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Catalytic reduction of organic pollutants using metal nanoparticles (MNPs) as nanocatalyst is a promising approach. The widespread application of MNPs have led to an increase demand for their production. The green biosynthesis of MNPs using microbes is becoming increasingly important as they are benign and environmentally friendly. In this work, Deinococcus wulumuqiensis (D. wulumuqiensis) with high resistance to stresses was applied for the green preparation of different MNPs, including palladium nanoparticles (Pd NPs), silver nanoparticles (Ag NPs), and Pd−Ag bimetallic nanoparticles (Pd−Ag BNPs). The resultant MNPs exhibited a face‐centered cubic structure, with average size of 6.70 (Pd NPs), 6.24 (Ag NPs), and 6.13 nm (Pd−Ag BNPs), respectively. FT‐IR spectra indicated that the proteins and polysaccharides expressed by D. wulumuqiensis could potentially contributed to the formation of MNPs. The application of as‐prepared biocatalyst in catalytic degradation of pollutant dyes demonstrated that Pd−Ag BNPs had greater degradation ability of 4‐nitrophenol (95.1 %) and Rhodamine B (85.1 %) than monometallic nanoparticles. The pseudo‐first‐order kinetics results further indicated the superior catalytic ability of Pd−Ag BNPs, with rate constant of 0.450±0.009 (4‐nitrophenol) and 1.54×10−2 min−1 (Rhodamine B), respectively. Thus, the present study confirms that D. wulumuqiensis represents a promising candidate for the biosynthesis of Pd‐based nanoparticles with small size, which exhibited potential applications as bionanocatalyst in the degradation of water pollutants. [ABSTRACT FROM AUTHOR]

Terrestrial organisms have systems to escape from desiccation stresses. For example, tardigrades (also known as water bears) can survive severe dried and other extreme environments by anhydrobiosis. Although their extraordinary ability has enchanted people, little is known about the detailed molecular mechanisms of anhydrobiosis. Here, we focused on the tardigrade Ramazzottius varieornatus, one of the toughest animals on Earth. A transcriptome database of R. varieornatus shows that genes encoding a Ferritin‐like protein are upregulated during desiccation or ultraviolet radiation. This protein shows sequence similarity to enigmatic proteins in desiccation‐tolerant bacteria and plants, which are hypothesized to be desiccation‐related. However, because these proteins lack detailed biological information, their functions are relatively unknown. We determined an atomic (1.05 Å) resolution crystal structure of a Ferritin‐like protein from R. varieornatus. The structure revealed a dinuclear metal binding site, and we showed that this Ferritin‐like protein has phosphatase activity toward several metabolite compounds including unusual nucleotide phosphates produced by oxidative or radiation damage. We also found that a homologous protein from a desiccation‐ and ultraviolet‐tolerant bacterium Deinococcus radiodurans is a metabolite phosphatase. Our results indicate that through cleaning up damaged metabolites or regulation of metabolite levels, this phosphatase family can contribute to stress tolerances. This study provides a clue to one of the universal molecular bases of desiccation‐stress tolerance. [ABSTRACT FROM AUTHOR]

Consuming microbiologically‐contaminated water is the primary cause of many water‐borne diseases and deaths worldwide. Governments aim at providing drinking water for vulnerable populations, especially through low‐cost interventions. Therefore, the solar disinfection (SODIS) of such pathogens provides a simple and cost‐effective way to obtain good quality water. In this procedure, PET bottles are filled with contaminated water and exposed to sunlight for 1–2 days. To accelerate decontamination, methylene blue (MB) dye added as a photocatalyst, boosts singlet oxygen generation upon absorbing red‐band sunlight. This study explores the use of a Sunlight Simulator (SSL) device to research and standardize the SODIS method with a vital dye as MB. PET bottles were filled with artificially‐contaminated water with Streptococcus epidermidis and Deinococcus radiodurans Gram‐positive bacteria, Escherichia coli and Salmonella typhimurium Gram‐negative bacteria, or bacteriophage λ as well. In all experiments, 50 ng/mL MB ensued a synergistic lethal effect after SSL exposure. The results indicate that bacterial and bacteriophage inactivation can be achieved in shorter times with MB‐SSL treatment compared to SSL without MB. In this sense, when compared to previous sunlight‐SODIS results, the SSL source is a reliable tool to study the parameters of both SODIS and MB‐SODIS protocols, and also a feasible tool to afford assays whenever there are unfavorable climate conditions. [ABSTRACT FROM AUTHOR]

The accumulation of manganese ions is crucial for scavenging reactive oxygen species and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well‐conserved domains – a DNA‐binding domain, a dimerisation domain, and an ancillary domain – the mechanisms underlying both, metal ion activation and DNA recognition remain elusive. In this study, we present biophysical analyses and the structure of the dimerisation and DNA‐binding domains of DR2539 in its holo‐form and in complex with the 21 base pair pseudo‐palindromic repeat of the dr1709 promoter region, shedding light on these activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein‐DNA interfaces elucidates the significance of Tyr59 and helix α3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small‐angle X‐ray scattering experiments and supported by AlphaFold modeling provides a model illustrating the conformational changes induced upon metal binding. [ABSTRACT FROM AUTHOR]

Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α‐hydroxyketones, such as (S)‐2‐hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine‐tuned gene expression was achieved using an expression plasmid under the control of the LacIQ/Ptrc system, and the cross‐protective role of PprI was assessed against multiple stress treatments. Moreover, the stress‐tolerant P. putida strain was tested for 2‐hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2O2 and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2‐hydroxypropiophenone more efficiently than the parental P. putida strain. 2‐Hydroxypropiophenone concentration reached 1.6 g L−1 upon a 3‐h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL−1 in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2‐HPP g−1 benzaldehyde and 0.089 g 2‐HPP g cell dry weight−1 h−1, respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2‐HPP production in P. putida ATCC 12633. [ABSTRACT FROM AUTHOR]

It has been proposed that transient and reversible phenotypic changes could modify the response of bacteria to germicidal radiation, eventually leading to tailing in the survival curves. If this were the case, changes in susceptibility to radiation would reflect variations in gene expression and should only occur in cells in which gene expression is active. To obtain experimental evidence supporting the involvement of phenotypic changes in the origin of tailing, we studied changes in the susceptibility to radiation of cells able to survive high fluences, using split irradiations. Stationary phase cells of Enterobacter cloacae and Deinococcus radiodurans, in which gene expression is active, and spores of Bacillus subtilis, which are dormant cells without active gene expression, were used as microbial models. While cells of E. cloacae and D. radiodurans became susceptible after surviving exposures to high fluences, tolerant spores exhibited unchanged response to radiation. The results can be interpreted assuming that noise in gene expression modifies bacterial susceptibility to radiation, and tailing is the result of intrinsic phenomena of bacterial physiology rather than a technical artifact. For either theoretical or practical purposes, deviations from simple exponential decay kinetics should be considered in estimations of the effects of germicidal radiation at high fluences. [ABSTRACT FROM AUTHOR]

