type cells. After irradiation, the absence of RecN reduced recombination between chromosomal and plasmid DNA, indicating that the deinococcal RecN protein is important for recombinational repair of DNA lesions. The transformation efficiency of genomic DNA was also reduced in the absence of the RecN protein. Here, we propose a model in which RecN, via its cohesin activity, might favor recombinational repair of DNA double strand breaks. This might increase, in irradiated cells, DNA constraints with PprA protein being required to resolve them via its ability to recruit DNA gyrase and to stimulate its decatenation activity.


Assuntos
Reparo do DNA , Enzimas de Restrição do DNA/deficiência , Deinococcus/genética , Raios gama/efeitos adversos , Reparo de DNA por Recombinação/efeitos da radiação , Proteínas de Bactérias , DNA Girase , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Deinococcus/citologia , Deinococcus/enzimologia , Deinococcus/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Mutação , Fenótipo , Tolerância a Radiação/genética , Reparo de DNA por Recombinação/genética , Inibidores da Topoisomerase II/farmacologia
2.
Nat Commun ; 8(1): 84, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28729688

RESUMO

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.Single-cell genomics can be used to study uncultured microorganisms. Here, Stepanauskas et al. present a method combining improved multiple displacement amplification and FACS, to obtain genomic sequences and cell size information from uncultivated microbial cells and viral particles in environmental samples.


Assuntos
Deinococcus/genética , Escherichia coli/genética , Genoma Bacteriano/genética , Genoma Viral/genética , Prochlorococcus/genética , Vírion/genética , Composição de Bases , Tamanho Celular , Deinococcus/citologia , Escherichia coli/citologia , Citometria de Fluxo , Técnicas de Amplificação de Ácido Nucleico , Prochlorococcus/citologia , Análise de Sequência de DNA , Análise de Sequência de RNA , Análise de Célula Única
3.
Biochemistry (Mosc) ; 80(10): 1201-16, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26567564

RESUMO

The bacterium Deinococcus radiodurans reveals extraordinary resistance to ionizing radiation, oxidative stress, desiccation, and other damaging conditions. In this review, we consider the main molecular mechanisms underlying such resistance, including the action of specific DNA repair and antioxidation systems, and transcription regulation during the anti-stress response.


Assuntos
Deinococcus/genética , Deinococcus/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Tolerância a Radiação , Parede Celular/efeitos da radiação , Reparo do DNA/efeitos da radiação , Deinococcus/citologia , Deinococcus/efeitos da radiação , Estresse Oxidativo/genética
4.
Mol Microbiol ; 97(4): 759-74, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25988355

RESUMO

Processes favoring the exceptional resistance to genotoxic stress of Deinococcus radiodurans are not yet completely characterized. It was postulated that its nucleoid and chromosome(s) organization could participate in the DNA double strand break repair process. Here, we investigated the organization of chromosome 1 by localization of three chromosomal loci including oriC, Ter and a locus located in its left arm. For this purpose, we used a ParB-parS system to visualize the position of the loci before and after exposure to γ-rays. By comparing the number of fluorescent foci with the number of copies of the studied loci present in the cells measured by quantitative polymerase chain reaction (qPCR), we demonstrated that the 4-10 copies of chromosome 1 per cell are dispersed within the nucleoid before irradiation, indicating that the chromosome copies are not prealigned. Chromosome segregation is progressive but not co-ordinated, allowing each locus to be paired with its sister during part of the cell cycle. After irradiation, the nucleoid organization is modified, involving a transient alignment of the loci in the late stage of DNA repair and a delay of segregation of the Ter locus. We discuss how these events can influence DNA double strand break repair.


