Engineering Deinococcus geothermalis for Bioremediation of High-Temperature Radioactive Waste Environments

Deinococcus geothermalis is an extremely radiation-resistant thermophilic bacterium closely related to the mesophile Deinococcus radiodurans, which is being engineered for in situ bioremediation of radioactive wastes. We report that D. geothermalis is transformable with plasmids designed for D. radiodurans and have generated a Hg(II)-resistant D. geothermalis strain capable of reducing Hg(II) at elevated temperatures and in the presence of 50 Gy/h. Additionally, D. geothermalis is capable of reducing Fe(III)-nitrilotriacetic acid, U(VI), and Cr(VI). These characteristics support the prospective development of this thermophilic radiophile for bioremediation of radioactive mixed waste environments with temperatures as high as 55°C.     

Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments

We have developed a radiation resistant bacterium for the treatment of mixed radioactive wastes containing ionic mercury. The high cost of remediating radioactive waste sites from nuclear weapons production has stimulated the development of bioremediation strategies using Deinococcus radiodurans, the most radiation resistant organism known. As a frequent constituent of these sites is the highly toxic ionic mercury (Hg) (II), we have generated several D. radiodurans strains expressing the cloned Hg (II) resistance gene (merA) from Escherichia coli strain BL308. We designed four different expression vectors for this purpose, and compared the relative advantages of each. The strains were shown to grow in the presence of both radiation and ionic mercury at concentrations well above those found in radioactive waste sites, and to effectively reduce Hg (II) to the less toxic volatile elemental mercury. We also demonstrated that different gene clusters could be used to engineer D. radiodurans for treatment of mixed radioactive wastes by developing a strain to detoxify both mercury and toluene. These expression systems could provide models to guide future D. radiodurans engineering efforts aimed at integrating several remediation functions into a single host.     

Actinide and metal toxicity to prospective bioremediation bacteria

Bacteria may be beneficial for alleviating actinide contaminant migration through processes such as bioaccumulation or metal reduction. However, sites with radioactive contamination often contain multiple additional contaminants, including metals and organic chelators. Bacteria-based bioremediation requires that the microorganism functions in the presence of the target contaminant, as well as other contaminants. Here, we evaluate the toxicity of actinides, metals and chelators to two different bacteria proposed for use in radionuclide bioremediation, Deinococcus radiodurans and Pseudomonas putida, and the toxicity of Pu(VI) to Shewanella putrefaciens. Growth of D. radiodurans was inhibited at metal concentrations ranging from 1.8 microM Cd(II) to 32 mM Fe(III). Growth of P. putida was inhibited at metal concentrations ranging from 50 microM Ni(II) to 240 mM Fe(III). Actinides inhibited growth at mM concentrations: chelated Pu(IV), U(VI) and Np(V) inhibit D. radiodurans growth at 5.2, 2.5 and 2.1 mM respectively. Chelated U(VI) inhibits P. putida growth at 1.7 mM, while 3.6 mM chelated Pu(IV) inhibits growth only slightly. Pu(VI) inhibits S. putrefaciens growth at 6 mM. These results indicate that actinide toxicity is primarily chemical (not radiological), and that radiation resistance does not ensure radionuclide tolerance. This study also shows that Pu is less toxic than U and that actinides are less toxic than other types of metals, which suggests that actinide toxicity will not impede bioremediation using naturally occurring bacteria.     

Characterization of adhesion threads of Deinococcus geothermalis as type IV pili

Deinococcus geothermalis E50051 forms tenuous biofilms on paper machine surfaces. Field emission electron microscopy analysis revealed peritrichous appendages which mediated cell-to-surface and cell-to-cell interactions but were absent in planktonically grown cells. The major protein component of the extracellular extract of D. geothermalis had an N-terminal sequence similar to the fimbrial protein pilin annotated in the D. geothermalis DSM 11300 draft sequence. It also showed similarity to the type IV pilin sequence of D. radiodurans and several gram-negative pathogenic bacteria. Other proteins in the extract had N-terminal sequences identical to D. geothermalis proteins with conservative motifs for serine proteases, metallophosphoesterases, and proteins whose function is unknown. Periodic acid-Schiff staining for carbohydrates indicated that these extracellular proteins may be glycosylated. A further confirmation for the presence of glycoconjugates on the cell surface was obtained by confocal laser scanning imaging of living D. geothermalis cells stained with Amaranthus caudatus lectin, which specifically binds to galactose residues. The results indicate that the thread-like appendages of D. geothermalis E50051 are glycosylated type IV pili, bacterial attachment organelles which have thus far not been described for the genus Deinococcus.     