Free‐living organisms frequently encounter unfavorable abiotic environmental factors. Those who adapt and cope with sudden changes in the external environment survive. Desiccation is one of the most common and frequently encountered stresses in nature. On the contrary, ionizing radiations are limited to high local concentrations of naturally occurring radioactive materials and related anthropogenic activities. Yet, resistance to high doses of ionizing radiation is evident across the tree of life. The evolution of desiccation resistance has been linked to the evolution of ionizing radiation resistance, although, evidence to support the idea that the evolution of desiccation tolerance is a necessary precursor to ionizing radiation resistance is lacking. Moreover, the presence of radioresistance in hyperthermophiles suggests multiple paths lead to radiation resistance. In this minireview, we focus on the molecular aspects of damage dynamics and damage response pathways comprising protective and restorative functions with a definitive survival advantage, to explore the serendipitous genesis of ionizing radiation resistance. [ABSTRACT FROM AUTHOR]

Desiccation impacts a suite of physiological processes in microbes by elevating levels of damaging reactive oxygen species and inducing DNA strand breaks. In response to desiccation‐induced stress, microbes have evolved specialized mechanisms to help them survive. Here, we performed a 128‐day lab desiccation experiment on nine strains from three clades of an abundant soil bacterium, Curtobacterium. We sequenced RNA from each strain at three time points to investigate their response. Curtobacterium was highly resistant to desiccation, outlasting both Escherichia coli and a famously DNA damage‐resistant bacterium, Deinococcus radiodurans. However, within the genus, there were also 10‐fold differences in survival rates among strains. Transcriptomic profiling revealed responses shared within the genus including up‐regulation of genes involved in DNA damage repair, osmolyte production, and efflux pumps, but also up‐regulation of pathways and genes unique to the three clades. For example, trehalose synthesis gene otsB, the chaperone groEL, and the oxygen scavenger katA were all found in either one or two clades but not the third. Here, we provide evidence of considerable variation in closely related strains, and further elucidation of the phylogenetic conservation of desiccation tolerance remains an important goal for microbial ecologists. [ABSTRACT FROM AUTHOR]

To better understand the characteristic properties of violacein biosynthesized by engineered Escherichia coli VioABCDE‐SD, a convenient and simplified method was designed to extract violacein and its stability, antimicrobial activity, and antioxidant capacity were analyzed. Different from the traditional extraction methods, our new method is easier and less time consuming and can directly obtain violacein dry powder product with a higher extraction rate. Low temperature, dark condition, neutral pH, reducing agents, Ba2+, Mn2+, Ni2+, Co2+, and some food additives of sucrose, xylose, and glucose were conducive to maintaining its stability. The violacein also exhibited surprisingly high bacteriostatic action against Gram‐positive Bacillus subtilis, Deinococcus radiodurans R1, and Staphylococcus aureus and Gram‐negative Pseudomonas aeruginosa, but no effect on E. coli. The violacein of VioABCDE‐SD exhibited strong antioxidant activity, with the scavenging rate of 1,1‐diphenyl‐2‐picrylhydrazyl free radicals reaching 60.33%, the scavenging efficiency of hydroxyl radical scavenging reaching 56.34%, and the total antioxidant capacity reaching 0.63 U/mL. Violacein from VioABCDE‐SD can be synthesized directionally with better stability, antibacterial, and antioxidant properties compared with that from the original strain Janthinobacterium sp. B9‐8. Therefore, our study indicated that violacein from engineered E. coli VioABCDE‐SD was a kind of new antibiotic with potential biological activities, which may have potential utility in multiple areas such as pharmacological, cosmetics, and healthy food industries. [ABSTRACT FROM AUTHOR]

The polymerization/depolymerization dynamics of FtsZ play a pivotal role in cell division in the majority of the bacteria. Deinococcus radiodurans, a radiation‐resistant bacterium, shows an arrest of growth in response to DNA damage with no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode SulA homologues of Escherichia coli, which attenuate FtsZ functions in response to DNA damage in other bacteria. A radiation‐responsive Ser/Thr quinoprotein kinase (RqkA), characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins, including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we reported the detailed characterization of S235 and S335 phosphorylation effects in the regulation of drFtsZ functions and demonstrated that the phospho‐mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans. Interestingly, the phospho‐ablative replacements were found to be nearly similar to drFtsZ, whereas the phospho‐mimetic mutant lost the wild‐type protein's signature characteristics, including its dynamics under normal conditions. The kinetics of post‐bleaching recovery for drFtsZ and phospho‐mimetic mutant were nearly similar at 2 h post‐irradiation recovery but were found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage, which is demonstrated for the first time, in any bacteria. [ABSTRACT FROM AUTHOR]

Uracil‐DNA glycosylase (UDG) initiates base excision repair (BER) by removing damaged or modified nucleobases during DNA repair or mammalian demethylation. The UDG superfamily consists of at least six families with a variety of catalytic specificities and functions. Deinococcus radiodurans, an extreme radiation resistant bacterium, contains multiple members of UDG enzymes within its genome. The present study reveals that the putative protein, DR0022, is a uracil‐DNA glycosylase that requires acidic conditions for its glycosylase activity, which is the first case of such an enzyme within the UDG superfamily. The key residues in the catalytic motifs are investigated by biochemical, enzyme kinetics, and de novo structural prediction, as well as molecular modeling analyses. The structural and catalytic roles of several distinct residues are discussed in light of predicted and modeled DR0022 glycosylase structures. The spontaneous mutation rate analysis performed in a dr0022 deficient D. radiodurans strain indicated that the dr0022 gene plays a role in mutation prevention. Furthermore, survival rate analysis in a dr0022 deficient D. radiodurans strain demonstrated its role in stress resistance, including γ‐irradiation. Additionally, the novel acid UDG activity in relationship to its in vivo roles is discussed. This work underscores the functional diversity in the UDG superfamily. [ABSTRACT FROM AUTHOR]

