Deinococcus wulumuqiensis R12 synthesized silver nanoparticles with peroxidase-like activity for synergistic antibacterial application.

The use of a combination of several antibacterial agents for therapy holds great promise in reducing the dosage and side effects of these agents, improving their efficiency, and inducing potential synergistic therapeutic effects. Herein, this study provides an innovative antibacterial treatment strategy by synergistically combining R12-AgNPs with H2O2 therapy. R12-AgNPs were simply produced with the supernatant of an ionizing radiation-tolerant bacterium Deinococcus wulumuqiensis R12 by one-step under room temperature. In comparison with chemically synthesized AgNPs, the biosynthesized AgNPs presented fascinating antibacterial activity and peroxidase-like properties, which endowed it with the capability to catalyze the decomposition of H2O2 to generate hydroxyl radical. After the combination of R12-AgNPs and H2O2, an excellent synergistic bacteriostatic activity was observed for both Escherichia coli and Staphylococcus aureus, especially at low concentrations. In addition, in vitro cytotoxicity tests showed R12-AgNPs had good biocompatibility. Thus, this work presents a novel antibacterial agent that exhibits favorable synergistic antibacterial activity and low toxicity, without the use of antibiotics or a complicated synthesis process.

Tolerance engineering in Deinococcus geothermalis by heterologous efflux pumps.

Producing industrially significant compounds with more environmentally friendly represents a challenging task. The large-scale production of an exogenous molecule in a host microfactory can quickly cause toxic effects, forcing the cell to inhibit production to survive. The key point to counter these toxic effects is to promote a gain of tolerance in the host, for instance, by inducing a constant flux of the neo-synthetized compound out of the producing cells. Efflux pumps are membrane proteins that constitute the most powerful mechanism to release molecules out of cells. We propose here a new biological model, Deinococcus geothermalis, organism known for its ability to survive hostile environment; with the aim of coupling the promising industrial potential of this species with that of heterologous efflux pumps to promote engineering tolerance. In this study, clones of D. geothermalis containing various genes encoding chromosomal heterologous efflux pumps were generated. Resistant recombinants were selected using antibiotic susceptibility tests to screen promising candidates. We then developed a method to determine the efflux efficiency of the best candidate, which contains the gene encoding the MdfA of Salmonella enterica serovar Choleraesuis. We observe 1.6 times more compound in the external medium of the hit recombinant than that of the WT at early incubation time. The data presented here will contribute to better understanding of the parameters required for efficient production in D. geothermalis.

Understanding the survival mechanisms of Deinococcus radiodurans against oxidative stress by targeting thioredoxin reductase redox system.

The principal objective of this study is to determine the resistance of Deinococcus radiodurans to hydrogen peroxide (H2O2) induced oxidative stress by inhibiting its thioredoxin reductase (TrxR) antioxidant system. Treatment of D. radiodurans with different TrxR inhibitors such as ebselen, epigallocatechin gallate and auranofin displayed this organism sensitivity to H2O2 treatment in a concentration-dependent manner. We observed that D. radiodurans showed greater resistance to H2O2 treatment. Further, it has also been noticed that TrxR redox system was suppressed by TrxR inhibitors and that this response might be associated with the oxidative stress-mediated cell death in D. radiodurans. Thus, TrxR inhibitors affect the resistance of the D. radiodurans through suppression of its thioredoxin redox pathway via the inhibition of TrxR. Results from this study proved that TrxR plays an important role as an antioxidant enzyme by scavenging intracellular ROS, and thus contributing to the resistance of D. radiodurans towards oxidative stress.

The interplay between Mn and Fe in Deinococcus radiodurans triggers cellular protection during paraquat-induced oxidative stress.

The bacterium Deinococcus radiodurans is highly resistant to several stress conditions, such as radiation. According to several reports, manganese plays a crucial role in stress protection, and a high Mn/Fe ratio is essential in this process. However, mobilization of manganese and iron, and the role of DNA-binding-proteins-under-starved-conditions during oxidative-stress remained open questions. We used synchrotron-based X-ray fluorescence imaging at nano-resolution to follow element-relocalization upon stress, and its dependency on the presence of Dps proteins, using dps knockout mutants. We show that manganese, calcium, and phosphorus are mobilized from rich-element regions that resemble electron-dense granules towards the cytosol and the cellular membrane, in a Dps-dependent way. Moreover, iron delocalizes from the septum region to the cytoplasm affecting cell division, specifically in the septum formation. These mechanisms are orchestrated by Dps1 and Dps2, which play a crucial role in metal homeostasis, and are associated with the D. radiodurans tolerance against reactive oxygen species.

Crystal structure of the highly radiation-inducible DinB/YfiT superfamily protein DR0053 from Deinococcus radiodurans R1.

Deinococcus radiodurans is an extremophilic bacterium well-known for its extraordinary resistance to ionizing radiation and other DNA damage- and oxidative stress-generating agents. In addition to its efficient DNA damage repair and oxidative stress resistance mechanisms, protein family expansions and stress-induced genes/proteins are also regarded as important components that add to the robustness of this bacterium. D. radiodurans encodes specific expansions of 13 DinB/YfiT homologs, which is a relatively large number when compared to those found in Gram-positive bacteria. In this study, we investigated the expression profiles of 13 dinB genes after γ-irradiation, mitomycin C and H2O2 treatment. dr0053 had the highest expression levels after DNA-damage inducing γ-irradiation and MMC treatment, increasing ∼200-fold and ∼16-fold, respectively. We also determined the crystal structure of DR0053 at 2.07 Šresolution. DR0053 adopted a typical four-helix bundle structure that is characteristic of DinB/YfiT proteins. A putative metal binding site was occupied by zinc even though the highly conserved His triad of DinB/YfiT proteins was replaced by Glu-Asn-His.

Calcium ion induces outer membrane fusion of Deinococcus grandis spheroplasts to generate giant spheroplasts with multiple cytoplasms.

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.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

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.

Targeting the Unique Mechanism of Bacterial 1-Deoxy-d-xylulose-5-phosphate Synthase.

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.

Deinococcus radiodurans HD-Pnk, a Nucleic Acid End-Healing Enzyme, Abets Resistance to Killing by Ionizing Radiation and Mitomycin C.

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.

Low concentrations of ethanol during irradiation drastically reduce DNA damage caused by very high doses of ionizing radiation.

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.

Phage Display on the Anti-infective Target 1-Deoxy-d-xylulose-5-phosphate Synthase Leads to an Acceptor-Substrate Competitive Peptidic Inhibitor.

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.

Methylation of Ir(iii)-tetrazolato complexes: an effective route to modulate the emission outputs and to switch to antimicrobial properties.

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.

A Bright Future for Antibiotics?

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.

Oxidative stress response of Deinococcus geothermalis via a cystine importer.

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.

[Antioxidant function of the iron binding protein DRA0258 in Deinococcus radiodurans]

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.

Autoinducer-2 signaling is involved in regulation of stress-related genes of Deinococcus radiodurans.

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.

Untargeted metabolite profiling reveals that nitric oxide bioynthesis is an endogenous modulator of carotenoid biosynthesis in Deinococcus radiodurans and is required for extreme ionizing radiation resistance.

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.

Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro.

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.

The Site-Directed A184S Mutation in the HTH Domain of the Global Regulator IrrE Enhances Deinococcus radiodurans R1 Tolerance to UV Radiation and MMC Shock.

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.

Characteristics of dr1790 disruptant and its functional analysis in Deinococcus radiodurans.

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.