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1.
FEMS Microbiol Lett ; 366(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30500893

RESUMO

Generally, enlarged spheroplasts of the Gram-negative bacterium Deinococcus grandis contain a single cytoplasm and a large periplasmic space. Enlargement of D. grandis spheroplasts requires the presence of divalent cation Ca2+ or Mg2+. In this study, we elucidated the effects of concentrations of these divalent cations on the enlargement of spheroplasts. We compared the cell sizes of the spheroplasts at five different concentrations (16.2, 62, 100, 200 and 333 mM) of CaCl2 or MgCl2. At the lowest concentration (16.2 mM) of CaCl2 or MgCl2, the inner membrane of D. grandis spheroplasts collapsed and the spheroplasts did not enlarge. At the highest concentration (333 mM) of CaCl2 or MgCl2, enlargement was inhibited. At 200 mM of CaCl2, the outer membranes of D. grandis spheroplasts were fused repeatedly, but the inner membranes were not fused. Thus, at 200 mM of CaCl2, giant cells that have multiple cytoplasms were observed and were ≥ 500 µm in diameter. However, cell fusions were not observed in any concentrations of MgCl2. This indicates that Ca2+ induces lipopolysaccharide dehydration more strongly than Mg2+ and outer membranes may be fused by hydrophobic bonding. Our findings show the different functions of Ca2+ and Mg2+ on the outer membrane stability.


Assuntos
Cálcio/farmacologia , Deinococcus/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Esferoplastos/efeitos dos fármacos , Citoplasma/metabolismo , Íons/farmacologia
2.
Microbiology ; 164(10): 1266-1275, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30052171

RESUMO

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.


Assuntos
Cloretos/metabolismo , Deinococcus/crescimento & desenvolvimento , Deinococcus/metabolismo , Compostos de Manganês/metabolismo , Manganês/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biomassa , Cloretos/química , Cloretos/farmacologia , Meios de Cultura/química , Deinococcus/química , Deinococcus/efeitos dos fármacos , Manganês/farmacologia , Compostos de Manganês/química , Compostos de Manganês/farmacologia , Nucleotídeos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/química , Proteoma/metabolismo
3.
Biochemistry ; 57(29): 4349-4356, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29944345

RESUMO

The bacterial metabolite 1-deoxy-d-xyulose 5-phosphate (DXP) is essential in bacterial central metabolism feeding into isoprenoid, thiamin diphosphate (ThDP), and pyridoxal phosphate de novo biosynthesis. Halting its production through the inhibition of DXP synthase is an attractive strategy for the development of novel antibiotics. Recent work has revealed that DXP synthase utilizes a unique random sequential mechanism that requires formation of a ternary complex among pyruvate-derived C2α-lactylthiamin diphosphate (LThDP), d-glyceraldehyde 3-phosphate (d-GAP), and enzyme, setting it apart from all other known ThDP-dependent enzymes. Herein, we describe the development of bisubstrate inhibitors bearing an acetylphosphonate (AP) pyruvate mimic and a distal negative charge mimicking the phosphoryl group of d-GAP, designed to target the unique form of DXP synthase that binds LThDP and d-GAP in a ternary complex. A d-phenylalanine-derived triazole acetylphosphonate (d-PheTrAP) emerged as the most potent inhibitor in this series, displaying slow, tight-binding inhibition with a Ki* of 90 ± 10 nM, forward ( k1) and reverse ( k2) isomerization rates of 1.1 and 0.14 min-1, respectively, and exquisite selectivity (>15000-fold) for DXP synthase over mammalian pyruvate dehydrogenase. d-PheTrAP is the most potent, selective DXP synthase inhibitor described to date and represents the first inhibitor class designed specifically to exploit the unique E-LThDP-GAP ternary complex in ThDP enzymology.