To investigate the properties of carotenoids from the extremophile Deinococcus xibeiensis R13, the factors affecting the stability of carotenoids extracted from D. xibeiensis R13, including temperature, illumination, pH, redox chemicals, metal ions, and food additives, were investigated. The results showed that low temperature, neutral pH, reducing agents, Mn2+, and food additives (xylose and glucose) can effectively improve the stability of Deinococcus carotenoids. The carotenoids of D. xibeiensis R13 exhibited strong antioxidant activity, with the scavenging rate of hydroxyl radicals reaching 71.64%, which was higher than the scavenging efficiency for 1,1‐diphenyl‐2‐picrylhydrazyl free radicals and 2,2'‐azino‐bis (3‐ethyl‐benzothiazoline‐6‐sulfonic acid) free radicals (44.55 and 27.65%, respectively). In addition, the total antioxidant capacity reached 0.60 U/ml, which was 2.61‐fold that of carotenoids from the model strain Deinococcus radiodurans R1. Finally, we predicted the gene clusters encoding carotenoid biosynthesis pathways in the genome of R13 and identified putative homologous genes. The key enzyme genes (crtE, crtB, crtI, crtLm, cruF, crtD, and crtO) in carotenoid synthesis of D. xibeiensis R13 were cloned to construct the multigene coexpression plasmids pET‐EBI and pRSF‐LmFDO. The carotenoid biosynthesis pathway was heterologously introduced into engineered Escherichia coli EBILmFDO, which exhibited a higher yield (7.14 mg/L) than the original strain. These analysis results can help us to better understand the metabolic synthesis of carotenoids in extremophiles. [ABSTRACT FROM AUTHOR]

α,ω‐Dicarboxylic acids are valuable precursors in various chemical industries and have recently been produced using biotechnological methods to overcome the limitations of chemical synthesis. Nonanedioic acid, decanedioic acid, undecanedioic acid, and dodecanedioic acid have been produced at high concentrations from ω‐hydroxycarboxylic acids using engineered biocatalysts. However, no study has been attempted on the efficient production of pentadecanedioic acid. Here, the production of pentadecanedioic acid from 15‐hydroxypentadecanoic acid was carried out with alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs), and NAD(P)H oxidases, including NAD(P)H flavin oxidoreductase (NFO), that used a co‐factor regeneration system, derived from several species and expressed in Escherichia coli. Among the enzymes, Kangiella koreensis ADH (KkADH), Geobacillus kaustophilus ALDH (GkALDH), and Deinococcus radiodurans NFO (DrNFO) were selected because they had the highest activity. E. coli expressing pRSF‐DrNFO and pACYC‐KkADH/GkALDH as the best distribution of three genes in two plasmids was used as a biocatalyst to produce pentadecanedioic acid. The optimal conditions for producing pentadecanedioic acid from 15‐hydroxypentadecanoic acid by the biocatalyst were pH 8.0, 35°C, 5% (v/v) methanol, 40 g L−1 cells, and 60 mM 15‐hydroxypentadecanoic acid with agitation at 250 rpm. Under these optimized conditions, 57.4 mM pentadecanedioic acid was produced after 3 h, with a molar yield of 95.6% and a productivity of 19.1 mM h−1. The molar yield and concentration of pentadecanedioic acid showed the highest values among the reported biotechnological production of pentadecanedioic acid. [ABSTRACT FROM AUTHOR]

Oxidative stress alters cell viability, from microorganism irradiation sensitivity to human aging and neurodegeneration. Deleterious effects of protein carbonylation by reactive oxygen species (ROS) make understanding molecular properties determining ROS susceptibility essential. The radiation‐resistant bacterium Deinococcus radiodurans accumulates less carbonylation than sensitive organisms, making it a key model for deciphering properties governing oxidative stress resistance. We integrated shotgun redox proteomics, structural systems biology, and machine learning to resolve properties determining protein damage by γ‐irradiation in Escherichia coli and D. radiodurans at multiple scales. Local accessibility, charge, and lysine enrichment accurately predict ROS susceptibility. Lysine, methionine, and cysteine usage also contribute to ROS resistance of the D. radiodurans proteome. Our model predicts proteome maintenance machinery, and proteins protecting against ROS are more resistant in D. radiodurans. Our findings substantiate that protein‐intrinsic protection impacts oxidative stress resistance, identifying causal molecular properties. Synopsis: Proteins differ in intrinsic vulnerability to carbonylation, an adaptation for oxidative stress tolerance. Here, an integrated proteomics, protein structure, and machine learning approach unravels properties determining damage by γ‐irradiation in two bacterial species at multiple biological scales. γ‐irradiation causes more‐targeted oxidative degradation of proteins in D. radiodurans than in E. coli, suggesting the evolution of protein‐specific protection mechanisms against oxidative stress.D. radiodurans proteins enriched in methionine and cysteine but depleted in lysine are more resistant to oxidative degradation.Protein‐intrinsic molecular properties including local solvent accessibility, electrostatic charge, and lysine enrichment predict site‐specific vulnerability to protein carbonylation by reactive oxygen species in E. coli and D. radiodurans.D. radiodurans ribosomal proteins, chaperones, and proteins involved in response to and detoxification of reactive oxygen species are more resistant to carbonylation than their E. coli orthologs. [ABSTRACT FROM AUTHOR]

Herein, we describe a new family of tris chelate homoleptic Ru (II) complexes, [Ru(N^N)3]2+, where the role of the diimine‐type ligands (N^N) was fulfilled by 2‐pyridyl (PTZ) or 2‐quinolyl tetrazole (QTZ) derivatives decorated with various alkyl substituents at the N‐2 position of the tetrazole ring. The new Ru (II) complexes with general formula [Ru (PTZ‐R)3]2+ and [Ru (QTZ‐R)3]2+, were obtained as mixtures of facial (fac) and meridional (mer) isomers, as suggested by NMR (1H, 13C) experiments, and confirmed in the case of mer‐[Ru (QTZ‐Me)3]2+, by X‐ray crystallography. The photophysical behavior of the tetrazole‐based [Ru(N^N)3]2+ type species was investigated by UV–vis absorption spectroscopy, providing trends typical of polypyridyl Ru (II) complexes. The new homoleptic complexes fac/mer‐[Ru (PTZ‐R)3]2+ and fac/mer‐[Ru (QTZ‐R)3]2+ have been assessed for any eventual antimicrobial activity towards two different bacteria such as Gram‐negative Escherichia coli and Gram‐positive Deinococcus radiodurans. Whereas being inactive toward E. coli, the response of agar disks diffusion tests suggested that some of the new fac/mer Ru (II) complexes could inhibit the growth of D. radiodurans. This effect was further investigated by determining the growth kinetics in liquid medium of D. radiodurans exposed to the fac/mer‐[Ru (PTZ‐R)3]2+ and fac/mer‐[Ru (QTZ‐R)3]2+ complexes at different concentrations. The outcome of these experiments highlighted that the turn‐on of the growth inhibitory effect took place as the linear hexyl chain was appended to the PTZ or QTZ scaffold, suggesting also how the inhibitory activity appeared more pronouncedly exerted by the facial isomers fac‐[Ru (PTZ‐Hex)3]2+ and fac‐[Ru (QTZ‐Hex)3]2+ (MIC = ca. 3.0 μg/ml) with respect to the corresponding meridional isomers (MIC = ca. 6.0 μg/ml). [ABSTRACT FROM AUTHOR]