Assuntos
Deinococcus/genética , Deinococcus/efeitos da radiação , Proteínas de Bactérias/genética , Montagem e Desmontagem da Cromatina , Mapeamento Cromossômico , Cromossomos Bacterianos , Quebras de DNA de Cadeia Dupla , Dano ao DNA/efeitos da radiação , Reparo do DNA , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Deinococcus/citologia , Deinococcus/metabolismo , Tolerância a Radiação/fisiologia
5.
J Appl Microbiol ; 119(1): 1-10, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25809882

RESUMO

Deinococcus spp are among the most radiation-resistant micro-organisms that have been discovered. They show remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation and oxidizing agents. Traditionally, Escherichia coli and Saccharomyces cerevisiae have been the two platforms of choice for engineering micro-organisms for biotechnological applications, because they are well understood and easy to work with. However, in recent years, researchers have begun using Deinococcus spp in biotechnologies and bioremediation due to their specific ability to grow and express novel engineered functions. More recently, the sequencing of several Deinococcus spp and comparative genomic analysis have provided new insight into the potential of this genus. Features such as the accumulation of genes encoding cell cleaning systems that eliminate organic and inorganic cell toxic components are widespread among Deinococcus spp. Other features such as the ability to degrade and metabolize sugars and polymeric sugars make Deinococcus spp. an attractive alternative for use in industrial biotechnology.


Assuntos
Deinococcus/genética , Microbiologia Industrial , Biofilmes , Biotecnologia , Parede Celular/química , Deinococcus/citologia , Deinococcus/fisiologia , Microbiologia Industrial/instrumentação , Microbiologia Industrial/métodos , Estresse Oxidativo
6.
J Phys Chem B ; 119(43): 13515-23, 2015 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-25730710

RESUMO

The ability to characterize the structure of metal centers beyond their primary ligands is important to understanding their chemistry. High-magnetic-field pulsed electron double resonance detected NMR (ELDOR-NMR) is shown to be a very sensitive approach to measuring the multinuclear NMR spectra of the nuclei surrounding Mn(II) ions. Resolved spectra of intact organisms with resonances arising from (55)Mn, (31)P, (1)H, (39)K, (35)Cl, (23)Na, and (14)N nuclei surrounding Mn(2+) centers were obtained. Naturally abundant cellular (13)C could be routinely measured as well. The amplitudes of the (14)N and (2)H ELDOR-NMR spectra were found to be linearly dependent on the number of nuclei in the ligand sphere. The evolution of the Mn(II) ELDOR-NMR spectra as a function of excitation time was found to be best described by a saturation phenomenon rather than a coherently driven process. Mn(II) ELDOR-NMR revealed details about not only the immediate ligands to the Mn(II) ions but also more distant nuclei, providing a view of their extended structures. This will be important for understanding the speciation and chemistry of the manganese complexes as well as other metals found in organisms.


Assuntos
Deinococcus/química , Escherichia coli/química , Manganês/química , Ressonância Magnética Nuclear Biomolecular , Compostos Organometálicos/química , Deinococcus/citologia , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/citologia , Modelos Moleculares , Estrutura Molecular , Teoria Quântica
7.
Phys Rev Lett ; 114(4): 048103, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25679911

RESUMO

We demonstrate nanoscale x-ray holographic imaging using optimized illumination wave fronts emitted by x-ray waveguide channels. Mode filtering minimizes wave-front distortions and artifacts encountered in most hard x-ray focusing schemes, enabling quantitative reconstruction of the projected density, as evidenced by a test pattern imaged with a field of view of about 20×40 µm and at 22 nm resolution. The dose efficiency and contrast sensitivity make the optical scheme compatible with samples of intrinsically low contrast, typical for hydrated soft matter. This is demonstrated by imaging bacteria in the hydrated and living state, with quantitative phase contrast revealing dense structures of the bacterial nucleoids associated with compactified DNA. In response to continued irradiation, characteristic changes in these dense structures are observed.


Assuntos
Deinococcus/citologia , Holografia/métodos , DNA Bacteriano/química , Holografia/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Soluções , Raios X
8.
Curr Microbiol ; 68(2): 247-53, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24121615

RESUMO

Deinococcus radiodurans R1 is a highly radio-tolerant bacterium. Depending on the nutrient availability D. radiodurans R1 exists in three morphologies viz. monococcal, diplococcal and tetracoccal. In this study, we examined whether nutrition-induced morphotypes of D. radiodurans showed similar DNA damage upon gamma radiation exposure. Total DNA damage after radiation exposure was estimated by comparing percent double-strand breaks (DSBs) in genomic DNA. It was found that all three morphotypes exhibited different radiation tolerances which were also dependent on the radiation dose given. Monococcal forms were found to be most radio-tolerant at most of the tested radiation doses. Results showed that these nutrient-starved-condition induced morphotypes show lesser DNA DSBs upon irradiation, hence show higher radio-tolerance.


