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 degrees C.     

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.     

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.     

Physiology of resistant Deinococcus geothermalis bacterium aerobically cultivated in low-manganese medium

This dynamic proteome study describes the physiology of growth and survival of Deinococcus geothermalis, in conditions simulating paper machine waters being aerobic, warm, and low in carbon and manganese. The industrial environment of this species differs from its natural habitats, geothermal springs and deep ocean subsurfaces, by being highly exposed to oxygen. Quantitative proteome analysis using two-dimensional gel electrophoresis and bioinformatic tools showed expression change for 165 proteins, from which 47 were assigned to a function. We propose that D. geothermalis grew and survived in aerobic conditions by channeling central carbon metabolism to pathways where mainly NADPH rather than NADH was retrieved from the carbon source. A major part of the carbon substrate was converted into succinate, which was not a fermentation product but likely served combating reactive oxygen species (ROS). Transition from growth to nongrowth resulted in downregulation of the oxidative phosphorylation observed as reduced expression of V-type ATPase responsible for ATP synthesis in D. geothermalis. The battle against oxidative stress was seen as upregulation of superoxide dismutase (Mn dependent) and catalase, as well as several protein repair enzymes, including FeS cluster assembly proteins of the iron-sulfur cluster assembly protein system, peptidylprolyl isomerase, and chaperones. Addition of soluble Mn reinitiated respiration and proliferation with concomitant acidification, indicating that aerobic metabolism was restricted by access to manganese. We conclude that D. geothermalis prefers to combat ROS using manganese-dependent enzymes, but when manganese is not available central carbon metabolism is used to produce ROS neutralizing metabolites at the expense of high utilization of carbon substrate.     

Cloning, purification and characterization of a thermostable amylosucrase from Deinococcus geothermalis

Amylosucrase is a transglucosidase that catalyses the synthesis of an amylose-type polymer from sucrose, an abundant agro-resource. Here we describe a novel thermostable amylosucrase from the moderate thermophile Deinococcus geothermalis (DGAS). The dgas gene was cloned and expressed in Escherichia coli . The encoded enzyme was purified and characterized. DGAS displays a specific activity of 44 U mg⁻¹, an optimal temperature of 50 °C and a half-life of 26 h at 50 °C. Moreover, it produces an α-glucan at 50 °C, with an average degree of polymerization of 45 and a polymerization yield of 76%. DGAS is thus the most active and thermostable amylosucrase known to date.     

Engineering of Deinococcus radiodurans R1 for Bioprecipitation of Uranium from Dilute Nuclear Waste

Genetic engineering of radiation-resistant organisms to recover radionuclides/heavy metals from radioactive wastes is an attractive proposition. We have constructed a Deinococcus radiodurans strain harboring phoN, a gene encoding a nonspecific acid phosphatase, obtained from a local isolate of Salmonella enterica serovar Typhi. The recombinant strain expressed an ~27-kDa active PhoN protein and efficiently precipitated over 90% of the uranium from a 0.8 mM uranyl nitrate solution in 6 h. The engineered strain retained uranium bioprecipitation ability even after exposure to 6 kGy of ⁶⁰Co gamma rays. The PhoN-expressing D. radiodurans offers an effective and eco-friendly in situ approach to biorecovery of uranium from dilute nuclear waste.     

Biosynthesis of (+)-catechin glycosides using recombinant amylosucrase from Deinococcus geothermalis DSM 11300

Amylosucrase (ASase, EC 2.4.1.4) is a glucosyltransferase that hydrolyzes sucrose into glucose and fructose and produces amylose-like glucan polymers from the released glucose. (+)-Catechin is a plant polyphenolic metabolite having skin-whitening and antioxidant activities. In this study, the ASase gene from Deinococcus geothermalis (dgas) was expressed in Escherichia coli, while the recombinant DGAS enzyme was purified using a glutathione S-transferase fusion system. The (+)-catechin glycoside derivatives were synthesized from (+)-catechin using DGAS transglycosylation activity. We confirmed the presence of two major transglycosylation products using TLC. The (+)-catechin transglycosylation products were isolated using silica gel open column chromatography and recycling-HPLC. Two (+)-catechin major transfer products were determined through ¹H and ¹³C NMR to be (+)-catechin-3′-O-α-d-glucopyranoside with a glucose molecule linked to (+)-catechin and (+)-catechin-3′-O-α-D-maltoside with a maltose linked to (+)-catechin. The presence of (+)-catechin maltooligosaccharides in the DGAS reaction was also confirmed via recycling-HPLC and enzymatic analysis. The effects of various reaction conditions (temperature, enzyme concentration, and molar ratio of acceptor and donor) on the yield and type of (+)-catechin glycosides were investigated.     