Firm but slippery attachment of Deinococcus geothermalis

Bacterial biofilms impair the operation of many industrial processes. Deinococcus geothermalis is efficient primary biofilm former in paper machine water, functioning as an adhesion platform for secondary biofilm bacteria. It produces thick biofilms on various abiotic surfaces, but the mechanism of attachment is not known. High-resolution field-emission scanning electron microscopy and atomic force microscopy (AFM) showed peritrichous adhesion threads mediating the attachment of D. geothermalis E50051 to stainless steel and glass surfaces and cell-to-cell attachment, irrespective of the growth medium. Extensive slime matrix was absent from the D. geothermalis E50051 biofilms. AFM of the attached cells revealed regions on the cell surface with different topography, viscoelasticity, and adhesiveness, possibly representing different surface layers that were patchily exposed. We used oscillating probe techniques to keep the tip-biofilm interactions as small as possible. In spite of this, AFM imaging of living D. geothermalis E50051 biofilms in water resulted in repositioning but not in detachment of the surface-attached cells. The irreversibly attached cells did not detach when pushed with a glass capillary but escaped the mechanical force by sliding along the surface. Air drying eliminated the flexibility of attachment, but it resumed after reimmersion in water. Biofilms were evaluated for their strength of attachment. D. geothermalis E50051 persisted 1 h of washing with 0.2% NaOH or 0.5% sodium dodecyl sulfate, in contrast to biofilms of Burkholderia cepacia F28L1 or the well-characterized biofilm former Staphylococcus epidermidis O-47. Deinococcus radiodurans strain DSM 20539(T) also formed tenacious biofilms. This paper shows that D. geothermalis has firm but laterally slippery attachment not reported before for a nonmotile species.     

Physiologic determinants of radiation resistance in Deinococcus radiodurans

Immense volumes of radioactive wastes, which were generated during nuclear weapons production, were disposed of directly in the ground during the Cold War, a period when national security priorities often surmounted concerns over the environment. The bacterium Deinococcus radiodurans is the most radiation-resistant organism known and is currently being engineered for remediation of the toxic metal and organic components of these environmental wastes. Understanding the biotic potential of D. radiodurans and its global physiological integrity in nutritionally restricted radioactive environments is important in development of this organism for in situ bioremediation. We have previously shown that D. radiodurans can grow on rich medium in the presence of continuous radiation (6,000 rads/h) without lethality. In this study we developed a chemically defined minimal medium that can be used to analyze growth of this organism in the presence and in the absence of continuous radiation; whereas cell growth was not affected in the absence of radiation, cells did not grow and were killed in the presence of continuous radiation. Under nutrient-limiting conditions, DNA repair was found to be limited by the metabolic capabilities of D. radiodurans and not by any nutritionally induced defect in genetic repair. The results of our growth studies and analysis of the complete D. radiodurans genomic sequence support the hypothesis that there are several defects in D. radiodurans global metabolic regulation that limit carbon, nitrogen, and DNA metabolism. We identified key nutritional constituents that restore growth of D. radiodurans in nutritionally limiting radioactive environments.     

Comparison of the genomes of deinococcal species using oligonucleotide microarrays

The bacterium Deinococcus radiodurans is one of the most resistant organisms to ionizing radiation and other DNAdamaging agents. Although, at present, 30 Deinococcus species have been identified, the whole-genome sequences of most species remain unknown, with the exception of D. radiodurans (DRD), D. geothermalis, and D. deserti. In this study, comparative genomic hybridization (CGH) microarray analysis of three Deinococcus species, D. radiopugnans (DRP), D. proteolyticus (DPL), and D. radiophilus (DRPH), was performed using oligonucleotide arrays based on DRD. Approximately 28%, 14%, and 15% of 3,128 open reading frames (ORFs) of DRD were absent in the genomes of DRP, DPL, and DRPH, respectively. In addition, 162 DRD ORFs were absent in all three species. The absence of 17 randomly selected ORFs was confirmed by a Southern blot. Functional classification showed that the absent genes spanned a variety of functional categories: some genes involved in amino acid biosynthesis, cell envelope, cellular processes, central intermediary metabolism, and DNA metabolism were not present in any of the three deinococcal species tested. Finally, comparative genomic data showed that 120 genes were Deinococcus-specific, not the 230 reported previously. Specifically, ddrD, ddrO, and ddrH genes, previously identified as Deinococcus-specific, were not present in DRP, DPL, or DRPH, suggesting that only a portion of ddr genes are shared by all members of the genus Deinococcus.     

Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics.

The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism.     

Isolation and characterization of Deinococcus geothermalis T27, a slightly thermophilic and organic solvent-tolerant bacterium able to survive in the presence of high concentrations of ethyl acetate

A solvent-tolerant, slightly thermophilic bacterium was isolated at 45 degrees C in the presence of toluene vapor provided as the sole carbon source. Strain T27 was identified as Deinococcus geothermalis T27. It could tolerate high concentrations of solvent provided as a nonaqueous layer (5% and 20%, v/v) to a cell suspension and had a remarkable ability to tolerate a broad range of solvents having log P(ow) values ranging from 5.6 of n-decane to as low as 0.7 of ethyl acetate. It was also able to utilize some of the solvents tested as a growth substrate at 45 degrees C. The addition of Ca(2+) ion, glucose and fructose partially promoted solvent tolerance. Cells exposed to ethyl acetate appeared to have a smaller size; however, the cell structure was not altered and was apparently well defined even after solvent shock. The tolerance of D. geothermalis T27 in the presence of high levels of toxic solvent stress at a comparatively high temperature indicated its potential use in biotechnological applications as well as bioremediation of xenobiotics.     

Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus radiodurans R1

Deinococcus radiodurans is an exceptionally radiation-resistant microorganism capable of surviving acute exposures to ionizing radiation doses of 15,000 Gy and previously described as having a strictly aerobic respiratory metabolism. Under strict anaerobic conditions, D. radiodurans R1 reduced Fe(III)-nitrilotriacetic acid coupled to the oxidation of lactate to CO(2) and acetate but was unable to link this process to growth. D. radiodurans reduced the humic acid analog anthraquinone-2,6-disulfonate (AQDS) to its dihydroquinone form, AH(2)DS, which subsequently transferred electrons to the Fe(III) oxides hydrous ferric oxide and goethite via a previously described electron shuttle mechanism. D. radiodurans reduced the solid-phase Fe(III) oxides in the presence of either 0.1 mM AQDS or leonardite humic acids (2 mg ml(-1)) but not in their absence. D. radiodurans also reduced U(VI) and Tc(VII) in the presence of AQDS. In contrast, Cr(VI) was directly reduced in anaerobic cultures with lactate although the rate of reduction was higher in the presence of AQDS. The results are the first evidence that D. radiodurans can reduce Fe(III) coupled to the oxidation of lactate or other organic compounds. Also, D. radiodurans, in combination with humic acids or synthetic electron shuttle agents, can reduce U and Tc and thus has potential applications for remediation of metal- and radionuclide-contaminated sites where ionizing radiation or other DNA-damaging agents may restrict the activity of more sensitive organisms.     

耐辐射球菌转录因子DdrO的基因克隆与生物信息学分析

目的：克隆耐辐射球菌ddrO基因,并对其进行生物信息学分析,预测其功能。方法：根据耐辐射球菌ddrO基因序列,由Primer Premier 5设计一对引物,以提取的耐辐射球菌基因组为模板,PCR扩增获得耐辐射球菌ddrO基因,序列测定并利用生物信息学软件对ddrO基因的理化性质、高级结构及生物学功能等进行分析与预测。结果：成功获得了ddrO基因。生物信息学分析发现,ddrO基因核苷酸序列长度为396bp,编码一个131aa组成的相对分子质量为14.993kD的预测的DdrO转录因子。核酸同源性搜索及比较分析仅在与耐辐射球菌同属的Deinococcus geothermalis和Deinococcus deserti中发现高度相似的序列;蛋白同源性搜索发现一些与DdrO显著同源的蛋白,如Deide_20570（95%）,Dgeo_0336（90%）,Deide_3p02170（82%）等;结构域分析发现DdrO含有HTH（helix-turn-helix）DNA结合结构域。结论：根据生物信息学结果预测DdrO蛋白可能具有转录调控作用,参与DNA修复和复制,在耐辐射球菌的DNA损伤修复过程中发挥一定作用。     