Assuntos
Deinococcus/efeitos da radiação , Fenótipo , Tolerância a Radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Deinococcus/citologia , Deinococcus/genética
9.
Prep Biochem Biotechnol ; 44(7): 645-52, 2014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-24215305

RESUMO

Decreases in cell division at the stationary phase in bacterial cultures are often due to the depletion of nutrients and/or accumulation of toxic waste products. Yet, during the stationary phase, the highly radiation-resistant bacterium Deinococcus radiodurans undergoes new rounds of cell division when Mn(II) is added to the medium in a phenomenon known as manganese-induced cell division (MnCD). When cells were cultured in medium without Mn(II)-enrichment, a heat-resistant, proteinase K-resistant factor (or factors) with a molecular mass less than 10 kD accumulated in the spent medium. Inclusion of the concentrated spent medium in fresh medium could inhibit the growth of D. radiodurans significantly, and the degree of inhibition was dose dependent. However, the relative stimulatory effect of MnCD was also dose dependent-the higher the inhibition, the stronger was the MnCD response. Previous studies have shown that nutrients were not limiting and deinococcal cells would continue metabolizing its nutrients at stationary phase. Cells became more sensitive to radiation when nutrients in the medium eventually became depleted. We speculated that D. radiodurans might produce this factor in the medium to control its population density. The reduction in cell population would conserve the nutrients that in turn might enhance the survival of the species.


Assuntos
Deinococcus/efeitos dos fármacos , Deinococcus/crescimento & desenvolvimento , Manganês/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Meios de Cultivo Condicionados/farmacologia , Deinococcus/citologia , Relação Dose-Resposta a Droga , Endopeptidase K , Peso Molecular
10.
PLoS One ; 8(7): e69007, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23840905

RESUMO

Mutants created by deleting the ddrA, ddrB, ddrC, ddrD, and pprA loci of Deinococcus radiodurans R1alone and in all possible combinations of pairs revealed that the encoded gene products contribute to this species' resistance to UV light and/or mitomycin C. Deleting pprA from an otherwise wild type cell sensitizes the resulting strain to UV irradiation, reducing viability by as much as eight fold relative to R1. If this deletion is introduced into a ΔddrA or ΔddrD background, the resulting strains become profoundly sensitive to the lethal effects of UV light. At a fluence of 1000 Jm⁻², the ΔddrA ΔpprA and ΔddrD ΔpprA strains are 100- and 1000-fold more sensitive to UV relative to the strain that has only lost pprA. Deletion of ddrA results in a 100 fold increase in strain sensitivity to mitomycin C, but in backgrounds that combine a deletion of ddrA with deletions of either ddrC or ddrD, mitomycin resistance is restored to wild type levels. Inactivation of ddrB also increases D. radiodurans sensitivity to mitomycin, but unlike the ddrA mutant deleting ddrC or ddrD from a ΔddrB background further increases that sensitivity. Despite the effect that loss of these gene products has on DNA damage resistance, none appear to directly affect either excision repair or homologous recombination suggesting that they participate in novel processes that facilitate tolerance to UV light and interstrand crosslinks in this species.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Deinococcus/efeitos dos fármacos , Deinococcus/efeitos da radiação , Genes Bacterianos , Mitomicina/farmacologia , Deinococcus/citologia , Deinococcus/genética , Deleção de Genes , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Raios Ultravioleta
11.
DNA Repair (Amst) ; 12(4): 265-72, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23403184

RESUMO

Deinococcus radiodurans, one of the most radioresistant organisms known to date is able to reconstruct an intact genome from hundreds of DNA fragments. Here, we investigate the in vivo role of PprA, a radiation-induced Deinococcus specific protein. We report that DNA double strand break repair in cells devoid of PprA and exposed to 3800Gy γ-irradiation takes place efficiently with a delay of only 1h as compared to the wild type, whereas massive DNA synthesis begins 90min after irradiation as in the wild type, a phenotype insufficient to explain the severe radiosensitivity of the ΔpprA mutant. We show that the slow kinetics of reassembly of DNA fragments in a ΔpprA ΔrecA double mutant was the same as that observed in a ΔrecA single mutant demonstrating that PprA does not play a major role in DNA repair through RecA-independent pathways. Using a tagged PprA protein and immunofluorescence microscopy, we show that PprA is recruited onto the nucleoid after γ-irradiation before DNA double strand break repair completion, and then is found as a thread across the septum in dividing cells. Moreover, whereas untreated cells devoid of PprA displayed a wild type morphology, they showed a characteristic cell division abnormality after irradiation not found in other radiosensitive mutants committed to die, as DNA is present equally in the two daughter cells but not separated at the division septum. We propose that PprA may play a crucial role in the control of DNA segregation and/or cell division after DNA double strand break repair.


