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

Bacterial strains and growth conditions. Deinococcus radiodurans strain R1 was grown aerobically in TGY (1% Bacto Tryptone, 0.1% glucose, and 0.5% yeast extract) liquid medium at 32°C ± 1°C under agitation (180 ± 5 rpm). E. coli DH5α cells were grown in Luria-Bertani (LB) medium at 37°C ± 1°C under agitation (180 ± 5 rpm). Bacterial growth was assessed by measuring turbidity (optical density at 600 nm [OD₆₀₀]) or by determining the number of CFU on TGY agar plates (1.5% Bacto Agar) after 48 h of incubation at 32°C ± 1°C in the case of D. radiodurans and on LB after 24 h of incubation at 37°C ± 1°C in the case of E. coli.

The antibiotic concentration used for the selection of transformants was 3 μg ml⁻¹ of chloramphenicol for Deinococcus and 100 μg ml⁻¹ of ampicillin for E. coli (21). For screening of recombinant PhoN-expressing clones, LB-TGY containing phenolphthalein diphosphate (PDP) (1 mg ml⁻¹) and methyl green (MG) (50 μg ml⁻¹) was used in the case of E. coli. For Deinococcus phoN transformants, the MG concentration was reduced to 5 μg ml⁻¹, since higher concentrations were found to be toxic.

PCR amplification, cloning, and transformation.The various plasmids used in this study are listed in Table 1. Plasmid DNA was isolated from Deinococcus and E. coli cells as described earlier (7, 27). The primers for PCR amplification were designed on the basis of the published sequence of the phoN gene of Salmonella enterica serovar Typhimurium (GenBank accession no. X59036) and the D. radiodurans groESL promoter (DR0606) in their respective genomes. The primers used for cloning and DNA sequencing were obtained from Bangalore Genei Pvt. Ltd., Bangalore, India, and are listed in Table 1. PCR products were purified using an AuPrep PCR purification kit, Life Technologies Pvt. Ltd., India.

The shuttle vector pRAD1 (21) was used for cloning of Salmonella enterica serovar Typhi phoN in Deinococcus. The phoN ORF was PCR amplified, using primers DAP-f and T3 (Table 1), from an existing plasmid (pASR1) in our laboratory (Table 1) that contains S. enterica serovar Typhi phoN with its native promoter cloned in the pBluescript SK(+) vector. The purified PCR-amplified DNA fragment was purified, digested, and ligated to the NruI-BamHI site of pRAD1. Upstream of the phoN ORF, the deinococcal groESL promoter, amplified using primers DAG-f and DAG-r (Table 1), was ligated at the XbAI/NdeI site, yielding plasmid pPN1. The recombinant plasmid was then used to transform competent E. coli DH5α cells, which were plated on LB agar containing ampicillin (100 μg ml⁻¹). The transformants were screened for PhoN expression on LB agar containing PDP and MG. On these plates, positive clones turn distinctly greenish due to the deposition of methyl green on the colonies as the medium turns acidic on cleavage of phenolphthalein diphosphate by PhoN-positive clones. Plasmids were isolated from a few of the positive clones and used to transform competent D. radiodurans R1 cells as described earlier (15). The transformants were plated on TGY containing chloramphenicol (3 μg ml⁻¹). Deinococcus colonies expressing PhoN were subsequently screened on TGY agar containing PDP and MG.

DNA sequencing.DNA sequencing was carried out using a standard dideoxy DNA sequencing protocol (28). Sequencing grade primers P5 and P6 (Table 1) bound upstream and downstream of the multiple cloning sites in pRAD1, thus amplifying the insert (1,248 bp) present in these sites. Computational analyses of DNA were performed using Internet-based programs. Similarity searches were carried out using the BLAST algorithms available at http://www.ncbi.nlm.nih.gov/BLAST/ . Multiple alignments comparing the sequences were performed using ClustalW (http://www.ebi.ac.uk/clustalw/ ). D. radiodurans genomic sequence data were obtained from the completed and annotated genome at The Institute for Genomic Research (http://www.tigr.org ). Sequences from other bacteria were obtained from GenBank (http://www.ncbi.nlm.nih.gov ).

Acid phosphatase expression: zymogram and activity assays. Salmonella enterica serovar Typhi, E. coli, and Deinococcus cultures were harvested in the early stationary phase, washed twice, and resuspended in chilled distilled water. To this, equal volumes of 2× nonreducing cracking buffer (13) were added, and samples were warmed to 50°C for 15 min. The suspension was clarified by centrifugation at 12, 000 × g for 10 min, and the protein concentration in the supernatant was estimated by a modified micro Lowry method (24). Equal amounts of protein (5 μg) were electrophoretically resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 75 V for 30 min, followed by 100 V for 90 min. In the case of Salmonella enterica serovar Typhi, 5 to 50 μg protein was loaded. The gel was rinsed briefly with water to remove surface sodium dodecyl sulfate and renatured using 1% Triton X-100 in 100 mM acetate buffer of pH 5.0 (two washes of 20 min each), followed by a wash with 100 mM acetate buffer without Triton. The gel was developed using nitroblue tetrazolium (NBT)-5-bromo-4-chloro-3-indolylphosphate (BCIP) mix in 100 mM acetate buffer at pH 5.0. The assay was terminated by rinsing the gel in distilled water.

