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Journal of Biotechnology 189 (2014) 88–93

Contents lists available at ScienceDirect

Journal of Biotechnology

j ourna l ho me pa ge: www.elsev ier .com/ locate / jb io tec

arnessing a radiation inducible promoter of Deinococcus radioduransor enhanced precipitation of uranium

hitra Seetharam Misra, Rita Mukhopadhyaya ∗, Shree Kumar Apteolecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India

r t i c l e i n f o

rticle history:eceived 6 June 2014eceived in revised form1 September 2014ccepted 15 September 2014vailable online 28 September 2014

eywords:

a b s t r a c t

Bioremediation is an attractive option for the treatment of radioactive waste. We provide a proof of prin-ciple for augmentation of uranium bioprecipitation using the radiation inducible promoter, Pssb fromDeinococcus radiodurans. Recombinant cells of D. radiodurans carrying acid phosphatase gene, phoN underthe regulation of Pssb when exposed to 7 kGy gamma radiation at two different dose rates of 56.8 Gy/minand 4 Gy/min, showed 8–9 fold increase in acid phosphatase activity. Highest whole cell PhoN activitywas obtained after 2 h in post irradiation recovery following 8 kGy of high dose rate radiation. Such cellsshowed faster removal of high concentrations of uranium than recombinant cells expressing PhoN under

einococcusadiation inducible promoterranium bioprecipitationadioactive waste

a radiation non-inducible deinococcal promoter, PgroESL and could precipitate uranium even after contin-uous exposure to 0.6 Gy/min gamma radiation for 10 days. Radiation induced recombinant D. radioduranscells when lyophilized retained high levels of PhoN activity and precipitated uranium efficiently. Theseresults highlight the importance of using a suitable promoter for removal of radionuclides from solution.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Bioremediation involves use of organisms for removal of toxicomponents present in the environment. This has been achievedoth by using natural or genetically engineered strains to aid wasteanagement (Valls and de Lorenzo, 2002). Choice of the appro-

riate organism depends on its ability to survive and efficientlyxpress desired genes under conditions prevailing in waste sites.his necessitates use of radioresistant organisms for bioremedia-ion of radioactive waste.

The Gram positive bacterium, Deinococcus radiodurans canolerate high doses of ionizing radiation. Members of the Deinococ-aceae family are vegetative, non-pathogenic, ubiquitous, andxhibit remarkable resistance to DNA damage caused by ioniz-ng radiation, desiccation, ultraviolet radiation and electrophilic

utagens (Battista, 1997; Minton, 1994, 1996; Slade and Radman,011; Venkateswaran et al., 2000). D. radiodurans, which canurvive 15 kGy ionizing radiation, is one of the most radiation-esistant members of this family. This has prompted its genetic

ngineering to express proteins capable of detoxifying organicolvents and heavy metals that prevail in radionuclide contam-nated wastes (Brim et al., 2000, 2003; Lange et al., 1998). The

∗ Corresponding author. Tel.: +91 2225592696; fax: +91 2225505326.E-mail addresses: ritam@barc.gov.in, rita45@gmail.com (R. Mukhopadhyaya).

ttp://dx.doi.org/10.1016/j.jbiotec.2014.09.013168-1656/© 2014 Elsevier B.V. All rights reserved.

organism has been extensively studied for its highly proficientDNA damage repair mechanisms that are aided by proteins rapidlyinduced upon irradiation (Misra et al., 2006; Zahradka et al., 2006).Radiation-enhanced gene expression in this organism appears tobe regulated by transcriptional activation and may be exploited forenhanced expression of desired genes. This approach can be use-ful for radioactive waste management at radioactive storage sites,nuclear facilities and waste holding tanks.