Assuntos
Proteínas de Bactérias/genética , Divisão Celular/genética , Deinococcus/genética , Raios gama , Proteínas de Bactérias/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Deinococcus/citologia , Deinococcus/efeitos da radiação , Deleção de Genes , Fenótipo , Tolerância a Radiação/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo
12.
J Ind Microbiol Biotechnol ; 39(9): 1367-75, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22584820

RESUMO

In this study we investigated the sensitivity of Deinococcus radiodurans to contact-free cold atmospheric plasma treatment as part of a project to establish new efficient procedures for disinfection of inanimate surfaces. The Gram-positive D. radiodurans is one of the most resistant microorganisms worldwide. Stationary phases of D. radiodurans were exposed to cold atmospheric plasma for different time intervals or to ultraviolet C (UVC) radiation at dose rates of 0.001-0.0656 J cm⁻², respectively. A methicillin-resistant Staphylococcus aureus strain (MRSA) served as control for Gram-positive bacteria. The surface microdischarge plasma technology was used for generation of cold atmospheric plasma. A plasma discharge was ignited using ambient air. Surprisingly, D. radiodurans was sensitive to the cold atmospheric plasma treatment in the same range as the MRSA strain. Survival of both bacteria decreased with increasing plasma exposure times up to 6 log10 cycles (>99.999 %) within 20 s of plasma treatment. In contrast, UVC radiation of both bacteria demonstrated that D. radiodurans was more resistant to UVC treatment than MRSA. Cold atmospheric plasma seems to be a promising tool for industrial and clinical purposes where time-saving is a critical point to achieve efficient disinfection of inanimate surfaces and where protection from corrosive materials is needed.


Assuntos
Deinococcus/efeitos dos fármacos , Desinfecção/métodos , Gases em Plasma/farmacologia , Deinococcus/citologia , Deinococcus/efeitos da radiação , Staphylococcus aureus Resistente à Meticilina/citologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos da radiação , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Raios Ultravioleta
13.
DNA Repair (Amst) ; 11(4): 349-56, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22301370

RESUMO

The single-stranded DNA-specific nuclease RecJ is found in most bacteria where it is involved in the RecFOR double-stranded break (DSBs) repair pathway. DSBs repair mainly occurs via the RecFOR pathway in Deinococcus radiodurans, a well-known radiation-resistant bacterium. A recJ null mutant was constructed to investigate the role of recJ in D. radiodurans. recJ inactivation caused growth defects and sensitivity to high temperatures. However, the radiation resistance of the recJ mutant was only moderately decreased. The full-length D. radiodurans RecJ (DrRecJ) protein was expressed and purified to further characterize its biochemical properties. DrRecJ possessed a Mn(2+) concentration-dependent nuclease activity where the optimal Mn(2+) concentration was 0.1mM. DrRecJ had a similar activity profile after adding 10mM Mg(2+) to reactions with different Mn(2+) concentrations, indicating that Mn(2+) is a RecJ regulator. Escherichia coli RecJ has no activity on 5' ssDNA tails shorter than 6-nt, but DrRecJ could effectively degrade DNA with a 4-nt 5' ssDNA tail, suggesting that DrRecJ may have a wider range of DNA substrates. Moreover, SSB in D. radiodurans stimulated the DrRecJ exonuclease activity, whereas DdrB inhibited it and provided protection to ssDNA. Overall, our results indicate that recJ is a nonessential gene in D. radiodurans and that the activity of DrRecJ is regulated by Mn(2+) and SSB-DdrB.