Acid phosphatase activity was estimated by the liberation of p-nitrophenol from disodium p-nitrophenyl phosphate (pNPP) as described earlier (3). PhoN activity of whole cells is reported as nmol of p-nitrophenol (pNP) liberated min⁻¹ mg⁻¹ bacterial protein. Protein concentration was determined by Lowry's method (17) using a protein estimation kit (Bangalore Genei Pvt. Ltd., India).

Radiation response of transformants.Early-stationary-phase cultures of both Deinococcus and E. coli clones were washed twice and resuspended in fresh TGY and LB broth, respectively, at an OD₆₀₀ of 3.0. The cultures were exposed to 6 to 21 kGy of ⁶⁰Co gamma rays at a dose rate of 5 kGy h⁻¹ (⁶⁰Co Gamma Cell 5000 irradiation unit; HIRUP, Bhabha Atomic Research Centre, Mumbai, India). An aliquot of the culture kept outside the radiation source served as the sham control. The irradiated and control cells were washed, serially diluted, plated in triplicate on TGY plates containing chloramphenicol in the case of Deinococcus clones and on LB containing ampicillin in the case of E. coli clones, and incubated under optimum growth conditions. Colonies were counted, and the survival curve was plotted. Irradiated cells were also assayed for their PhoN activity as described earlier.

Uranium precipitation assays.Uranium precipitation assays were performed as previously described (20) with certain modifications. Overnight grown cultures (OD₆₀₀ of 0.3) of Deinococcus and E. coli clones were independently incubated with 0.8 mM uranyl nitrate in 2 mM acetate buffer (pH 5.0), supplemented with 5 mM β-glycerophosphate at 25°C ± 2°C under static conditions. Aliquots were taken at different time intervals and subjected to centrifugation at 12,000 × g for 10 min. Residual uranium in the supernatant and uranium associated with the cell pellet were estimated using Arsenazo III by a modification of the method of Fritz and Bradford (9). The metal in the pellet fraction was estimated after the pellet was digested with concentrated HCl. A 0.1% solution of Arsenazo III was prepared by dissolution of 0.2 g of Arsenazo III in 180 ml of 0.01 N HCl and 20 ml of absolute ethanol (30 min of stirring) and filtration through Whatman No. 1 filter paper. Twenty microliters of the spent supernatant was diluted to a total volume of 600 μl. The samples were acidified (200 μl of 0.01 N HCl), followed by the addition of 200 μl of Arsenazo III. The resultant purple-colored metal-Arsenazo III complex was estimated spectrophotometrically at 655 nm using uranyl nitrate hexahydrate (Merck) as the standard. Metal precipitation was expressed as the percentage of the initial metal present in the supernatant.

For comparison of uranium precipitation by Deinococcus and E. coli cells harboring cloned phoN, cells containing equivalent PhoN activities (as measured by a pNPP assay and corresponding to OD₆₀₀ values of 0.3 for E. coli and 1.5 for D. radiodurans) were incubated with 0.8 mM uranyl nitrate. In another experiment, Deinococcus and E. coli cells harboring phoN were irradiated at 1 and 6 kGy and the irradiated cells were then assessed for their uranium precipitation ability.

Cloning of Salmonella enterica serovar Typhi phoN in D. radiodurans R1.The phoN gene was PCR amplified from a previously cloned phoN gene of Salmonella enterica serovar Typhi (plasmid pASR1) already available in our laboratory (Table 1). The 0.887-kb PCR product was digested with NdeI and BamHI. The resultant 0.815-kb digested product was ligated to the NruI-BamHI site of the E. coli-Deinococcus shuttle vector pRAD1 (21). Upstream of the phoN ORF, the 0.256-kb deinococcal groESL promoter was ligated at the XbaI-NdeI site to obtain pPN1 (Table 1). The E. coli transformants carrying plasmid pPN1 were first selected on ampicillin and then on plates containing a modified version of a histochemical medium, described previously (26), for selecting phosphatase-positive clones. A few phosphatase-positive E. coli colonies were grown, plasmids were isolated, and the inserts therein were confirmed by PCR amplification. A typical result is shown in Fig. 1. This plasmid was then used to transform D. radiodurans R1 and transformants screened on the modified histochemical plate containing chloramphenicol (3 μg μl⁻¹). DNA inserts (1,244 bp) from four phoN-positive Deinococcus clones were sequenced using pRAD1 primers P5 and P6 (Table 1), which, bind upstream and downstream, respectively, of the multiple cloning sites, and revealed no changes.

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FIG. 1.

DNA fragments were electrophoretically resolved on a 1% agarose gel at 8 V cm⁻¹ in 0.5× Tris-borate-EDTA. The different lanes contained a 1-kb ladder (lane 1), groESL PCR product amplified using primers DAG-f and DAG-r (lane 2), phoN PCR product amplified using primers DAP-f and T3 (lane 3), pRAD1 digested with NdeI (lane 4), pPN1 digested with NdeI (lane 5), pPN1 digested with XbaI and BamHI (lane 6), PCR-amplified (using primers P5 and P6) cloned insert from pPN1 (lane 7), and a 100-bp ladder (lane 8).