One of the ways to augment levels of recombinant gene expres-sion is to use appropriate promoters, which are recruited bythe bacterium under specific growth conditions. Use of stress-inducible promoters provides a choice of gene expression controlunder defined stimuli. Bacterial promoters that are triggered byorganic contaminants and regulate expression of proteins involvedin degradation of the concomitant pollutants have found sev-eral applications (Diaz and Prieto, 2000; Girotti et al., 2008; Liuet al., 2010; Wise and Kuske, 2000). Likewise, promoters of genesinvolved in metal transport, sequestration or detoxification areinduced in the presence of the target metal itself (Lloyd and Lovley,2001; Summers, 2009; Yagi, 2007). To exploit the radiation resis-tant property of D. radiodurans for bioremediation, promoters thatcontrol expression of radiation inducible genes appear to be attrac-

tive candidates.

In bacteria, a majority of promoters possess consensussequences of hexamer boxes, −10 (TATAAT) and −35 (TTGACA)with which the �-factors interact and facilitate RNA polymerase

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inding (Burgess and Anthony, 2001) and transcription. In D. radio-urans, the upstream regions of 29 radiation-inducible genes oftenack a consensus promoter sequence but contain a 17 bp palin-romic sequence known as the Radiation/Dessication Responseotif (RDRM) (Makarova et al., 2007). The radiation inducible

romoter for the ssb gene encoding the single stranded DNA-inding protein (SSB) harbors both consensus motifs and two RDRMequences (Ujaoney et al., 2010). Upstream region of groESL gene,hich codes for a chaperone protein and is not inducible by radi-

tion, has the consensus promoter sequence and does not possessny RDRMs (Meima et al., 2001; Ujaoney et al., 2010).

In the past, groESL promoter, PgroESL, from D. radiodurans wassed to constitutively over-express phoN gene of Salmonella enter-

ca serovar Typhi that encodes a non-specific acid phosphataseNSAP) (Appukuttan et al., 2006). This enzyme can cleave a phos-homonoester substrate to release the phosphate moiety, which

n turn can cause precipitation of metals such as uranium andadmium from solutions (Misra et al., 2012). Recombinant D. radio-urans strain that carries a PgroESL-phoN construct on plasmidPN1 was shown to bring about metal precipitation even after beingubjected to 6 kGy gamma radiation under non-growing condi-ions, while Escherichia coli cells carrying the same construct failedo do so (Appukuttan et al., 2006). Further, Deinococcus (pPN1)ells retained PhoN activity and uranium precipitation ability uponyophilization and exhibited a shelf life of 6 months at room tem-erature with only 6% loss of activity (Appukuttan et al., 2011).hoN was also expressed in D. radiodurans under regulation ofhe ssb gene promoter region containing two RDRMs (Pssb) thats inducible by radiation (Ujaoney et al., 2010). This recombinanttrain, Deinococcus (pSN4), showed 6–8 fold higher PhoN activitypon irradiation, compared to unirradiated cells (Ujaoney et al.,010).

In the present study, the radiation inducibility of PhoN ofeinococcus (pSN4) strain was evaluated and optimized forranium bioprecipitation in radiation environment. The resultsrovide a proof of concept of enhanced bioremediation by exploita-ion of a radiation inducible promoter.

. Materials and methods

.1. Bacterial strains and growth conditions

D. radiodurans strain R1 transformed with plasmids pSN4 orPN1 carrying the constructs Pssb-phoN or PgroESL-phoN on pRAD1lasmid respectively (Appukuttan et al., 2006; Ujaoney et al., 2010)ere used. D. radiodurans cells carrying pRAD1 vector alone served

s control. The strains were grown in TGY (1% Tryptone, 0.1%lucose, 0.5% yeast extract) medium supplemented with or with-ut 3 �g/ml chloramphenicol (TGY-Cm) at 32 ◦C with agitation at50 rpm. Bacterial growth was measured spectrophotometricallys turbidity (OD600).