Architecture of Deinococcus geothermalis biofilms on glass and steel: a lectin study

Deinococcus geothermalis is resistant to chemical and physical stressors and forms tenuous biofilms in paper industry. The architecture of its biofilms growing on glass and on stainless acid proof steel was studied with confocal laser scanning microscopy and fluorescent lectins and nanobeads as in situ probes. Hydrophobic nanobeads adhered to the biofilms but did not penetrate to biofilm interior. In contrast, the biofilms were readily permeable towards many different lectins. A skeletal network of glycoconjugates, reactive with Dolichos biflorus and Maclura pomifera lectins, was prominent in the space inside the biofilm colony core but absent on the exterior. Cells in the core space of the biofilm were interconnected by a network of adhesion structures, reactive with Amaranthus caudatus lectin but with none of the 65 other tested lectins. The glycoconjugates connecting the individual cells to steel reacted with Phaseolus vulgaris lectin whereas those connecting to glass mainly reacted with A. caudatus lectin. Envelopes of all cells in the D. geothermalis biofilm reacted with several other lectins, with many different specificities. We conclude that numerous different glycoconjugates are involved in the adhesion and biofilm formation of D. geothermalis , possibly contributing its unique survival capacity when exposed to dehydration, biocidal chemicals and other extreme conditions.     

Experimental and statistical analysis of nutritional requirements for the growth of the extremophile Deinococcus geothermalis DSM 11300

Few studies concerning the nutritional requirements of Deinococcus geothermalis DSM 11300 have been conducted to date. Three defined media compositions have been published for the growth of this strain but they were found to be inadequate to achieve growth without limitation. Furthermore, growth curves, biomass concentration and growth rates were generally not available. Analysis in Principal Components was used in this work to compare and consequently to highlight the main compounds which differ between published chemically defined media. When available, biomass concentration, and/or growth rate were superimposed to the PCA analysis. The formulations of the media were collected from existing literature; media compositions designed for the growth of several strains of Deinococcaceae or Micrococcaceae were included. The results showed that a defined medium adapted from Holland et al. (Appl Microbiol Biotechnol 72:1074–1082, 2006) was the best basal medium and was chosen for further studies. A growth rate of 0.03 h^?1 and a final OD_600nm of 0.55 were obtained, but the growth was linear. Then, the effects of several medium components on oxygen uptake and biomass production by Deinococcus geothermalis DSM 11300 were studied using a respirometry-based method, to search for the nutritional limitation. The results revealed that the whole yeast extract in the medium with glucose is necessary to obtain a non-limiting growth of Deinococcus geothermalis DSM 11300 at a maximum growth rate of 0.64 h^?1 at 45 °C.     

Destruction of Deinococcus geothermalis biofilm by photocatalytic ALD and sol-gel TiO2 surfaces

The aim of the present work was to explore possibilities of photocatalytic TiO₂ coating for reducing biofilms on non-living surfaces. The model organism, Deinococcus geothermalis, known to initiate growth of durable, colored biofilms on machine surfaces in the paper industry, was allowed to form biofilms on stainless steel, glass and TiO₂ film coated glass or titanium. Field emission electron microscopy revealed that the cells in the biofilm formed at 45°C under vigorous shaking were connected to the surface by means of numerous adhesion threads of 0.1--0.3 μm in length. Adjacent cells were connected to one another by threads of 0.5--1 μm in length. An ultrastructural analysis gave no indication for the involvement of amorphous extracellular materials (e.g., slime) in the biofilm. When biofilms on photocatalytic TiO₂ surfaces, submerged in water, were exposed to 20 W h m⁻² of 360 nm light, both kinds of adhesion threads were completely destroyed and the D. geothermalis cells were extensively removed (from >10⁷ down to below 10⁶ cells cm⁻²). TiO₂ films prepared by the sol-gel technique were slightly more effective than those prepared by the ALD technique. Doping of the TiO₂ with sulfur did not enhance its biofilm-destroying capacity. The results show that photocatalytic TiO₂ surfaces have potential as a self-cleaning technology for warm water using industries.     