Phylogenetic and disruption analyses of aspartate kinase of Deinococcus radiodurans

The extremely radioresistant bacterium Deinococcus radiodurans is evolutionarily closely related to the extremely thermophilic bacterium Thermus thermophilus. These bacteria have a single gene encoding an aspartate kinase (AK) that catalyzes the phosphorylation of L-aspartate. T. thermophilus has an aminoadipate pathway for lysine biosynthesis that does not use AK for lysine biosynthesis. Phylogenetic analysis in this study indicated that D. radiodurans AK has a different protein structure and a different evolutionary history from T. thermophilus AK. Disruption analysis of D. radiodurans AK indicated that D. radiodurans AK was not used for lysine biosynthesis but for threonine and methionine biosyntheses. A D. radiodurans AK disruption mutant exhibited a phenotype similar to a T. thermophilus AK disruption mutant, which indicates that these two AKs have different evolutionary origins, though their functions are not different.     

Identification of signature proteins that are distinctive of the Deinococcus-Thermus phylum.

The members of the Deinococcus-Thermus phylum, which include many species that are resistant to extreme radiation, as well as several thermophiles, have been recognized solely on the basis of their branching patterns in 16S rRNA and other phylogenetic trees. No biochemical or physiological characteristic is currently known that is unique to this group of species. To identify genes/proteins that are exclusive of this group of species, systematic protein basic local alignment tool (Blastp) searches were carried out on each open reading frame (ORF) in the genome of Deinococcus radiodurans. These studies identified 65 proteins that were only found in all three sequenced Deinococcus-Thermus genomes (viz. D. radiodurans, D. geothermalis and Thermus thermophilus), but not in any other bacteria. In addition, these studies also identified 206 proteins that are exclusively found in the two Deinocococci species, and 399 proteins that are unique to D. radiodurans. The identified proteins, which represent a genetic repertoire distinctive to the Deinococcus-Thermus group, or to Deinococci species, provide novel molecular markers for their identification and characterization. The cellular functions of most of these proteins are not known and their studies should prove useful in identifying novel biochemical and physiological characteristics that are exclusive of these groups of bacteria and also those responsible for the extreme radiation resistance of Deinococci.     

Lesion bypass activity of DNA polymerase A from the extremely radioresistant organism Deinococcus radiodurans

The bacterium Deinococcus radiodurans survives extremely high exposure to ionizing radiation and extended periods of desiccation. Radiation at the survival doses is known to cause numerous DNA damage, such as hundreds of double strand breaks and single strand breaks, as well as damage of the nucleobases. The mechanisms of D. radiodurans to survive the depicted threats are still only beginning to be understood. DNA polymerase A (PolA) has been shown to be crucially involved in irradiation resistance mechanisms of D. radiodurans. We expressed and characterized the DNA polymerase domain of PolA for the first time in vitro. The obtained enzyme is able to efficiently catalyze DNA-dependent DNA synthesis requiring Mg(II) as divalent metal ion. Additionally, strand displacement synthesis of the DNA polymerase, which is required in several repair processes, could be detected. We further found that DNA polymerase function of PolA is modulated by the presence of Mn(II). Whereas proceeding DNA synthesis of PolA was blocked by certain DNA damage that occurs through radiation of DNA, bypass was facilitated by Mn(II). Our results suggest an enzyme modulator function of Mn(II). These observations parallel reports that D. radiodurans accumulates intracellular Mn(II) in cases of irradiation and that the level of irradiation protection correlates with Mn(II) concentrations.     