.2. Radiation exposure and recovery

Recombinant D. radiodurans cells were exposed to gamma radi-tion from two 60Co sources that permitted two different doseates. Dose at the rate of 4 Gy/min (Gamma Chamber GC-220,tomic Energy of Canada, Canada) and at 56.8 Gy/min (Gammahamber GC-5000, Board of Radiation and Isotope Technology,umbai, India) were applied to cell suspensions (OD600 of 5.0) in

GY medium. Unirradiated cells were kept at room temperature in

ark during this period. Control and irradiated cells were subse-uently inoculated (OD600 of 0.5) in TGY and allowed to recover at2 ◦C with agitation at 150 rpm for up to 16 h. This period is referredo as post irradiation recovery (PIR). For studying dose-dependent

hnology 189 (2014) 88–93 89

induction of promoter activity, recombinant cells were irradiatedat 56.8 Gy/min dose rate for 19 min to 3.16 h in order to deliver totalgamma radiation doses between 1 and 10 kGy. Deinococcus (pSN4)cells, washed free of medium and re-suspended in distilled waterwere also irradiated at a dose rate of 0.6 Gy/min (Blood Irradiator,Board of Radiation and Isotope Technology, Mumbai, India) for 10days and then used in uranium precipitation assay.

2.3. Assays for reporter gene activity

PhoN activity expressed from the Pssb and PgroESL pro-moters was monitored in three ways. (a) Histochemical plateassay, briefly 5 × 106 cells/ml were spotted on TGY-Cm platescontaining phenolphthalein diphosphate (PDP) substrate andmethyl green (MG) pH indicator dye and incubated at 32 ◦Covernight. Green PhoN-expressing colonies on histochemicalplates appeared as a consequence of precipitation of methylgreen upon acidification caused by released phosphoric acid(Appukuttan et al., 2006); (b) zymogram analyses in gels for acidphosphatase activity, using NBT/BCIP (nitroblue tetrazolium–5-bromo-4-chloro-3-indolylphosphate) as substrate (Appukuttanet al., 2006); (c) assay for cell bound PhoN activity by spectro-photometric (OD405) measurement of p-nitrophenol (pNP) releasedfrom p-nitrophenylphosphate (pNPP) as described earlier (Boltonand Dean, 1972). Briefly, aliquots containing 5 × 106 cells/ml wereused in 1.2 ml assay containing 100 mM acetate buffer pH 5.0. Reac-tion was stopped after half an hour by addition of 2 ml of 0.2N NaOHand OD at 405 nm was determined.

2.4. Western blot for PhoN detection

Whole cell protein extracts from recombinant Deinococcus cellswere prepared by incubating cells at OD600 of 20 cells in Laemmli’sbuffer for 5 min in a boiling water bath. Fifty micrograms of proteinfrom the soluble lysate was separated on 10% SDS–PAGE, followedby blotting on nitrocellulose membrane. Anti-PhoN polyclonal anti-body (Rabbit IgG) was used at 1:500 dilution for detection byAnti-rabbit secondary antibody IgG conjugated to alkaline phos-phatase (Sigma, St Louis, MO).

2.5. Uranium precipitation assay

Phosphatase mediated uranium precipitation was assessed asdescribed earlier (Appukuttan et al., 2006). Briefly, irradiatedrecombinant Deinococcus cells, which had undergone PIR, werewashed in distilled water and re-suspended in 2 mM acetate buffer,pH 5.0 containing uranyl nitrate and �-glycerophosphate, as spec-ified in each experiment. Aliquots were removed at fixed intervals,subjected to centrifugation at 10,000 rpm for 3 min and the amountof uranium in the supernatant was determined using Arsenazo IIIreagent as described earlier.

For testing uranium precipitation using lyophilized cells, 80 mgof lyophilized powder was used in 50 ml solution containing 2 mMuranyl nitrate and 15 mM �-glycerophosphate in 2 mM acetatebuffer (pH 5). Appropriate controls were included to rule out spon-taneous precipitation of uranium, and to exclude biosorption of themetal to cells or sorption to the walls of the container, as described(Appukuttan et al., 2011).