Assuntos
Proteínas de Bactérias/metabolismo , Deinococcus/enzimologia , Exodesoxirribonucleases/metabolismo , Proteínas de Bactérias/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Deinococcus/citologia , Deinococcus/genética , Deinococcus/efeitos da radiação , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Exodesoxirribonucleases/deficiência , Exodesoxirribonucleases/genética , Técnicas de Inativação de Genes , Manganês/farmacologia , Tolerância a Radiação/genética
14.
Phys Rev Lett ; 107(20): 208101, 2011 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-22181778

RESUMO

The unique strengths of x-ray microscopy are high penetration depth and near-edge resonances that provide chemical information. We use ptychography, a coherent diffractive imaging technique that disposes of the requirement for isolated specimens, and demonstrate resonant imaging by exploiting resonances near the oxygen K edge to differentiate between two oxygen-containing materials. To highlight a biological system where resonant ptychography might be used for chemical mapping of unsliced cells, reconstructions of freeze-dried Deinococcus radiodurans cells at an energy of 517 eV are shown.


Assuntos
Difração de Raios X/métodos , Deinococcus/citologia , Oxigênio/química , Polimetil Metacrilato/química , Dióxido de Silício/química
15.
Astrobiology ; 11(9): 875-82, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22059692

RESUMO

In this experimental study, cells of the radiation-resistant bacterium Deinococcus radiodurans were exposed to several different sources of radiation chosen to replicate the charged particles found in the solar wind. Naked cells or cells mixed with dust grains (basalt or sandstone) differing in elemental composition were exposed to electrons, protons, and ions to determine the probability of cell survival after irradiation. Doses necessary to reduce the viability of cell population to 10% (LD(10)) were determined under different experimental conditions. The results of this study indicate that low-energy particle radiation (2-4 keV), typically present in the slow component of the solar wind, had no effect on dehydrated cells, even if exposed at fluences only reached in more than 1000 years at Sun-Earth distance (1 AU). Higher-energy ions (200 keV) found in solar flares would inactivate 90% of exposed cells after several events in less than 1 year at 1 AU. When mixed with dust grains, LD(10) increases about 10-fold. These results show that, compared to the highly deleterious effects of UV radiation, solar wind charged particles are relatively benign, and organisms protected under grains from UV radiation would also be protected from the charged particles considered in this study.


Assuntos
Simulação por Computador , Deinococcus/citologia , Deinococcus/efeitos da radiação , Laboratórios , Viabilidade Microbiana , Luz Solar , Vento , Carbono , Deinococcus/ultraestrutura , Elétrons , Viabilidade Microbiana/efeitos da radiação , Prótons , Silicatos/química
16.
DNA Repair (Amst) ; 9(11): 1151-61, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-20817622

RESUMO

Deinococcus radiodurans is one of the most radiation-resistant organisms known. It can repair hundreds of radiation-induced double-strand DNA breaks without loss of viability. Genome reassembly in heavily irradiated D. radiodurans is considered to be an error-free process since no genome rearrangements were detected after post-irradiation repair. Here, we describe for the first time conditions that frequently cause erroneous chromosomal assemblies. Gross chromosomal rearrangements have been detected in recA mutant cells that survived exposure to 5kGy γ-radiation. The recA mutants are prone also to spontaneous DNA rearrangements during normal exponential growth. Some insertion sequences have been identified as dispersed genomic homology blocks that can mediate DNA rearrangements. Whereas the wild-type D. radiodurans appears to repair accurately its genome shattered by 5kGy γ-radiation, extremely high γ-doses, e.g., 25kGy, produce frequent genome rearrangements among survivors. Our results show that the RecA protein is quintessential for the fidelity of repair of both spontaneous and γ-radiation-induced DNA breaks and, consequently, for genome stability in D. radiodurans. The mechanisms of decreased genome stability in the absence of RecA are discussed.