In vitro and in vivo acid phosphatase activity of E. coli and Deinococcus clones harboring PhoN.Cell extracts of both E. coli and Deinococcus showed distinct PhoN phosphatase activities, visualized in the zymograms as an ∼27-kDa activity band (Fig. 2). This indicated good expression of an active enzyme from the deinococcal groESL promoter. The cell-based activity assays, performed using pNPP as the substrate, showed much less activity in the case of Deinococcus clones than in the case of E. coli clones, though nearly equal activities were seen in the zymogram (Fig. 2). On an equal protein amount basis (5 μg), protein extract from the parent Salmonella enterica serovar Typhi did not show detectable activity in the zymogram. However, when 10 times more protein was loaded, a low intensity band matching the expected molecular weight of PhoN (Fig. 2) was observed. This can be attributed to a single copy of the phoN gene in the Salmonella genome. Figure 2 clearly shows significant overexpression of PhoN in the E. coli and Deinococcus clones.

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FIG. 2.

Zymogram showing relative in-gel PhoN activities. The different lanes contained protein extracts from 5 μg Salmonella enterica serovar Typhi (lane 1), 50 μg Salmonella enterica serovar Typhi (lane 2), and 5 μg each of E. coli pRAD1 (lane 3), E. coli pPN1 (lane 4), D. radiodurans pRAD1 (lane 5), and D. radiodurans pPN1 (lane 6). Protein extracts were coelectrophoresed with prestained molecular weight standards (lane M). The values below each lane show the whole-cell PhoN activities of various clones expressed as nmol of pNP liberated min⁻¹ mg⁻¹ protein.

Radiation response of Deinococcus transformants carrying the phoN gene.The radiation resistances of different Deinococcus clones were determined by evaluating their D₁₀ values (dose causing 90% lethality or allowing 10% survival). The D₁₀ value for E. coli clones was ∼200 Gy. In comparison, Deinococcus clones harboring the empty vector pRAD1 and Deinococcus phoN clones showed D₁₀ values of 18.0 and 17.4 kGy, respectively (Fig. 3A), indicating that the cloned S. enterica serovar Typhi phoN gene did not affect the radioresistance of the host cell. There was also no change in the whole-cell PhoN activity of D. radiodurans clones after up to 12 kGy of exposure. However, beyond an 18-kGy dose a decrease of 20% was seen (Fig. 3B).

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FIG. 3.

Radioresistance of phoN transformants. (A) Stationary-phase cultures adjusted to an OD₆₀₀ of 3.0 were exposed to different doses of ⁶⁰Co gamma radiation. Survival was measured in terms of the number of CFU ml⁻¹. (B) Postirradiation PhoN activity of D. radiodurans (D. rad.) clones expressed as nmol of pNP liberated min⁻¹ mg⁻¹ protein.

Uranium precipitation by transformed E. coli and Deinococcus cells. E. coli as well as Deinococcus clones harboring phoN precipitated uranium from uranyl nitrate solutions. On an equal cell number basis, Deinococcus clones were slow (∼70% precipitation in 26 h) compared to E. coli clones (∼70% precipitation in 2 h) (Fig. 4A). However, when cells carrying equal PhoN activities were used, the time courses of uranium precipitation were nearly identical and both E. coli and D. radiodurans clones could precipitate >90% uranium in approximately 6 h (Fig. 4B). Metal loss from the supernatant was shown to be in conformity with the precipitated metal in the pellet fraction (Fig. 5) in both E. coli and D. radiodurans clones.

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FIG. 4.

Comparison of uranium precipitation by E. coli and Deinococcus (D. rad.) phoN transformants. (A) Comparable cell numbers (OD₆₀₀ of 0.3). (B) Cells with equivalent PhoN activities.

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FIG. 5.

Uranium bioprecipitation by E. coli and Deinococcus (D. rad.) phoN clones. The percent loss of uranium from the supernatant and the corresponding gain in pellet are shown.

Postirradiation uranium precipitation activities. E. coli and Deinococcus clones with equivalent PhoN activities were subjected to different doses of ionizing ⁶⁰Co gamma radiation (1 to 6 kGy) and immediately allowed to precipitate uranium from dilute (<1 mM) uranyl nitrate solution. Under these conditions, Deinococcus phoN clones could precipitate uranium much more efficiently than the corresponding E. coli phoN clones. Deinococcus phoN clones retained nearly 90% of their uranium-precipitating ability after up to 6 kGy of irradiation, whereas E. coli clones showed limitations even at 1 kGy and were severely inhibited at higher doses (Fig. 6).

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FIG. 6.

Postirradiation uranium precipitation ability of E. coli and Deinococcus (D. rad) clones harboring phoN. E. coli phoN and Deinococcus phoN cultures with equivalent PhoN activities (as determined by the p-NPP method) were used.