2.6. Lyophilization

Irradiated cells after PIR and control cells grown for the same

duration were rinsed twice with distilled water. Thick cell sus-pensions (∼OD600 of 100) were poured into plastic Petri platesand frozen in liquid nitrogen. Frozen cells were lyophilized in aLyospeed (Genevac, UK) at 0.07 mbar for 18 h (Appukuttan et al.,

9 Biotechnology 189 (2014) 88–93

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Fig. 1. Zymogram analysis of acid phosphatase activity expressed from thedeinococcal ssb or groESL promoters during post irradiation recovery. The recom-binant Deinococcus (pSN4) and Deinococcus (pPN1) strains were exposed to 7 kGygamma radiation at two different dose rates, 56.8 Gy/min (a) and 4 Gy/min (b), andallowed to recover in fresh TGY medium for 3 h. Proteins from control (C) andirradiated (I) cells were extracted in non-reducing buffer and 30 �g of each was

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011). Lyophilized cells were scraped off from the Petri plates andtored in plastic vials at room temperature until used for uraniumrecipitation experiments. Protein content of 0.33 mg lyophilizedells re-suspended in 1 ml distilled water was equivalent to 1 mlf OD600 1.0 fresh cells (Appukuttan et al., 2011). Lyophilized cellsere allowed to form a uniform suspension in acetate buffer before

he PhoN activity and uranium precipitation assays were carriedut.

. Results

.1. Effect of dose rate of gamma radiation on PhoN activity andranium precipitation by recombinant Deinococcus strains

Acid phosphatase activity of irradiated and unirradiated recom-inant D. radiodurans cells was assayed to determine effect of highnd low dose rates of radiation on Pssb and PgroESL promoter drivenhoN gene expression and activity. In-gel enzyme activity showedhat 7 kGy of 60Co gamma rays strongly induced acid phosphatasectivity in irradiated Deinococcus (pSN4) cells, at both the dose ratesested (Fig. 1). In Deinococcus (pPN1) cells, the PhoN activity was

arginally induced under low dose rate irradiation, but severelyeduced under high dose rate irradiation (Fig. 1). Compared to unir-adiated cells, irradiated cells of Deinococcus (pSN4) also showedarker green colonies on histochemical plate assay (Fig. 2a), and

ig. 2. Enhanced acid phosphatase activity and uranium precipitation driven by deinococeinococcus (pSN4) were exposed to 7 kGy gamma radiation at two different dose rates

liquots containing equal cell densities of Deinococcus (pSN4) were spotted on histochemubstrate (b). For uranium precipitation, OD600 6.0 of Deinococcus (pSN4) cells and Deino

mM �-glycerophosphate in 2 mM acetate buffer (pH 5). Timed aliquots of the cell suspstimated (c) and (d).

electrophoretically resolved by 10% SDS–PAGE. Activity bands were developed byincubating the gel with NBT-BCIP in acetate buffer, pH 5.

8–10 fold higher whole cell acid phosphatase activity as determinedby pNPP assay (Fig. 2b).

Whether increased acid phosphatase activity seen in irradiatedDeinococcus (pSN4) cells leads to more efficient uranium precip-itation was investigated. Deinococcus (pSN4) cells were able to

precipitate uranium faster than control cells at both the doserates tested. Under high dose rate radiation, Deinococcus (pSN4)cells precipitated 100 mg uranium/g dry biomass in 3 h as com-pared to 30 mg uranium/g dry cell biomass by un-irradiated cells

cal ssb promoter during post irradiation recovery. Cultures of recombinant cells ofof 56.8 Gy/min and 4 Gy/min, and allowed to recover in fresh TGY medium for 3 h.ical plate containing PDP-MG (a), or used in acid phosphatase assay using pNPP ascoccus (pPN1) cells were used in 5 ml assays containing, 1 mM uranyl nitrate andensions were subjected to centrifugation and the uranium in the supernatant was