Assuntos
Reparo do DNA , Deinococcus/enzimologia , Deinococcus/genética , Instabilidade Genômica , Recombinases Rec A/metabolismo , Proliferação de Células/efeitos da radiação , Quebras de DNA/efeitos da radiação , Fragmentação do DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Deinococcus/citologia , Deinococcus/efeitos da radiação , Relação Dose-Resposta à Radiação , Raios gama , Rearranjo Gênico/efeitos da radiação , Genoma Bacteriano/genética , Instabilidade Genômica/efeitos da radiação , Mutação/efeitos da radiação , Recombinases Rec A/genética
17.
Phys Rev Lett ; 103(19): 198102, 2009 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-20365956

RESUMO

X-ray diffraction microscopy (XDM) is well suited for nondestructive, high-resolution biological imaging, especially for thick samples, with the high penetration power of x rays and without limitations imposed by a lens. We developed nonvacuum, cryogenic (cryo-) XDM with hard x rays at 8 keV and report the first frozen-hydrated imaging by XDM. By preserving samples in amorphous ice, the risk of artifacts associated with dehydration or chemical fixation is avoided, ensuring the imaging condition closest to their natural state. The reconstruction shows internal structures of intact D. radiodurans bacteria in their natural contrast.


Assuntos
Deinococcus/citologia , Congelamento , Microscopia/métodos , Difração de Raios X/métodos , Cristalização , Deinococcus/ultraestrutura , Água/metabolismo
18.
Mol Cell Proteomics ; 8(3): 481-94, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18953020

RESUMO

Preliminary findings indicate that PprI is a regulatory protein that stimulates transcription and translation of recA and other DNA repair genes in response to DNA damage in the extremely radioresistant bacterium Deinococcus radiodurans. To define the repertoire of proteins regulated by PprI and investigate the in vivo regulatory mechanism of PprI in response to gamma radiation, we performed comparative proteomics analyses on wild type (R1) and a pprI knock-out strain (YR1) under conditions of ionizing irradiation. Results of two-dimensional electrophoresis and MALDI-TOF MS or MALDI-TOF/TOF MS indicated that in response to low dose gamma ray exposure 31 proteins were significantly up-regulated in the presence of PprI. Among them, RecA and PprA are well known for their roles in DNA replication and repair. Others are involved in six different pathways, including stress response, energy metabolism, transcriptional regulation, signal transduction, protein turnover, and chaperoning. The last group consists of many proteins with uncharacterized functions. Expression of an additional four proteins, most of which act in metabolic pathways, was down-regulated in irradiated R1. Additionally phosphorylation of two proteins was under the control of PprI in response to irradiation. The different functional roles of representative PprI-regulated genes in extreme radioresistance were validated by gene knock-out analysis. These results suggest a role, either directly or indirectly, for PprI as a general switch to efficiently enhance the DNA repair capability and extreme radioresistance of D. radiodurans via regulation of a series of pathways.


Assuntos
Proteínas de Bactérias/metabolismo , Dano ao DNA , Deinococcus/citologia , Deinococcus/efeitos da radiação , Raios gama , Genes de Troca , Viabilidade Microbiana/efeitos da radiação , Proteínas de Bactérias/genética , Proliferação de Células/efeitos da radiação , Deinococcus/genética , Deleção de Genes , Genes Bacterianos , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteômica , Tolerância a Radiação/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Fatores de Tempo
19.
Crit Rev Biochem Mol Biol ; 43(3): 221-38, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18568848

RESUMO

Deinococcus radiodurans, one of the most radioresistant organisms known to date, is able to repair efficiently hundreds of DNA double- and single-strand breaks as well as other types of DNA damages promoted by ionizing or ultraviolet radiation. We review recent discoveries concerning several aspects of radioresistance and survival under high genotoxic stress. We discuss different hypotheses and possibilities that have been suggested to contribute to radioresistance and propose that D. radiodurans combines a variety of physiological tools that are tightly coordinated. A complex network of regulatory proteins may be discovered in the near future that might allow further understanding of radioresistance.


Assuntos
Deinococcus/citologia , Deinococcus/efeitos da radiação , Reparo do DNA/efeitos da radiação , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/efeitos da radiação , Deinococcus/genética , Deinococcus/metabolismo , Dessecação , Genoma Bacteriano/genética , Genoma Bacteriano/efeitos da radiação , Viabilidade Microbiana/efeitos da radiação
20.
Biophys J ; 91(7): 2699-707, 2006 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16829564

RESUMO

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.


Assuntos
Deinococcus/crescimento & desenvolvimento , Deinococcus/citologia , Deinococcus/fisiologia , Luz , Microscopia Confocal , Espalhamento de Radiação
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