Summary: The GC‐rich genome of Deinococcus radiodurans contains a very high density of putative guanine quadruplex (G4) DNA motifs and its RecQ (drRecQ) was earlier characterized as a 3′→5′ dsDNA helicase. We saw that N‐Methyl mesoporphyrin IX (NMM), a G4 DNA binding drug affected normal growth as well as the gamma radiation resistance of the wild‐type bacterium. Interestingly, NMM treatment and recQ deletion showed additive effect on normal growth but there was no effect of NMM on gamma radiation resistance of recQ mutant. The recombinant drRecQ showed ~400 times higher affinity to G4 DNA (Kd = 11.74 ± 1.77 nM) as compared to dsDNA (Kd = 4.88 ± 1.30 µM). drRecQ showed ATP independent helicase function on G4 DNA, which was higher than ATP‐dependent helicase activity on dsDNA. Unlike wild‐type cells that sparingly stained for G4 structure with Thioflavin T (ThT), recQ mutant showed very high‐density of ThT fluorescence foci on DNA indicating an important role of drRecQ in regulation of G4 DNA structure dynamics in vivo. These results together suggested that drRecQ is an ATP independent G4 DNA helicase that plays an important role in the regulation of G4 DNA structure dynamics and its impact on radioresistance in D. radiodurans. [ABSTRACT FROM AUTHOR]

Summary: Late embryogenesis abundant (LEA) proteins play a protective role during desiccation and oxidation stresses. LEA3 proteins are a major group characterized by a hydrophilic domain (HD) with a highly conserved repeating 11‐amino acid motif. We compared four different HD orthologs from distant organisms: (i) DrHD from the extremophilic bacterium Deinococcus radiodurans; (ii) CeHD from the nematode Caenorhabditis elegans; (iii) YlHD from the yeast Yarrowia lipolytica; and (iv) BnHD from the plant Brassica napus. Circular dichroism spectroscopy showed that all four HDs were intrinsically disordered in phosphate buffer and then folded into α‐helical structures with the addition of glycerol or trifluoroethanol. Heterologous HD expression conferred enhanced desiccation and oxidation tolerance to Escherichia coli. These four HDs protected the enzymatic activities of lactate dehydrogenase (LDH) by preventing its aggregation under desiccation stress. The HDs also interacted with LDH, which was intensified by the addition of hydrogen peroxide (H2O2), suggesting a protective role in a chaperone‐like manner. Based on these results, the HDs of LEA3 proteins show promise as protectants for desiccation and oxidation stresses, especially DrHD, which is a potential ideal stress‐response element that can be applied in synthetic biology due to its extraordinary protection and stress resistance ability. [ABSTRACT FROM AUTHOR]

Red‐light photosensory proteins, phytochromes, link light activation to biological functions by interconverting between two conformational states. For this, they undergo large‐scale secondary and tertiary changes which follow small‐scale Z to E bond photoisomerization of the covalently bound bilin chromophore. The complex network of amino acid interactions in the chromophore‐binding pocket plays a central role in this process. Highly conserved Y263 and H290 have been found to be important for the photoconversion yield, while H260 has been identified as important for bilin protonation and proton transfer steps. Here, we focus on the roles these amino acids are playing in preserving the chemical properties of bilin in the resting Pr state of the photosensory unit of a bacteriophytochrome from Deinococcus radiodurans. By using pH‐dependent UV‐Vis spectroscopy and spectral decomposition modeling, we confirm the importance of H260 for biliverdin protonation. Further, we demonstrate that in the canonical bacteriophytochromes, the pKa value of the phenol group of the Y263 is uncommonly low. This directly influences the protonation of the bilin molecule and likely the functional properties of the protein. Our study expands the understanding of the tight interplay between the nearby amino acids and bilin in the phytochrome family. [ABSTRACT FROM AUTHOR]

Pyrrolidone‐carboxylate peptidase (PCP) catalyzes the removal of an unusual amino acid, l‐pyroglutamate (pG), from the N‐termini of peptides and proteins. It has implications in the functional regulation of different peptides in both prokaryotes and eukaryotes. However, the pG‐recognition mechanism of the PCP enzyme remains largely unknown. Here, crystal structures of PCP I from Deinococcus radiodurans (PCPdr) are reported in pG‐free and pG‐bound forms at resolutions of 1.73 and 1.55 Å, respectively. Four protomers in PCPdr form a tetrameric structure. The residues responsible for recognizing the pG residue are mostly contributed by a flexible loop (loop A) that is present near the active site. These residues are conserved in all known PCPs I, including those from mammals. Phe9 and Phe12 of loop A form stacking interactions with the pyrrolidone ring of pG, while Asn18 forms a hydrogen bond to OE of pG. The main chain of a nonconserved residue, Leu71, forms two hydrogen bonds to NH and OE of pG. Thus, pG is recognized in the S1 substrate subsite of the enzyme by both van der Waals and polar interactions, which provide specificity for the pG residue of the peptide. In contrast to previously reported PCP I structures, the PCPdr tetramer is in a closed conformation with an inaccessible active site. The structures show that the active site can be accessed by the substrates via disordering of loop A. This disordering could also prevent product inhibition by releasing the bound pG product from the S1 subsite, thus allowing the enzyme to engage a fresh substrate. The crystal structure of pyroglutamate (pG) bound PCP I enzyme reveals the mechanism of its pG specificity. The residues of a conserved active‐site loop interact with pG via phenylalanine stacking interactions and hydrogen bonds. [ABSTRACT FROM AUTHOR]

Deinococcus radiodurans R1 is extremely resistant to ionizing radiation and oxidative stress. In this study, we characterized DR0846, a candidate peroxiredoxin in D. radiodurans. DR0846 is a peroxiredoxin Q containing two conserved cysteine residues. DR0846 exists mainly in monomeric form with an intramolecular disulfide bond between the two cysteine residues. We found that DR0846 functions as a molecular chaperone as well as a peroxidase. A mutational analysis indicates that the two cysteine residues are essential for enzymatic activity. A double‐deletion mutant lacking DR0846 and catalase DR1998 exhibits decreased oxidative and heat shock stress tolerance with respect to the single mutants or the wild‐type cells. These results suggest that DR0846 contributes to resistance against oxidative and heat stresses in D. radiodurans. [ABSTRACT FROM AUTHOR]