C.S. Misra et al. / Journal of Biotechnology 189 (2014) 88–93 91

Fig. 3. Enhanced acid phosphatase activity and PhoN protein production inDeinococcus (pSN4) cells after induction by high dose irradiation. (a) Cultures ofrecombinant Deinococcus (pSN4) were exposed to increasing doses of gamma radi-ation at a rate of 56.8 Gy/min, and allowed to recover in fresh TGY medium for 3 h.Equal cell densities were used for assaying acid phosphatase activity with pNPPas substrate. (b) Western blot of recombinant Deinococcus cells exposed to 8 kGyga

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Table 1Acid phosphatase activity of Deinococcus (pSN4) cultures during post irradiationrecovery (PIR). Cultures were subjected to 8 kGy irradiation at 56.8 Gy/min dose rateand allowed to recover under standard growth conditions. The fold induction of acidphosphatase activity was determined by comparison with unirradiated cultures.

Time of PIR (h) Fold induction in PhoN activity*

0 1.07 ± 0.0252 10.5 ± 0.43 7.9 ± 0.8056 3.75 ± 0.35

Heavy metals present in radioactive waste are a cause of envi-

amma irradiation (I) and unexposed cells (C) followed by 2 h PIR using anti-PhoNntibody.

Fig. 2c), and under low dose rate irradiation they precipitated20 mg uranium as compared to 50 mg uranium/g dry biomass byn-irradiated cells (Fig. 2d). Deinococcus (pPN1) cells, on the otherand, showed a reduction in uranium precipitation capacity afterigh dose rate radiation (Fig. 2c). Under low dose rate radiation,he precipitation profile of irradiated Deinococcus (pPN1) cells wasimilar to that of control cells (Fig. 2d); around 70 mg uranium/gry cell biomass precipitated under both conditions. Importantly,ranium precipitation by irradiated Deinococcus (pSN4) cells wasaster than by Deinococcus (pPN1) cells.

As seen in Fig. 2c and d, uranium precipitation by DeinococcuspSN4) was slightly better under low dose rate (4 Gy/min) irradi-tion compared with high dose rate irradiation. However, 29.1 hxposure was required to deliver 7 kGy under low dose rate condi-ion, while the same was achieved in 2.05 h at high dose rate. Thus,ubsequent experiments were done with high dose rate gammaadiation.

.2. Optimization for maximum acid phosphatase activity ineinococcus (pSN4) cells

Acid phosphatase activity was measured in Deinococcus (pSN4)ultures that were subjected to 1–10 kGy radiation at 56.8 Gy/minnd allowed to recover for up to 3 h. The activity gradually increasedn a dose dependent manner from 1 kGy to 8 kGy beyond whicht started to decrease at 9 and 10 kGy dose (Fig. 3a). Between 0 hnd 2 h of PIR, at 8 kGy dose, phosphatase activity increased from.07 to 10.5 fold compared with unirradiated control (Table 1), butecreased thereafter. Thus, cells subjected to 8 kGy of radiation atigh dose rate, followed by 2 h recovery displayed the highest PhoNctivity.

A Western blot using anti-PhoN antibody was carried out to

onfirm the amount of PhoN protein produced in recombinanteinococcus cells after irradiation under the above conditions. Ashown in Fig. 3b, at 8 kGy irradiation followed by 2 h PIR, the PhoN

16 2.5 ± 0.25

* As determined by pNPP assay.

protein levels in irradiated Deinococcus (pSN4) cells far exceededthat in Deinococcus (pPN1) cells (both control and irradiated).

3.3. Metal precipitation ability of Deinococcus (pSN4) cells athigher uranium concentrations and under chronic radiationexposure

Recombinant Deinococcus cells were evaluated for their abil-ity to precipitate higher concentrations of uranium (5–20 mMuranyl nitrate) with twice the corresponding concentration of �-glycerophosphate. Deinococcus (pSN4) cells could precipitate all of5 mM and 10 mM uranium in 1 day and 2 days respectively, result-ing in a loading of 1.8 and 3.6 g uranium/g dry biomass. At 20 mMuranium concentration, Deinococcus (pSN4) cells could precipitate70% uranium in 4 days while in the same time Deinococcus (pPN1cells) could precipitate only 20% uranium. To achieve a loading of1.7 g uranium/g dry biomass at this concentration, the former tookonly 6 h while the latter took 3 days (Fig. 4a).