Assuntos
Acetaldeído/análogos & derivados , Deinococcus/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Transferases/antagonistas & inibidores , Acetaldeído/química , Acetaldeído/farmacologia , Deinococcus/efeitos dos fármacos , Desenho de Drogas , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Pentosefosfatos/metabolismo , Transferases/metabolismo
4.
J Bacteriol ; 200(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29891641

RESUMO

5'- and 3'-end healing are key steps in nucleic acid break repair in which 5'-OH and 3'-PO4 or 2',3'-cyclic-PO4 ends are converted to 5'-PO4 and 3'-OH termini suitable for sealing by polynucleotide ligases. Here, we characterize Deinococcus radiodurans HD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5'-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3'-phosphatase and 2',3'-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis of p-nitrophenylphosphate. HD-Pnk is encoded by the LIG-PARG-HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential for Deinococcus growth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ΔHD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitize Deinococcus to killing by mitomycin C.IMPORTANCE End healing is a process whereby nucleic acid breaks with "dirty" 3'-PO4 or 2',3'-cyclic-PO4 and 5'-OH ends are converted to 3'-OH and 5'-PO4 termini that are amenable to downstream repair reactions. Deinococcus radiodurans is resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlights Deinococcus HD-Pnk as a bifunctional 3'- and 5'-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.


Assuntos
Deinococcus/enzimologia , Farmacorresistência Bacteriana , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Mitomicina/farmacologia , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , Radiação Ionizante , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deinococcus/efeitos dos fármacos , Deinococcus/efeitos da radiação , Genoma Bacteriano , Mutação , Óperon , Polinucleotídeo 5'-Hidroxiquinase/genética
5.
J Biosci ; 43(1): 15-23, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29485112

RESUMO

Presence of low concentrations (1-2%) of ethanol during irradiation exhibited significant protection against DNA damage caused by very high doses (2-12 kGy) of 60 Co-gamma-rays in vitro. Radiation-induced DNA damage was substantially reduced in different types of DNA molecules (chromosomal DNA from Anabaena 7120 or Deinococcus radiodurans or bacteriophage Lambda, and plasmid pBluescript DNA) when irradiated in the presence of ethanol, thus indicating the generic nature of ethanol protection. The radioprotection appeared to be a consequence of the well known ability of ethanol to scavenge hydroxyl radicals. Addition of ethanol during 6 kGy irradiation also reduced DNA damage in vivo and improved post-irradiation growth recovery of Anabaena 7120 cultures. To our knowledge, this is the first instance of ability of very low ethanol concentrations to protect DNA from damage triggered by extremely high doses of 60 Co-gamma rays.


Assuntos
Anabaena/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , Deinococcus/efeitos dos fármacos , Etanol/farmacologia , Depuradores de Radicais Livres/farmacologia , Protetores contra Radiação/farmacologia , Anabaena/efeitos da radiação , Dano ao DNA , DNA Bacteriano/efeitos da radiação , Deinococcus/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Etanol/química , Raios gama/efeitos adversos , Radical Hidroxila/antagonistas & inibidores , Radical Hidroxila/metabolismo , Plasmídeos/efeitos da radiação
6.
Chembiochem ; 19(1): 58-65, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29119720

RESUMO

Enzymes of the 2-C-methyl-d-erythritol-4-phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1-deoxy-d-xylulose-5-phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false-positive hits. The enriched peptide binder P12 emerged as a substrate (d-glyceraldehyde-3-phosphate)-competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor- and acceptor-substrate-binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition.


Assuntos
Anti-Infecciosos/metabolismo , Proteínas de Bactérias/metabolismo , Biblioteca de Peptídeos , Transferases/metabolismo , Sequência de Aminoácidos , Anti-Infecciosos/química , Proteínas de Bactérias/antagonistas & inibidores , Ligação Competitiva , Deinococcus/efeitos dos fármacos , Deinococcus/enzimologia , Escherichia coli/metabolismo , Cinética , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Especificidade por Substrato , Transferases/antagonistas & inibidores
7.
Dalton Trans ; 46(36): 12328-12338, 2017 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-28891573