Identification and characterization of a novel bacterial sulfite oxidase with no heme binding domain from Deinococcus radiodurans

An open reading frame (draSO) encoding a putative sulfite oxidase (SO) was identified in the sequence of chromosome II of Deinococcus radiodurans; the predicted gene product showed significant amino acid sequence homology to several bacterial and eukaryotic SOs, such as the biochemically and structurally characterized enzyme from Arabidopsis thaliana. Cloning of the Deinococcus SO gene was performed by PCR amplification from the bacterial genomic DNA, and heterologous gene expression of a histidine-tagged polypeptide was obtained in a molybdopterin-overproducing strain of Escherichia coli. The recombinant protein was purified to homogeneity by nickel chelating affinity chromatography, and its main kinetic and chemical physical parameters were determined. Northern blot and enzyme activity analyses indicated that draSO gene expression is constitutive in D. radiodurans and that there is no increase upon exposure to thiosulfate and/or molybdenum(II).     

Cold sensitivity of thermophilic and mesophilic RNA polymerases

RNA polymerase from mesophilic Deinococcus radiodurans displays the same cold sensitivity of promoter opening as RNA polymerase from the closely related thermophilic Thermus aquaticus. This suggests that, contrary to the accepted view, cold sensitivity of promoter opening by thermophilic RNA polymerases may not be a consequence of their thermostability.     

A comparative proteomic approach to better define Deinococcus nucleoid specificities

Compared to radiation-sensitive bacteria, the nucleoids of radiation-resistant Deinococcus species show a higher degree of compaction. Such a condensed nucleoid may contribute to the extreme radiation resistance of Deinococcus by limiting dispersion of radiation-induced DNA fragments. Architectural proteins may play a role in this high degree of nucleoid compaction, but comparative genomics revealed only a limited number of Deinococcus homologs of known nucleoid-associated proteins (NAPs) from other species such as Escherichia coli. A comparative proteomic approach was used to identify potentially novel proteins from isolated nucleoids of Deinococcus radiodurans and Deinococcus deserti. Proteins in nucleoid enriched fractions were identified and semi-quantified by shotgun proteomics. Based on normalized spectral counts, the histone-like DNA-binding protein HU appeared to be the most abundant among candidate NAPs from both micro-organisms. By immunofluorescence microscopy, D. radiodurans HU and both DNA gyrase subunits were shown to be distributed throughout the nucleoid structure and absent from the cytoplasm. Taken together, our results suggest that D. radiodurans and D. deserti bacteria contain a very low diversity of NAPs, with HU and DNA gyrase being the main proteins involved in the organization of the Deinococcus nucleoids.     

How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress

We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumulate exceptionally high intracellular manganese and low iron levels. In comparison, the dissimilatory metal-reducing bacterium Shewanella oneidensis accumulates Fe but not Mn and is extremely sensitive to radiation. We have proposed that for Fe-rich, Mn-poor cells killed at radiation doses which cause very little DNA damage, cell death might be induced by the release of Fe(II) from proteins during irradiation, leading to additional cellular damage by Fe(II)-dependent oxidative stress. In contrast, Mn(II) ions concentrated in D. radiodurans might serve as antioxidants that reinforce enzymic systems which defend against oxidative stress during recovery. We extend our hypothesis here to include consideration of respiration, tricarboxylic acid cycle activity, peptide transport and metal reduction, which together with Mn(II) transport represent potential new targets to control recovery from radiation injury.     

Duplication insertion of drug resistance determinants in the radioresistant bacterium Deinococcus radiodurans.

Escherichia coli drug resistance plasmids were introduced into Deinococcus radiodurans by cloning D. radiodurans DNA into the plasmids prior to transformation. The plasmids were integrated into the chromosome of the transformants and flanked by a direct repeat of the cloned D. radiodurans segment. The plasmid and one copy of the flanking chromosomal segment constituted a unit ("amplification unit") which was found repeated in tandem at the site of chromosomal integration. Up to 50 copies of the amplification unit were present per chromosome, accounting for approximately 10% of the genomic DNA. Circular forms of the amplification unit were also present in D. radiodurans transformants. These circles were introduced into E. coli, where they replicated as plasmids. The drug resistance determinants which have been introduced into D. radiodurans in this fashion are cat (from Tn9) and aphA (from Tn903). Transformation of D. radiodurans to drug resistance was efficient when the donor DNA was from D. radiodurans or E. coli, but was greatly reduced when the donor DNA was linearized with restriction enzymes prior to transformation. In the course of the study, a plasmid, pS16, was discovered in D. radiodurans R1, establishing that all Deinococcus strains so far examined contain plasmids.