Structural investigation of the thermostability and product specificity of amylosucrase from the bacterium Deinococcus geothermalis

Amylosucrases are sucrose-utilizing α-transglucosidases that naturally catalyze the synthesis of α-glucans, linked exclusively through α1,4-linkages. Side products and in particular sucrose isomers such as turanose and trehalulose are also produced by these enzymes. Here, we report the first structural and biophysical characterization of the most thermostable amylosucrase identified so far, the amylosucrase from Deinoccocus geothermalis (DgAS). The three-dimensional structure revealed a homodimeric quaternary organization, never reported before for other amylosucrases. A sequence signature of dimerization was identified from the analysis of the dimer interface and sequence alignments. By rigidifying the DgAS structure, the quaternary organization is likely to participate in the enhanced thermal stability of the protein. Amylosucrase specificity with respect to sucrose isomer formation (turanose or trehalulose) was also investigated. We report the first structures of the amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea in complex with turanose. In the amylosucrase from N. polysaccharea (NpAS), key residues were found to force the fructosyl moiety to bind in an open state with the O3' ideally positioned to explain the preferential formation of turanose by NpAS. Such residues are either not present or not similarly placed in DgAS. As a consequence, DgAS binds the furanoid tautomers of fructose through a weak network of interactions to enable turanose formation. Such topology at subsite +1 is likely favoring other possible fructose binding modes in agreement with the higher amount of trehalulose formed by DgAS. Our findings help to understand the inter-relationships between amylosucrase structure, flexibility, function, and stability and provide new insight for amylosucrase design.     

Quantitative contributions of bacteria and of Deinococcus geothermalis to deposits and slimes in paper industry

Deinococcus geothermalis has frequently been isolated from pink colored deposits of paper industry processes. Laboratory studies have shown that D. geothermalis is capable of forming on nonliving surfaces patchy biofilms that are resistant to adverse agents such as extreme pH, desiccation, solubilising detergents and biocides. This study was done to quantitatively assess the role of D. geothermalis as a biofouler in paper industry. Colored deposits were collected from 24 European and North American paper and board machines and the densities of the bacterial 16S rRNA genes and those of the red slime producers D. geothermalis and Meiothermus spp. were measured by QPCR (quantitative real time PCR). D. geothermalis was found at nine machines, usually from splash area deposits, but its contribution was minor, 0.001–1%, to the total bacterial burden of 8.3 to log 10.5 log units per gram wet-weight of the deposits. When D. geothermalis was found in a measurable quantity, Meiothermus spp. also was found, often in bulk quantity (7–100% of the total bacteria). The data are in line with the properties of D. geothermalis known from laboratory biofilm studies, indicating this species is a pioneer coloniser of machine surfaces and may help other bacteria to adhere and grown into biofilms, rather than competing with them.     

Mechanisms of biofilm formation in paper machine by Bacillus species: the role of Deinococcus geothermalis

Mechanisms for the undesired persistence of Bacillus species in paper machine slimes were investigated. Biofilm formation was measured for industrial Bacillus isolates under paper machine wet-end-simulating conditions (white water, pH 7, agitated at 45°C for 1–2 days). None of the 40 tested strains of seven Bacillus species formed biofilm on polished stainless steel or on polystyrene surfaces as a monoculture. Under the same conditions, Deinococcus geothermalis E50051 covered all test surfaces as a patchy thick biofilm. The paper machine bacilli, however, formed mixed biofilms with D. geothermalis E50051 as revealed by confocal microscopy. Biofilm interactions between the bacilli and the deinococci varied from synergism to antagonism. Synergism in biofilm formation of D. geothermalis E50051 was strongest with Bacillus coagulans D50192, and with the type strains of B. coagulans, B. amyloliquefaciens or B. pumilus. Two B. licheniformis, one B. amyloliquefaciens, one B. pumilus and four B. cereus strains antagonized biofilm production by D. geothermalis. B. licheniformis D50141 and the type strain of B. licheniformis were the strongest antagonists. These bacteria inhibited deinococcal growth by emitting heat-stable, methanol-soluble metabolite(s). We conclude that the persistence of Bacillus species in paper machine slimes relates to their ability to conquer biofilms formed by primary colonizers, such as D. geothermalis. Journal of Industrial Microbiology & Biotechnology (2001) 27, 343–351. Received 17 April 2001/ Accepted in revised form 16 July 2001     