RESUMO

Two neutral cyclometalated Ir(iii)-tetrazolato complexes that differ by variations of the substituents on either the phenylpyridine or the tetrazolate ligand have been converted into the corresponding methylated and cationic analogues. NMR (1H and 13C) characterization of the Ir(iii) complexes provided the results in agreement with the chemo- and regioselective character of methylation at the N-3 position of the Ir(iii)-coordinated tetrazolato ring. This evidence was further corroborated by the analysis of the molecular structures of the cationic complexes obtained by X-ray diffraction. In view of the photophysical properties, the addition of a methyl moiety to neutral Ir(iii) tetrazolates, which behave as sky-blue or orange phosphors, caused a systematic red shift of their phosphorescence output. The transformation of neutral Ir(iii) tetrazolates into cationic Ir(iii)-tetrazole complexes was screened for any eventual antimicrobial activity in vitro against Gram negative (E. coli) and Gram positive (D. radiodurans) microorganisms. While both kinds of complexes were not active against E. coli, the conversion of the neutral Ir(iii) tetrazolates into the corresponding methylated and cationic Ir(iii)tetrazole derivatives determined the turn-on of a good to excellent antimicrobial activity toward Gram positive Deinococcus radiodurans, a non-pathogenic bacterium that is listed as one of the toughest microorganisms in light of its outstanding resistance to radiation and oxidative stress.


Assuntos
Anti-Infecciosos/química , Complexos de Coordenação/química , Irídio/química , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Complexos de Coordenação/síntese química , Complexos de Coordenação/farmacologia , Cristalografia por Raios X , Deinococcus/efeitos dos fármacos , Deinococcus/crescimento & desenvolvimento , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Ligantes , Metilação , Conformação Molecular , Tetrazóis/química
8.
Annu Rev Biochem ; 86: 567-583, 2017 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-28654325

RESUMO

Multidrug resistance is a global threat as the clinically available potent antibiotic drugs are becoming exceedingly scarce. For example, increasing drug resistance among gram-positive bacteria is responsible for approximately one-third of nosocomial infections. As ribosomes are a major target for these drugs, they may serve as suitable objects for novel development of next-generation antibiotics. Three-dimensional structures of ribosomal particles from Staphylococcus aureus obtained by X-ray crystallography have shed light on fine details of drug binding sites and have revealed unique structural motifs specific for this pathogenic strain, which may be used for the design of novel degradable pathogen-specific, and hence, environmentally friendly drugs.


Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/química , Desenho de Drogas , Ribossomos/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Cristalografia por Raios X , Deinococcus/efeitos dos fármacos , Deinococcus/genética , Deinococcus/metabolismo , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Modelos Moleculares , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
9.
J Microbiol ; 55(2): 137-146, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28120190

RESUMO

A cystine-dependent anti-oxidative stress response is characterized in Deinococcus geothermalis for the first time. Nevertheless, the same transcriptional directed Δdgeo_1985F mutant strain was revealed to have an identical phenotype to the wild-type strain, while the reverse transcriptional directed Δdgeo_1985R mutant strain was more resistant to oxidative stress at a certain concentration of H2O2 than the wild-type strain. The wild-type and mutant strains expressed equal levels of superoxide dismutase and catalase under H2O2-induced stress. Although the expression levels of the general DNA-damage response-related genes recA, pprA, ddrA, and ddrB were up-regulated by more than five-fold in the wild-type strain relative to the Δdgeo_1985R mutant strain, the mutant strain had a higher survival rate than the wild-type under H2O2 stress. The Δdgeo_1985R mutant strain highly expressed a cystine-transporter gene (dgeo_1986), at levels 150-fold higher than the wild-type strain, leading to the conclusion that this cystine transporter might be involved in the defensive response to H2O2 stress. In this study, the cystine transporter was identified and characterized through membrane protein expression analysis, a cystine-binding assay, and assays of intracellular H2O2, cysteine, and thiol levels. The genedisrupted mutant strain of the cystine importer revealed high sensitivity to H2O2 and less absorbed cystine, resulting in low concentrations of total thiol. Thus, the absorbed cystine via this cystine-specific importer may be converted into cysteine, which acts as a primitive defense substrate that non-enzymatically scavenges oxidative stress agents in D. geothermalis.