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. The resistance mechanism is unknown, but an efficient reactive oxygen species (ROS) scavenging system and DNA‐repair and DNA‐protection mechanisms are believed to play important roles. Here, the cloning and small‐ and medium‐scale expression tests of a novel dye‐decolourizing peroxidase from D. radiodurans (DrDyP) using three different Escherichia coli strains and three different temperatures in order to identify the optimum conditions for the expression of recombinant DrDyP are presented. The best expression conditions were used for large‐scale expression and yielded ∼10 mg recombinant DrDyP per litre of culture after purification. Initial characterization experiments demonstrated unusual features with regard to the haem spin state, which motivated the crystallization experiment. The obtained crystals were used for data collection and diffracted to 2.2 Å resolution. The crystals belonged to the trigonal space group P31 or P32, with unit‐cell parameters a = b = 64.13, c = 111.32 Å, and are predicted to contain one DrDyP molecule per asymmetric unit. Structure determination by molecular replacement using previously determined structures of dye‐decolourizing peroxidases with ∼30% sequence identity at ∼2 Å resolution as templates are ongoing. [ABSTRACT FROM AUTHOR]

Xaa-Pro peptidases (XPP) are dinuclear peptidases of MEROPS M24B family that hydrolyze Xaa-Pro iminopeptide bond with a trans-proline at the second position of the peptide substrate. XPPs specific towards dipeptides are called prolidases while those that prefer longer oligopeptides are called aminopeptidases P. Though XPPs are strictly conserved in bacterial and archaeal species, the structural and sequence features that distinguish between prolidases and aminopeptidases P are not always clear. Here, we report 1.4 Å resolution crystal structure of a novel XPP from Deinococcus radiodurans (XPPdr). XPPdr forms a novel dimeric structure via unique dimer stabilization loops of N-terminal domains such that their C-terminal domains are placed far apart from each other. This novel dimerization is also the consequence of a different orientation of N-terminal domain in XPPdr monomer than those in other known prolidases. The enzymatic assays show that it is a prolidase with broad substrate specificity. Our structural, mutational, and molecular dynamics simulation analyses show that the conserved Arg46 of N-terminal domain is important for the dipeptide selectivity. Our BLAST search found XPPdr orthologs with conserved sequence motifs which correspond to unique structural features of XPPdr, thus identify a new subfamily of bacterial prolidases. [ABSTRACT FROM AUTHOR]

RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a recF-dr1088 operon, which is highly conserved in the Deinococcus-Thermus phylum, was identified in Deinococcus radiodurans. Interaction between DRRecF and DR1088 was confirmed by yeast two-hybrid and pull-down assays. DR1088 exhibited some RecO-like biochemical properties including single/double-stranded DNA binding activity, ssDNA binding protein (SSB) replacement ability and ssDNA (with or without SSB) annealing activity. However, unlike other recombination proteins, dr1088 is essential for cell viability. These results indicate that DR1088 might play a role in DNA replication and DNA repair processes. [ABSTRACT FROM AUTHOR]

Trehalose synthase (TS) catalyzes the reversible conversion of maltose to trehalose and belongs to glycoside hydrolase family 13 (GH13). Previous mechanistic analysis suggested a rate-limiting protein conformational change, which is probably the opening and closing of the active site. Consistently, crystal structures of Deinococcus radiodurans TS (DrTS) in complex with the inhibitor Tris displayed an enclosed active site for catalysis of the intramoleular isomerization. In this study, the apo structure of the DrTS N253F mutant displays a new open conformation with an empty active site. Analysis of these structures suggests that substrate binding induces a domain rotation to close the active site. Such a substrate-induced domain rotation has also been observed in some other GH13 enzymes. [ABSTRACT FROM AUTHOR]

2′,3′-Cyclic phosphodiesterase (CPDase) homologues have been found in all domains of life and are involved in diverse RNA and nucleotide metabolisms. The CPDase from Deinococcus radiodurans was crystallized and the crystals diffracted to 1.6 Å resolution, which is the highest resolution currently known for a CPDase structure. Structural comparisons revealed that the enzyme is in an open conformation in the absence of substrate. Nevertheless, the active site is well formed, and the representative motifs interact with sulfate ion, which suggests a conserved catalytic mechanism. [ABSTRACT FROM AUTHOR]

Lineage-specific Gfh factors from the radioresistant bacterium Deinococcus radiodurans, which bind within the secondary channel of RNA polymerase, stimulate transcriptional pausing at a wide range of pause signals (elemental, hairpin-dependent, post-translocated, backtracking-dependent, and consensus pauses) and increase intrinsic termination. Universal bacterial factor NusA, which binds near the RNA exit channel, enhances the effects of Gfh factors on termination and hairpin-dependent pausing but do not act on other pause sites. It is proposed that NusA and Gfh target different steps in the pausing pathway and may act together to regulate transcription under stress conditions. Thus, transcription factors that interact with nascent RNA in the RNA exit channel can communicate with secondary channel regulators to modulate RNA polymerase activities. [ABSTRACT FROM AUTHOR]

DR2231 from Deinococcus radiodurans was previously functionally and structurally characterized as an all-α NTP pyrophosphohydrolase with specific d UTPase activity. d UTPases have a central role in the regulation of d UTP intracellular levels and d TTP nucleotide metabolism. DR2231 presents a conserved dimetal catalytic site, similar to all-α dimeric d UTPases, but contrary to these enzymes, it is unable to process d UDP. In this article, we present functional and structural evidence of single-point mutations that affect directly or indirectly the enzyme catalysis and provide a complete description of the all-α NTP pyrophosphohydrolase mechanism. Activity assays, isothermal titration calorimetry and the crystal structures of these mutants obtained in complex with d UMP or a d UTP analogue aid in probing the reaction mechanism. Our results demonstrate that the two metals are necessary for enzyme processing and also important to modulate the substrate binding affinity. Single-point mutations located in a structurally mobile lid-like loop show that the interactions with the nucleoside monophosphate are essential for induction of the closed conformation and ultimately for substrate processing. β- and γ-phosphates are held in place through coordination with the second metal, which is responsible for the substrate 'gauche' orientation in the catalytic position. The lack of sufficient contacts to orient the d UDP β-phosphate for hydrolysis explains DR2231 preference towards d UTP. Sequence and structural similarities with MazG proteins suggest that a similar mechanism might be conserved within the protein family. Database Structural data are available in the PDB under the accession numbers , , , , , , . [ABSTRACT FROM AUTHOR]