Deinococcus recombinant cells irradiated with 8 kGy dose fol-lowed by 2 h PIR were placed for 10 days under conditions thatmimic the doses typically prevalent in radioactive waste sites(0.6 Gy/min). Such cells could precipitate 160 mg uranium/g drybiomass which was only marginally lower than 200 mg uranium/gdry biomass precipitated by cells kept in the dark, un-exposed tocontinuous radiation for same time period (Fig. 4b). Deinococcus(pPN1), on the other hand could precipitate no more than 90 and60 mg uranium/g dry biomass under control and chronic radiationconditions respectively. Under all the above experimental condi-tions, Deinococcus (pSN4) cells continued to show superior uraniumprecipitation capability as compared to Deinococcus (pPN1) cells.

3.4. PhoN activity and uranium precipitation ability oflyophilized recombinant strains

Cells irradiated with 8 kGy dose followed by 2 h PIR, werelyophilized and stored at room temperature. Acid phosphataseactivity of Deinococcus (pSN4) cells was lowered by about 20%in freshly lyophilized cells compared to wet cells (Fig. 5a).Lyophilized Deinococcus (pSN4) cells could precipitate uraniummuch faster than lyophilized pPN1 cells. For example, after 1 h,irradiated Deinococcus (pSN4) cells had precipitated 200 mg ura-nium/g dry biomass while unirradiated Deinococcus (pPN1) cellscould only precipitate 50 mg uranium/g dry biomass (Fig. 5b). Fur-ther, lyophilized Deinococcus (pSN4) cells retained 83% of their acidphosphatase activity up to 30 days of storage at room temperature(Fig. 5a).

4. Discussion

ronmental concern and removal of these metals by bioprecipitationis a viable option (Appukuttan et al., 2006; Choudhary and Sar,2011; Kulkarni et al., 2013; Martinez et al., 2007). This work

92 C.S. Misra et al. / Journal of Biotechnology 189 (2014) 88–93

Fig. 4. Uranium precipitation by recombinant D. radiodurans. Deinococcus (pSN4) cells (closed symbols) were exposed to 8 kGy gamma irradiation followed by 2 h recoveryand (a) challenged with 5–20 mM of uranyl nitrate using 10–40 mM �-glycerophosphate in 2 mM acetate buffer (pH 5) along with (�) un-irradiated Deinococcus (pPN1) cellsi llengea re as

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ncluded for comparison and (♦) D. radiodurans (pRAD1) cells as control or (b) chafter exposure to radiation environment of 0.6 Gy/min for 10 days. Other details wen 6 h. (C) Refers to cells unexposed to high dose rate gamma radiation and (I) refer

xploited the radiation inducibility of D. radiodurans ssb promotero drive expression of a gene encoding a NSAP, PhoN, from S. entericaerovar Typhi in D. radiodurans, to significantly augment precipita-ion of uranium.

In wild type D. radiodurans, ssb is expressed at all times at lowevel but its promoter is strongly induced several fold in responseo DNA damage signals particularly double strand breaks (DSBs)Ujaoney et al., 2010). In this study, different dose rates of gammaadiation could enhance PhoN gene expression by activation ofhe Pssb promoter cloned upstream on a plasmid. Thus, a highevel of PhoN activity was achieved in Deinococcus (pSN4) cells,

n spite of low copy numbers (7–10) of the pRAD1 plasmid inuch host cells (Meima and Lidstrom, 2000). Other studies havemployed constitutive promoter in tandem duplication integrative

ig. 5. PhoN activity and uranium precipitation ability of lyophilized recombinant D. radiamma radiation at a dose rate of 56.8 Gy/min (I), or kept outside the irradiator (C) follohoN activity and uranium precipitation. (a) PhoN activity (nmoles pNP released/mg wholt room temperature. Freshly harvested cells of the same strain served as controls. (b) Cof Deinococcus (pSN4) and Deinococcus (pPN1) strains.