Assuntos
Proteínas de Bactérias/genética , Cisteína/metabolismo , Cistina/metabolismo , Deinococcus/genética , Deinococcus/metabolismo , Proteínas de Membrana Transportadoras/genética , Estresse Oxidativo , Proteínas de Bactérias/metabolismo , Catalase/genética , Catalase/metabolismo , Dano ao DNA , Deinococcus/efeitos dos fármacos , Genes Bacterianos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Família Multigênica , Mutação , Oxirredução , Estresse Oxidativo/genética , Compostos de Sulfidrila/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
10.
Wei Sheng Wu Xue Bao ; 57(1): 140-53, 2017 Jan 04.
Artigo em Chinês | MEDLINE | ID: mdl-29746768

RESUMO

Objective: The complete genome of the extreme environmental resistant bacterium Deiococcus radiodurans R1 was analyzed by sequence comparative method and putative ferritin-like protein DRA0258 was screened. Molecular techniques were applied to validate and analyze its function. Methods: We applied sequence alignment to analyze amino acid sequence of the hypothetical protein DRA0258 and detected its iron binding activity after purification. We used triple-fraction-ligation method to construct dra0258 null mutant and detected its survival rate under H2O2 treatment, catalase activity and total antioxidant capacity, using QRT-PCR to examine the relative transcriptional level change of the antioxidant relative enzymes and iron transport relative proteins. Resutls: We confirmed DRA0258 obtained a certain iron binding activity. The survival rate assay with H2O2 treatment suggested that deletion of dra0258 reduced the cellular antioxidant activity of D. radiodurans. The attenuation of catalase activity, total antioxidant capacity as well as the reduction of relative transcriptional levels of antioxidant related genes verified that both the oxidative stress response systems and the iron regulation network were damaged. Conclusion: This study verified DRA0258 is an iron-binding protein. Deletion of this gene would affect cellular iron transport system and reduce cellular antioxidant capability.


Assuntos
Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Deinococcus/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Bactérias/genética , Catalase/genética , Catalase/metabolismo , Deinococcus/efeitos dos fármacos , Deinococcus/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
11.
Arch Biochem Biophys ; 589: 38-52, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26550929

RESUMO

Deinococcus radiodurans (Drad) is the most radioresistant organism known. Although mechanisms that underlie the extreme radioresistance of Drad are incompletely defined, resistance to UV irradiation-induced killing was found to be greatly attenuated in an NO synthase (NOS) knockout strain of Drad (Δnos). We now show that endogenous NO production is also critical for protection of Drad against γ-irradiation (3000 Gy), a result of accelerated growth recovery, not protection against killing. NO-donor treatment rescued radiosensitization in Δnos Drad but did not influence radiosensitivity in wild type Drad. To discover molecular mechanisms by which endogenous NO confers radioresistance, metabolite profiling studies were performed. Untargeted LC-MS-based metabolite profiling in Drad quantified relative abundances of 1425 molecules and levels of 294 of these were altered by >5-fold (p < 0.01). Unexpectedly, these studies identified a dramatic perturbation in carotenoid biosynthetic intermediates in Δnos Drad, including a reciprocal switch in the pathway end-products from deoxydeinoxanthin to deinoxanthin. NO supplementation rescued these nos deletion-associated changes in carotenoid biosynthesis, and fully-restored radioresistance to wildtype levels. Because carotenoids were shown to be important contributors to radioprotection in Drad, our findings suggest that endogenously-produced NO serves to maintain a spectrum of carotenoids critical for Drad's ability to withstand radiation insult.


Assuntos
Carotenoides/biossíntese , Deinococcus/metabolismo , Deinococcus/efeitos da radiação , Metabolômica , Óxido Nítrico/biossíntese , Tolerância a Radiação , Antioxidantes/metabolismo , Carotenoides/química , Deinococcus/efeitos dos fármacos , Deinococcus/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Técnicas de Inativação de Genes , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Tolerância a Radiação/efeitos dos fármacos
12.
Arch Microbiol ; 198(1): 43-51, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26510931