Here, we show that AHLs can be employed by Deinococcus radiodurans, which belongs to the unique phylum Deinococcus-Thermus and is known for its cellular resistance to environmental stresses. An AHL-mediated quorum-sensing system (DqsI/DqsR) was identified in D. radiodurans. We found that under non-stress conditions, the AHL level was 'shielded' by quorum quenching enzymes, whereas AHLs accumulated when D. radiodurans was exposed to oxidative stress. Upon exposure to H2O2, AHL synthetic enzymes (DqsI) were immediately induced, while the expression of quorum-quenching enzymes began to increase approximately 30 min after exposure to H2O2, as shown by time-course analyses of gene expression. Both dqsI mutant (DMDqsI) and dqsR mutant (MDqsR) were more sensitive to oxidative stress compared with the wild-type strain. Exogenous AHLs (5 μM) could completely restore the survival fraction of DMDqsI under oxidative stress. RNA-seq analysis showed that a number of genes involved in stress-response, cellular cleansing, and DNA repair had altered transcriptional levels in MDqsR. The DqsR, acting as a regulator of quorum sensing, controls gene expression along with AHLs. Hence, the DqsIR-mediated quorum sensing that mediates gene regulation is an adaptive strategy for D. radiodurans in response to oxidative stresses and is conserved in the extremophilic Deinococcus bacteria. [ABSTRACT FROM AUTHOR]

The DNA binding proteins from starved cells from Deinococcus radiodurans, Dps1-DR2263 and Dps2-DRB0092, have a common overall structure of hollow spherical dodecamers. Their involvement in the homeostasis of intracellular metal and DNA protection was addressed. Our results show that DrDps proteins are able to oxidize ferrous to ferric iron by oxygen or hydrogen peroxide. The iron stored inside the hollow sphere cavity is fully released. Furthermore, these proteins are able to store and release manganese, suggesting they can play a role in manganese homeostasis as well. The interaction of DrDps with DNA was also addressed. Even though DrDps1 binds both linear and coiled DNA, DrDps2 preferentially binds to coiled DNA, forming different protein-DNA complexes, as clearly shown by atomic force microscopy. DrDps1 (dimer and dodecamer) and DrDps2 can protect DNA against reactive oxygen species, although the protection occurs at different Fe to protein ratios. The difference between DrDps could be the result of the DrDps1 higher iron oxidation rate in the presence of hydrogen peroxide and its higher affinity to bind DNA than in DrDps2. Using cellular extracts obtained from D. radiodurans cultures, we showed that DrDps1 oligomers observed in in vitro conditions are also present in vivo. This indicates that DrDps1 has a structural dynamic plasticity that allows its oligomeric state to change between dimer, trimer and dodecamer. This in turn suggests the existence of a regulation mechanism that modulates the oligomer equilibrium and is dependent on growth stages and environmental conditions. [ABSTRACT FROM AUTHOR]

Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans ( DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues in the N-terminus of a symmetry-related molecule and the complementary DNA strand facing away from the active site were also observed which seem to stabilize the enzyme-DNA complex. However, the significance of this observation remains to be investigated. The results provide new insights into the current knowledge about DNA damage recognition and repair by uracil-DNA glycosylases. [ABSTRACT FROM AUTHOR]

Carotenoids are among the key biomarkers in the search for life on other planets, and non-destructive Raman spectroscopy on future rover missions is a potential sensitive detection method, especially under resonant conditions. In this research, reflectance spectra of minerals and microorganisms were measured using ultraviolet/visible diffuse reflectance spectroscopy in order to evaluate potential resonance Raman conditions and the possible degree of sample damage during laser irradiation. We report a photodegradation and semi-quantitative Raman study of β-carotene and the carotenoid-containing extremophile Deinococcus radiodurans mixed with calcite at excitation wavelengths of 440 nm, 532 nm and 785 nm. A different type of carotenoid was detected in a culture of Chroococcidiopsis. Carotenoids embedded in bacterial membranes were found to be less sensitive to photodegradation than in a mineral matrix. Corrections for self-absorption effects were performed using the 1085 cm−1 peak of calcite as an internal standard. Carotenoid-type signals from 1 mg g−1 D. radiodurans in calcite could be detected, corresponding to about 5 µg g−1 β-carotene in calcite (≈0.5% cell weight). This research emphasizes the potential suitability of Raman spectroscopy in the detection of organic biomarkers in future planetary exploration. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

D einococcus radiodurans is known for its extreme radioresistance. Comparative genomics identified a radiation-desiccation response ( RDR) regulon comprising genes that are highly induced after DNA damage and containing a conserved motif ( RDRM) upstream of their coding region. We demonstrated that the RDRM sequence is involved in cis-regulation of the RDR gene ddrB in vivo. Using a transposon mutagenesis approach, we showed that, in addition to ddrO encoding a predicted RDR repressor and irrE encoding a positive regulator recently shown to cleave DdrO in D einococcus deserti, two genes encoding α-keto-glutarate dehydrogenase subunits are involved in ddrB regulation. In wild-type cells, the DdrO cell concentration decreased transiently in an IrrE-dependent manner at early times after irradiation. Using a conditional gene inactivation system, we showed that DdrO depletion enhanced expression of three RDR proteins, consistent with the hypothesis that DdrO acts as a repressor of the RDR regulon. DdrO-depleted cells loose viability and showed morphological changes evocative of an apoptotic-like response, including membrane blebbing, defects in cell division and DNA fragmentation. We propose that DNA repair and apoptotic-like death might be two responses mediated by the same regulators, IrrE and DdrO, but differently activated depending on the persistence of IrrE-dependent DdrO cleavage. [ABSTRACT FROM AUTHOR]

DR0248 is a protein identified in the Deinococcus radiodurans (DR) genome that is predicted to encompass two domains: an N-terminal minimal nucleotidyl transferase domain (MNT) and a C-terminal higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN). These two domains, usually encoded in two ORFs, have been suggested to play the role of a toxin-antitoxin (TA) system in prokaryotes. Recombinant DR0248 was overexpressed and purified from Escherichia coli and diffraction-quality crystals were obtained in the presence of the detergent molecules dodecyldimethylamine oxide (DDAO) and octaethylene glycol monododecyl ether (C12E8), which were used as crystallization additives. Crystals grown with DDAO diffracted to a resolution of 2.24 Å and belonged to space group C2221, with unit-cell parameters a = 98.4, b = 129.9, c = 59.2 Å. Crystals grown with C12E8 diffracted to a resolution of 1.83 Å and belonged to space group P212121, with unit-cell parameters a = 51.6, b = 87.2, c = 108.2 Å. The structure was solved by multiwavelength anomalous dispersion from zinc bound to the protein using a single crystal obtained in the presence of DDAO. [ABSTRACT FROM AUTHOR]

Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: Ssb N (N-terminal without connector), Ssb NC (N-terminal with connector) and Ssb C (C-terminal), each harboring one OB fold. Both Ssb N and Ssb NC displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (Ssb FL ). The level of ssDNA binding activity displayed by Ssb C was intermediate between Ssb FL and Ssb N . Ssb C and Ssb FL predominantly existed as homo-dimers while Ssb NC /Ssb N formed different oligomeric forms. In vitro , Ssb NC or Ssb N formed a binary complex with Ssb C that displayed enhanced ssDNA binding activity. Unlike Ssb FL , Ssb variants were able to differentially modulate topoisomerase-I activity, but failed to stimulate Deinococcal RecA-promoted DNA strand exchange. The results suggest that the C-terminal OB fold is primarily responsible for ssDNA binding. The N-terminal OB fold binds weakly to ssDNA but is involved in multimerization. [ABSTRACT FROM AUTHOR]

Trehalose synthase catalyzes the simple conversion of the inexpensive maltose into trehalose with a side reaction of hydrolysis. Here, the crystal structures of the wild type and the N253A mutant of Deinococcus radiodurans trehalose synthase (DrTS) in complex with the inhibitor Tris are reported. DrTS consists of a catalytic (β/α)8 barrel, subdomain B, a C-terminal β domain and two TS-unique subdomains (S7 and S8). The C-terminal domain and S8 contribute the majority of the dimeric interface. DrTS shares high structural homology with sucrose hydrolase, amylosucrase and sucrose isomerase in complex with sucrose, in particular a virtually identical active-site architecture and a similar substrate-induced rotation of subdomain B. The inhibitor Tris was bound and mimics a sugar at the −1 subsite. A maltose was modelled into the active site, and subsequent mutational analysis suggested that Tyr213, Glu320 and Glu324 are essential within the +1 subsite for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through the replacement of Arg148 and Asn253 with alanine resulted in a decrease in isomerase activity by 8-9-fold and an increased hydrolase activity by 1.5-1.8-fold. The N253A structure showed a small pore created for water entry. Therefore, our DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis. [ABSTRACT FROM AUTHOR]

Endonuclease III is a bifunctional DNA glycosylase that removes a wide range of oxidized bases in DNA. Deinococcus radiodurans is an extreme radiation-resistant and desiccation-resistant bacterium and possesses three genes encoding endonuclease III enzymes in its genome: DR2438 (EndoIII-1), DR0289 (EndoIII-2) and DR0982 (EndoIII-3). Here, EndoIII-1 and an N-terminally truncated form of EndoIII-3 (EndoIII-3Δ76) have been expressed, purified and crystallized, and preliminary X-ray crystallographic analyses have been performed to 2.15 and 1.31 Å resolution, respectively. The EndoIII-1 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 181.38, b = 38.56, c = 37.09 Å, β = 89.34° and one molecule per asymmetric unit. The EndoIII-3Δ76 crystals also belonged to the monoclinic space group C2, but with unit-cell parameters a = 91.47, b = 40.53, c = 72.47 Å, β = 102.53° and one molecule per asymmetric unit. The EndoIII-1 structure was determined by molecular replacement, while the truncated EndoIII-3Δ76 structure was determined by single-wavelength anomalous dispersion phasing. Refinement of the structures is in progress. [ABSTRACT FROM AUTHOR]

Deinococcus radiodurans is an aerobic organism with the ability to survive under conditions of high radiation doses or desiccation. As part of its protection system against oxidative stress, this bacterium encodes three monofunctional catalases. The DR1998 catalase belongs to clade 1, and is present at high levels under normal growth conditions. The crystals of DR1998 diffracted very weakly, and the merged diffraction data showed an Rsym of 0.308. Its crystal structure was determined and refined to 2.6 Å. The four molecules present in the asymmetric unit form, by crystallographic symmetry, two homotetramers with 222 point-group symmetry. The overall structure of DR1998 is similar to that of other monofunctional catalases, showing higher structural homology with the catalase structures of clade 1. Each monomer shows the typical catalase fold, and contains one heme b in the active site. The heme is coordinated by the proximal ligand Tyr369, and on the heme distal side the essential His81 and Asn159 are hydrogen-bonded to a water molecule. A 25-Å-long channel is the main channel connecting the active site to the external surface. This channel starts with a hydrophobic region from the catalytic heme site, which is followed by a hydrophilic region that begins on Asp139 and expands up to the protein surface. Apart from this channel, an alternative channel, also near the heme active site, is presented and discussed. [ABSTRACT FROM AUTHOR]

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium, Deinococcus radiodurans (APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space group P21, with unit-cell parameters a = 77.6, b = 189.6, c = 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å3 Da−1 corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme in D. radiodurans. [ABSTRACT FROM AUTHOR]

The article provides information on a study which determined the ammonia (NH3)-dependent Deinococcus radiodurans (dra) nicotinamide adenine dinucleotide+ synthetase (NADS) in apo form and the substrate binding sites of nicotinic acid adenine dinucleotide (NaAD) and adenosine triphosphate (ATP). Topics discussed include crystallization, protein expression and purification, and crystal structure of the draNADS.

A search for lipases was conducted in the radiophile of Deinococcus radiodurans R1. Four putative lipase genes, encoding DR0334, DR2078, DR1485, and DR2522, were cloned and expressed in Escherichia coli. The recombinant enzymes were subsequently purified and characterized. The results showed DR0334 and DR2078 had the ability to hydrolyze long-chain length p-nitrophenyl esters (C12-C16), while DR1485 and DR2522 hydrolyzed short- and medium-chain length p-nitrophenyl esters (C2-C10). DR0334, DR1485, DR2078, and DR2522 showed optimum pH at 8.5, and optimum temperature at 40, 50, 60, and 60 °C, respectively. DR0334 almost lost its whole activity after 60 min pretreatment at 60 °C, while DR1485, DR2078, and DR2522 retained more than 70% of their original activities after 6 h incubation at 80 °C. The activities of DR2078, DR1485, and DR2522 were enhanced by Mg2+, Ba2+, and Mn2+, but strongly inhibited by EDTA. Nevertheless, DR2078, DR1485, and DR2522 showed moderate stability in organic solvents and detergents. Phylogenetic analysis revealed that DR0334 and DR2078, respectively belong to family IV and family IX, while each of DR1485 and DR2522 forms a new separate branch. The unique properties of DR2078, DR1485, and DR2522, thermostability and organic solvent tolerance, make them useful in industrial applications. [ABSTRACT FROM AUTHOR]