d with 1 mM uranyl nitrate and 5 mM �-glycerophosphate in 2 mM acetate bufferdescribed in legend to Fig. 2c and d. Graph shows amount of uranium precipitatedlls irradiated at 8 kGy dose followed by 2 h PIR.

vectors to increase copy numbers of heterologous genes (Brimet al., 2000, 2003; Lange et al., 1998) in D. radiodurans for degra-dation of toluene, and detoxification of Hg and Cr. The induciblesystem of protein expression described here, where the externalinducer is radiation itself, achieves the same result and is desir-able in high radiation environment. In addition, the incrementalinduction of the Pssb promoter in response to increasing doses(1–8 kGy) of gamma radiation allows scope for fine tuning ofheterologous protein expression in this organism.

Conditions for maximum PhoN activity were optimized in thissystem. To begin with, 7 kGy of radiation dose was chosen to eval-

uate the effect of dose rate of radiation on Pssb induction, basedon our earlier work (Ujaoney et al., 2010). Having shown that irra-diated cells of Deinococcus (pSN4) are better suited for uranium

odurans cells. Cultures of recombinant D. radiodurans strains were exposed to 8 kGywed by 2 h recovery, and lyophilized. Such cells were reconstituted and tested fore cell protein/min) was determined in Deinococcus (pSN4) cells after 30 d of storagemparison of uranium precipitation by irradiated and unirradiated, lyophilized cells

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C.S. Misra et al. / Journal of

recipitation than Deinococcus (pPN1), we went on to optimizerradiation conditions for maximum PhoN activity (Fig. 3, Table 1)

hich was found to occur at high dose rate radiation of 8 kGyollowed by 2 h PIR. Phosphatase activity of irradiated Deinococ-us (pSN4) cells was higher than that of recombinant DeinococcuspPN1) cells expressing PhoN from a radiation non-inducible, con-titutive promoter, PgroESL as seen in zymograms. That this was

direct effect of increased PhoN production was corroboratedy Western blotting (Fig. 3b) which showed much higher levelsf protein in Deinococcus (pSN4) than Deinococcus (pPN1) cells,hus implicating higher induction of PhoN by radiation induciblessb promoter. The decrease in phosphatase activity in irradiatedecombinant Deinococcus cells following lyophilization could be

result of irradiated cells sustaining additional damage duringyophilization.

Most importantly, the PhoN activity of Deinococcus (pSN4) cellsas sustained after radiation exposure while Deinococcus (pPN1)

uffered a loss of PhoN activity upon irradiation at high dose rateFigs. 1, 2c and Fig. 5b). Radiation induced Deinococcus (pSN4)ells could rapidly precipitate uranium in few hours comparedith Deinococcus (pPN1) which took days. This would likely per-it operation of a flow-through process at high flow rates, and

educe holding time in a batch system. Deinococcus (pSN4) cellsould precipitate uranium even after 10 days of chronic exposuret 0.6 Gy/min gamma radiation showing that the system couldustain under radiation dose rates typically found in waste sitesKryshev and Sazykina, 1998; Pitonzo et al., 1999). Further, sincehosphatase-activity-based metal precipitation is essentially non-pecific in nature, a wide range of radionuclides can be removedrom contained radioactive waste solution using this approachMacaskie et al., 1994; Misra et al., 2012).

. Conclusions

The inherent radiation tolerance of the organism together withadiation enhanced PhoN activity enables Deinococcus (pSN4) toackle high uranium concentrations and prolonged radiation expo-ure making this a robust bioremediation agent. Lyophilization ofhese cells converted them into an easy to handle and store formu-ation, which retained uranium precipitation ability.

cknowledgments

The authors are thankful to Mr. Priyoda Abhishek for help-ng with maintenance and irradiation of the bacterial culturesnd to Dr. Anuradha Alahari for her helpful comments on theanuscript.

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