RESUMO

Autoinducer-2 (AI-2) serves as a quorum-sensing signaling molecule that mediates both intraspecies and interspecies communication among bacteria, and plays critical roles in regulating various bacterial behaviors. In the present study, we investigated the functions of AI-2 signaling in the extremophilic bacterium Deinococcus radiodurans R1 by construction of the LuxS gene disruption mutant, survival phenotype assay and gene transcription assay. The gene mutant (DRΔLuxS), which was unable to produce AI-2, was significantly more sensitive to both gamma radiation and H2O2 compared with the wild-type strain. Addition of the wild-type-derived spent medium into the cell culture of DRΔLuxS fully restored the radioresistance of D. radiodurans. A higher level of reactive oxygen species accumulated in the mutant compared with the wild type under normal or oxidative stress. Quantitative real-time PCR assays showed that transcriptional levels of stress-related proteins, including catalase, extracellular nuclease, Dps-1 and ABC transporters, were decreased in DRΔLuxS, indicating that AI-2 is involved in regulation of stress-related genes of D. radiodurans. Hence, AI-2 signaling may contribute to the extreme resistance of D. radiodurans to radiation and oxidative stresses.


Assuntos
Deinococcus/genética , Regulação Bacteriana da Expressão Gênica , Homosserina/análogos & derivados , Lactonas/metabolismo , Estresse Oxidativo/genética , Transdução de Sinais , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Deinococcus/efeitos dos fármacos , Deinococcus/efeitos da radiação , Raios gama , Homosserina/metabolismo , Peróxido de Hidrogênio/farmacologia , Percepção de Quorum/genética , Espécies Reativas de Oxigênio/metabolismo
13.
J Biosci ; 40(5): 833-43, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26648029

RESUMO

Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase Ib of D. radiodurans (DraTopoIB) could change the electrophoretic mobility of fast migrating intramolecular recF-G4 DNA into the slow migrating species. DraTopoIB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular recF-G4 DNA structures stabilized by K+. On the contrary, when DraTopoIB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopoIB. This implies that DraTopoIB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopoIB activity on intramolecular G4 DNA structures. These results suggested that DraTopoIB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Bacteriano/química , Deinococcus/enzimologia , Quadruplex G , Proteínas de Bactérias/genética , Camptotecina/farmacologia , Dicroísmo Circular , DNA Topoisomerases Tipo I/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Deinococcus/efeitos dos fármacos , Deinococcus/genética , Cloreto de Potássio/química , Inibidores da Topoisomerase I/farmacologia
14.
J Microbiol Biotechnol ; 25(12): 2125-34, 2015 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-26370803

RESUMO

IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad- IrrE in the D. radiodurans irrE-deletion mutant (ΔirrE), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the ΔirrE mutant complemented strain with Drad-irrE, designated ΔirrE-E. The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA, and ddrB, were remarkably induced after UV radiation and MMC shock in the ΔirrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.


Assuntos
Deinococcus/fisiologia , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Mitomicina/metabolismo , Mutação de Sentido Incorreto , Fatores de Transcrição/metabolismo , Raios Ultravioleta , Substituição de Aminoácidos , Análise Mutacional de DNA , Enzimas Reparadoras do DNA/genética , Deinococcus/efeitos dos fármacos , Deinococcus/genética , Deinococcus/efeitos da radiação , Deleção de Genes , Perfilação da Expressão Gênica , Mutagênese Sítio-Dirigida , Estresse Fisiológico , Fatores de Transcrição/genética
15.
Braz J Microbiol ; 46(2): 601-11, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26273280

RESUMO

Deinococcus radiodurans (DR) is an extremophile that is well known for its resistance to radiation, oxidants and desiccation. The gene dr1790 of D. radiodurans was predicted to encode a yellow-related protein. The primary objective of the present study was to characterize the biological function of the DR1790 protein, which is a member of the ancient yellow/major royal jelly (MRJ) protein family, in prokaryotes. Fluorescence labeling demonstrated that the yellow-related protein encoded by dr1790 is a membrane protein. The deletion of the dr1790 gene decreased the cell growth rate and sensitivity to hydrogen peroxide and radiation and increased the membrane permeability of D. radiodurans. Transcript profiling by microarray and RT-PCR analyses of the dr1790 deletion mutant suggested that some genes that are involved in protein secretion and transport were strongly suppressed, while other genes that are involved in protein quality control, such as chaperones and proteases, were induced. In addition, the expression of genes with predicted functions that are involved in antioxidant systems, electron transport, and energy metabolism was significantly altered through the disruption of dr1790. Moreover, the results of proteomic analyses using 2-DE and MS also demonstrated that DR1790 contributed to D. radiodurans survival. Taken together, these results indicate that the DR1790 protein from the ancient yellow protein family plays a pleiotropic role in the survival of prokaryotic cells and contributes to the extraordinary resistance of D. radiodurans against oxidative and radiation stresses.