The protective mechanisms of Deinococcus radiodurans against primary reactive oxygen species involve nonenzymatic scavengers and a powerful enzymatic antioxidant system including catalases, peroxidases and superoxide dismutases that prevents oxidative damage. Catalase is an enzyme that is responsible for the conversion of H2O2 to O2 and H2O, protecting the organism from the oxidative effect of H2O2. This study reports the purification and crystallization of the DR1998 catalase from D. radiodurans. The crystals diffracted to 2.6 Å resolution and belonged to space group C2221, with unit-cell parameters a = 97.33, b = 311.88, c = 145.63 Å, suggesting that they contain four molecules per asymmetric unit. The initial phases were determined by molecular replacement and the obtained solution shows the typical catalase quaternary structure. A preliminary model of the protein structure has been built and refinement is currently in progress. [ABSTRACT FROM AUTHOR]

Highlights: [•] The structure of DrRecQ HRDC1 was determined by NMR techniques. [•] DrRecQ HRDC1 is a potential DNA binding domain. [•] DrRecQ HRDC1 cooperates with other domains to enhance full-length DrRecQ interactions with DNA. [ABSTRACT FROM AUTHOR]

ABSTRACT Deinococcus radiodurans is known for its extraordinary resistance to various DNA damaging agents including γ-radiation and desiccation. The pqqE:cat and Δ dr2518 mutants making these cells devoid of pyrroloquinoline quinone (PQQ) and a PQQ inducible Ser/Thr protein kinase, respectively, became sensitive to γ-radiation. Transcriptome analysis of these mutants showed differential expression of the genes including those play roles in oxidative stress tolerance and (DSB) repair in D. radiodurans and in genome maintenance and stress response in other bacteria. Escherichia coli cells expressing DR2518 and PQQ showed improved resistance to γ-radiation, which increased further when both DR2518 and PQQ were present together. Although, profiles of genes getting affected in these mutants were different, there were still a few common genes showing similar expression trends in both the mutants and some others as reported earlier in oxyR and pprI mutant of this bacterium. These results suggested that PQQ and DR2518 have independent roles in γ-radiation resistance of D. radiodurans but their co-existence improves radioresistance further, possibly by regulating differential expression of the genes important for bacterial response to oxidative stress and DNA damage. [ABSTRACT FROM AUTHOR]

This paper tries to focus on pertinent issues in health care and analyses how cold atmospheric plasmas can contribute towards the long-standing problems identified. It starts quite generally by looking at the major elements that make up our current health care system, the aims that govern these and the corresponding requirements on health care products. It then proceeds to identify areas where current research promises contributions from the new cold plasma technologies. It turns out that there are indeed many areas where investments in plasma health care could pay off, both in terms of cost and in reduction of human suffering (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [ABSTRACT FROM AUTHOR]

3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584). Dr_2074 is a homologue of human AlkA (MPG or AAG) and Dr_2584 is a homologue of bacterial AlkAs. Here, the three-dimensional structure of Dr_2584 (DrAlkA2) is presented and compared with the previously determined structure of Escherichia coli AlkA (EcAlkA). The results show that the enzyme consists of two helical-bundle domains separated by a wide DNA-binding cleft and contains an HhH motif. Overall, the protein fold is similar to the two helical-bundle domains of EcAlkA, while the third N-terminal mixed α/β domain observed in EcAlkA is absent. Substrate-specificity analyses show that DrAlkA2, like EcAlkA, is able to remove both 3-methyladenine (3meA) and 7-methylguanine (7meG) from DNA; however, the enzyme possesses no activity towards 1, N6-ethenoadenine (ɛA) and hypoxanthine (Hx). In addition, it shows activity towards the AlkB dioxygenase substrates 3-methylcytosine (3meC) and 1-methyladenine (1meA). Thus, the enzyme seems to preferentially repair methylated bases with weakened N-glycosidic bonds; this is an unusual specificity for a bacterial AlkA protein and is probably dictated by a combination of the wide DNA-binding cleft and a highly accessible specificity pocket. [ABSTRACT FROM AUTHOR]

Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd2+) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity. [ABSTRACT FROM AUTHOR]

Deinococcus radiodurans mutant lacking pyrroloquinoline-quinone (PQQ) synthesis shows sensitivity to γ-rays and impairment of DNA double strand break repair. The genome of this bacterium encodes five putative proteins having multiple PQQ binding motifs. The deletion mutants of corresponding genes were generated, and their response to DNA damage was monitored. Only the Δ dr2518 mutant exhibited higher sensitivity to DNA damage. Survival of these cells decreased by 3-log cycle both at 6 kGy γ-rays and 1200 J m−2 UV (254 nm) radiation, and 2.5-log cycle upon 14 days desiccation at 5% humidity. The Δ dr2518 mutant showed complete inhibition of DSB repair until 24 h PIR and disappearance of a few phosphoproteins. The Δ dr2518pqqE:cat double mutant showed γ-ray sensitivity similar to Δ dr2518 indicating functional interaction of these genes in D. radiodurans. DR2518 contains a eukaryotic type Ser/Thr kinase domain and structural topology suggesting stress responsive transmembrane protein. Its autokinase activity in solution was stimulated by nearly threefold with PQQ and twofold with linear DNA, but not with circular plasmid DNA. More than 15-fold increase in dr2518 transcription and several-fold enhanced in vivo phosphorylation of DR2518 were observed in response to γ irradiation. These results suggest that DR2518 as a DNA damage-responsive protein kinase plays an important role in radiation resistance and DNA strand break repair in D. radiodurans. [ABSTRACT FROM AUTHOR]

The gene product of histidine kinase DR2244 (putative phoR) encoded by Deinococcus radiodurans has been suggested to be involved in the PhoR-PhoB two-component regulatory system. This two-component signalling system is activated upon phosphate starvation in several bacteria, including D. radiodurans. Single crystals were obtained from a recombinant preparation of the catalytic/ATP-binding (CA) domain of D. radiodurans PhoR (79-224) overexpressed in Escherichia coli. The crystals belonged to space group P212121, with unit-cell parameters a = 46.9, b = 81.8, c = 204.6 Å. The crystals contained six molecules in the asymmetric unit. Diffraction data were collected to 2.4 Å resolution on beamline ID23-2 of the European Synchrotron Radiation Facility. [ABSTRACT FROM AUTHOR]

A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 &Angstrom; resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested. [ABSTRACT FROM AUTHOR]