Anion Scavengers for Low-Level Radioactive Waste Repository Backfills

Minimization of ¹²⁹/^? and ⁹⁹ TcO₄ ^? transport to the biosphere is critical to the success of low level radioactive waste (LLRW) storage facilities. Here we experimentally identify and classify potential sorbent materials for inclusion in LLRW backfills. For low pH conditions (pH 4-5), Cu-sulfides and possibly imogolite-rich soils provide K_d's (distribution coefficients) of roughly 10³?mL g^?1 for /^?, and 10²?mL g^?1 for TcO₄ ^?. At near neutral pH, hydrotalcites, Cu-oxides, Cu-sulfides, and lignite coal possess K_d's on the order of 10²?mL g^?1 for both /^? and TcO₄ ^?. At high pH (pH>10), such as might occur in a cementitious LLRW facility, calcium monosulfate aluminate K_d's are calculated to be roughly 10²?mL g^?1 for both both /? and TcO₄ ^?.     

Microbial Transformations of Selenium and the Bioremediation of Seleniferous Environments

Selenium (Se) exists in many natural soil and water environments around the world, but anthropological activities such as irrigated agriculture on Se-laden soils has created many ecological problems with respect to this element. Seleniferous agricultural drainage water in California's San Joaquin Valley has been linked to the death and deformity of waterfowl. In the environment, microbial reduction, oxidation, methylation, and demethylation reactions predominantly control the oxidation state of Se and its subsequent behavior. In an effort to remediate these Se-contaminated environments a number of biological technologies have been investigated. Biological transformations of toxic Se oxyanions into less toxic or biologically unavailable forms, such as elemental Se or volatile Se compounds, has received much attention over the last decade. In this literature review, a major emphasis is placed on Se reduction and methylation/volatilization reactions because these processes are currently the most promising techniques being investigated for the bioremediation of seleniferous soil and water.     

High-temperature cultivation and 5' mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli

Overexpression of genes from thermophiles in Escherichia coli is an attractive approach towards the large-scale production of thermostable biocatalysts. However, various factors can challenge efficient heterologous protein expression--one example is the formation of stable 5' mRNA secondary structures that can impede an efficient translation initiation. In this work, we describe the expression optimization of purine nucleoside phosphorylase from the thermophilic microbe Deinococcus geothermalis in E. coli. Poor expression levels caused by stable secondary 5' mRNA structure formation were addressed by two different approaches: (i) increasing the cultivation temperature above the range used typically for recombinant protein expression and (ii) optimizing the 5' mRNA sequence for reduced secondary structures in the translation initiation region. The increase of the cultivation temperature from 30°C to 42°C allowed a more than 10-fold increase of activity per cell and optimizing the 5' mRNA gene sequence further increased the activity per cell 1.7-fold at 42°C. Thus, the combination of high-temperature cultivation and 5' sequence optimization is described as an effective approach to overcome poor expression levels resulting from stable secondary 5' mRNA structure formation. We suggest that this method is especially suitable for improving the expression of proteins derived from thermophiles in E. coli.     

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.     

Engineering of Deinococcus radiodurans R1 for bioprecipitation of uranium from dilute nuclear waste

Genetic engineering of radiation-resistant organisms to recover radionuclides/heavy metals from radioactive wastes is an attractive proposition. We have constructed a Deinococcus radiodurans strain harboring phoN, a gene encoding a nonspecific acid phosphatase, obtained from a local isolate of Salmonella enterica serovar Typhi. The recombinant strain expressed an approximately 27-kDa active PhoN protein and efficiently precipitated over 90% of the uranium from a 0.8 mM uranyl nitrate solution in 6 h. The engineered strain retained uranium bioprecipitation ability even after exposure to 6 kGy of 60Co gamma rays. The PhoN-expressing D. radiodurans offers an effective and eco-friendly in situ approach to biorecovery of uranium from dilute nuclear waste.