Assuntos
Deinococcus/genética , Genes Bacterianos , Pleiotropia Genética , Mutagênese Insercional , Proteínas de Bactérias/genética , Membrana Celular/fisiologia , Deinococcus/efeitos dos fármacos , Deinococcus/crescimento & desenvolvimento , Deinococcus/efeitos da radiação , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Peróxido de Hidrogênio/toxicidade , Proteínas de Membrana/genética , Análise em Microsséries , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Permeabilidade , Radiação Ionizante , Reação em Cadeia da Polimerase em Tempo Real
16.
PLoS One ; 10(6): e0131015, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26115538

RESUMO

The phylum Deinococcus-Thermus is a deeply-branching lineage of bacteria widely recognized as one of the most extremophilic. Members of the Thermus genus are of major interest due to both their bioremediation and biotechnology potentials. However, the molecular mechanisms associated with these key metabolic pathways remain unknown. Reverse-transcription quantitative PCR (RT-qPCR) is a high-throughput means of studying the expression of a large suite of genes over time and under different conditions. The selection of a stably-expressed reference gene is critical when using relative quantification methods, as target gene expression is normalized to expression of the reference gene. However, little information exists as to reference gene selection in extremophiles. This study evaluated 11 candidate reference genes for use with the thermophile Thermus scotoductus when grown under different culture conditions. Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB. The commonly-employed rpoC does not serve as a reliable reference gene in thermophiles, due to its expression instability across all culture conditions tested here. As extremophiles exhibit a tendency for polyploidy, absolute quantification was employed to determine the ratio of transcript to gene copy number in a subset of the genes. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes reflect transcript copy number, and not gene copy number, fluctuations. Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification indicate that relative quantification is appropriate for RT-qPCR studies with this thermophile.


Assuntos
Deinococcus/crescimento & desenvolvimento , Deinococcus/genética , Perfilação da Expressão Gênica/normas , Regulação Bacteriana da Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Técnicas Bacteriológicas/métodos , Meios de Cultura/farmacologia , Deinococcus/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos/efeitos dos fármacos , Genes Essenciais/efeitos dos fármacos , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
17.
Braz. j. microbiol ; 46(2): 601-611, Apr-Jun/2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-749726

RESUMO

Deinococcus radiodurans (DR) is an extremophile that is well known for its resistance to radiation, oxidants and desiccation. The gene dr1790 of D. radiodurans was predicted to encode a yellow-related protein. The primary objective of the present study was to characterize the biological function of the DR1790 protein, which is a member of the ancient yellow/major royal jelly (MRJ) protein family, in prokaryotes. Fluorescence labeling demonstrated that the yellow-related protein encoded by dr1790 is a membrane protein. The deletion of the dr1790 gene decreased the cell growth rate and sensitivity to hydrogen peroxide and radiation and increased the membrane permeability of D. radiodurans. Transcript profiling by microarray and RT-PCR analyses of the dr1790 deletion mutant suggested that some genes that are involved in protein secretion and transport were strongly suppressed, while other genes that are involved in protein quality control, such as chaperones and proteases, were induced. In addition, the expression of genes with predicted functions that are involved in antioxidant systems, electron transport, and energy metabolism was significantly altered through the disruption of dr1790. Moreover, the results of proteomic analyses using 2-DE and MS also demonstrated that DR1790 contributed to D. radiodurans survival. Taken together, these results indicate that the DR1790 protein from the ancient yellow protein family plays a pleiotropic role in the survival of prokaryotic cells and contributes to the extraordinary resistance of D. radiodurans against oxidative and radiation stresses.


Assuntos
Deinococcus/genética , Genes Bacterianos , Pleiotropia Genética , Mutagênese Insercional , Proteínas de Bactérias/genética , Membrana Celular/fisiologia , Deinococcus/efeitos dos fármacos , Deinococcus/crescimento & desenvolvimento , Deinococcus/efeitos da radiação , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Peróxido de Hidrogênio/toxicidade , Análise em Microsséries , Proteínas de Membrana/genética , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Permeabilidade , Radiação Ionizante , Reação em Cadeia da Polimerase em Tempo Real
18.
PLoS One ; 10(4): e0124358, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25884619

RESUMO

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.


Assuntos
Deinococcus/genética , Genes Bacterianos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Dano ao DNA , Reparo do DNA/genética , Elementos de DNA Transponíveis , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , DNA Bacteriano/efeitos da radiação , Deinococcus/efeitos dos fármacos , Deinococcus/efeitos da radiação , Fosfatos de Dinucleosídeos/fisiologia , Raios gama , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/genética , Biblioteca Gênica , Redes Reguladoras de Genes , Teste de Complementação Genética , Peróxido de Hidrogênio/farmacologia , Mitomicina/farmacologia , Mutagênese Insercional , Mutação , Fases de Leitura Aberta/genética , Estresse Oxidativo , Tolerância a Radiação/genética , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Transposases/genética , Raios Ultravioleta
19.
PLoS One ; 10(2): e0118275, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25706748

RESUMO

In order to understand the mechanism governing radiation resistance in Deinococcus radiodurans, current efforts are aimed at identifying potential candidates from a large repertoire of unique Deinococcal genes and protein families. DR0053 belongs to the DinB/YfiT protein family, which is an over-represented protein family in D. radiodurans. We observed that dr0053 transcript levels were highly induced in response to gamma radiation (γ-radiation) and mitomycin C (MMC) exposure depending on PprI, RecA and the DrtR/S two-component signal transduction system. Protein profiles demonstrated that DR0053 is a highly induced protein in cultures exposed to 10 kGy γ-radiation. We were able to determine the transcriptional start site of dr0053, which was induced upon irradiation, and to assign the 133-bp promoter region of dr0053 as essential for radiation responsiveness through primer extension and promoter deletion analyses. A dr0053 mutant strain displayed sensitivity to γ-radiation and MMC exposure, but not hydrogen peroxide, suggesting that DR0053 helps cells recover from DNA damage. Bioinformatic analyses revealed that DR0053 is similar to the Bacillus subtilis protein YjoA, which is a substrate of bacterial protein-tyrosine kinases. Taken together, the DNA damage-inducible (din) gene dr0053 may be regulated at the transcriptional and post-translational levels.


Assuntos
Proteínas de Bactérias/genética , Deinococcus/genética , Regulação Bacteriana da Expressão Gênica/genética , Mutação/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Análise Mutacional de DNA/métodos , Deinococcus/efeitos dos fármacos , Raios gama , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Mitomicina/farmacologia , Mutação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/genética
20.
Proteomics ; 15(1): 89-97, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25331933

RESUMO

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD50 = 40 µM tellurite, 40 min exposure). The organism reduced 70% of 40 µM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status. The level of ROS and protein carbonyl contents increased while the cellular reduction potential substantially decreased following tellurite exposure. Cellular thiols levels initially increased (within 30 min) of tellurite exposure but decreased at later time points. At proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes (superoxide dismutase and thioredoxin reductase), and protein folding chaperones (DnaK, EF-Ts, and PPIase) displayed increased abundance in tellurite-stressed cells. However, remarkably decreased levels of key metabolic enzymes (aconitase, transketolase, 3-hydroxy acyl-CoA dehydrogenase, acyl-CoA dehydrogenase, electron transfer flavoprotein alpha, and beta) involved in carbon and energy metabolism were observed upon tellurite stress. The results demonstrate that depletion of reduction potential in intensive tellurite reduction with impaired energy metabolism lead to tellurite toxicity in D. radiodurans.


Assuntos
Deinococcus/efeitos dos fármacos , Deinococcus/enzimologia , Telúrio/toxicidade , Proteínas de Bactérias/metabolismo , Deinococcus/metabolismo , Metabolismo Energético , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/metabolismo
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