Genetic Engineering of D. radiodurans for uranium bioremediation

Genetic Engineering of D. radiodurans for uranium bioremediation

Genetic Engineering of D. radiodurans for uranium bioremediation from high radiation environment Shree Kumar Apte Molecular Biology Division Bhabha Atomic Research Centre, Mumbai-400085, India th Uranium resources Primary Secondary High grade ore: 2% U (20,000 ppm) Low grade ore: 0.1% U (1000 ppm) [India : 0.03-0.06% U (300-600 ppm)] Rock phosphate: 100-200 ppm Monazite: 50-200 ppm Carbonaceous matter: 300 ppm Uranium in spent fuel Acidic waste < 1mM uranyl nitrate, pH 3-7 Alkaline waste Uranium in sea water 3ppb / 13 nM, pH 7.5-7.8 Di-/Tetravalent uranyl carbonate complex, [UO2(CO3 )3] 2-/ [UO2(CO3)3] 4- < 1mM uranyl carbonate, pH 7-10

Total U in sea water: 4.5 billion tonnes (1000 X of terrestial ores) Dilute solutions with 1- 4 mM uranium at pH 5-10 need to be addressed Mechanism of metal precipitation by PhoN Localised high concentration of Pi HUO2PO4 (insoluble and membrane bound) UO22+ (soluble) Outer membrane Pi Periplasm PhoN -Phosphoglycerate Inner membrane Cytoplasm Engineering E. coli for PhoN Overexpression Samonella phoN gene with its native promoter works well in E. coli (Seetharam, Soundarajan, Udas, Rao and Apte, Proc. Biochem. 44: 246-250, 2009) Over expression of PhoN S.typhi 5g

50g E. coli Controls 5g E.coli PhoN 5g Multicopy plasmid (pUC19) based PhoN overexpression URANIUM BIO-PRECIPITATION with BACTERIA >95% URANIUM (1mM) IS PRECIPITATED FROM AQUEOUS SOLUTIONS BY GM E. coli % Uranium precipitated 100 80 60 unbuffered citrate buffer acetate buffer 40 20 0 0

1 2 3 4 Time (hrs) VIABLE CELLS ARE NOT NEEDED Post-lyophilisation Performance of E. coli bearing phoN Cells retained their integrity and activities, but lost viability System used Resuspended fresh E. coli cells bearing phoN PhoN Activity Cadmium Metal removed (%) 932 43 83 5 Lyophilized cells following storage for

0 month 989 59 83 7 1 month 850 89 ND 3 months 832 16 ND 6 months 781 20 79 4 (Seetharam, Soundarajan, Udas, Rao and Apte, Proc. Biochem. 44: 246-250, 2009) Column based uranium precipitation by E. coli-phoN clones E. coli-pRAD1 E. coli-pPN1 INPUT SOLUTION 5 mM Uranyl Nitrate with10 mM -glycerophosphate in 1.5 litres

of 2 mM Acetate Buffer COLUMN 200 mg of lyophilized cells immobilized in 15% polyacrylamide gel. GEL VOLUME : 100 ml Run time : 56 h Total Loading on the column 7.6 g of U/g dry wt. of cells The Extreme Radioresistance of Deinococcus radiodurans Provides opportunities for novel basic research and applications Engineering phoN in Deinococcus radiodurans DAG-f groESL promoter D. rad. groESL ORF D. rad. DAG-r Restriction digested Restriction digested with NdeI

with XbaI XbaI and and NdeI 256 bps 815 bps Restriction digested Restriction digested with and BamHI BamHI with NdeI NdeI and DAP-f (pASR1) phoN promoter phoN ORF BamHI (pASR1) T3 (Appukuttan , Rao & Apte, Appl. Env. Microbiol. 72: 7873-7878, 2006) Genetic engineering of phoN gene into D. radiodurans Deinococcus radiodurans Wild type Engineered 1 - E. coli - pRAD1 2 - E. coli - groESL+phoN (GN) 3 - E. coli - full phoN (CL#50) 4 - D. rad - pRAD1

5 - D. rad - full phoN (CL#29) 6 - D. rad - groESL+phoN (DN) (Appukuttan , Rao & Apte, Appl. Env. Microbiol. 72: 7 Uranium precipitation by E. coli and Deinococcus clones under 6kGy dose of irradiation % Uranium precipitated 100 E. coli (c) D. radiodurans (c) D. radiodurans (i) 80 60 40 20 E. coli (i) 0 0 2 4 6

8 10 Time (h) (Appukuttan, Rao & Apte, Appl. Env. Microbiol. 72: 7873-7878, 2006) Cell-surface bound uranyl phosphate precipitate (SEM) Seeing is believing . Cell-surface bound uranyl phosphate precipitate (TEM) A Radiation responsive Deinococcal Promoter (Pssb) rpsF 6 rpsF 18 ssb -102bp phoN pSN2 -132bp pSN3 -351bp pSN4 RDRM2 C RDRM 1

S pSN2 pRN1 H2O2 -ray 7 kGy 50 mM C S C S Mito-C 20 g ml-1 C S UV 5 kJ m-2 C S pSN3 pSN4 pSN2 C T pSN3 C T

pSN4 C T pRN1 60 Co, -ray (7 kGy) 60 Co, -ray (7 kGy) (Ujaoney, Potnis, Dani, Mukhopadhyay & Apte, J.Bacteriol., 2011) (a) (b) Control Irradiated 4 Gy/min 56.8 Gy/min nmoles pNP released / mg cell protein / min Use of radiation-induced Pssb promoter for U bioprecipitation 500 450 400 350 300

250 200 150 100 0 2 4 6 8 10 400 350 (d) 56.8 Gy/min 4 Gy/min 300 250 200 150 100 50 0 Control

Irradiated Control Irradiated Uranium precipitated (mg) / g dry biomass (c) nanomoles pNP released / mg cell protein / min Dose (kGy) 500 Deinococcus (pSN4) C Deinococcus (pSN4) I Deinococcus (pPN1) C Deinococcus (pPN1) I 400 300 200 100 0 0 1 2 Time (h) 3

An Alkaline Phosphatase Over-producer Bacterial Isolate nmoles p-NP/min Genetic Basis of this Enzyme Activity was investigated and cloned 9 10 8 6 4 5 2 0 0 1 2 3 4 5 6 7 8 9 10 11 12 pH pH optima for acid and alkaline phosphatase of Novosphingobium sp. BSAR-1 175kDa Zymogram for alkaline phosphatase analysis (Nilgiriwala, Alahari, Rao and Apte, Appl. Env. Microbiol. 74: 5516-5523, 2008) Phenotype of various native and recombinant PhoK expressing strains

(Nilgiriwala, Alahari, Rao and Apte, Appl. Env. Microbiol. 74: 5516-5523, 2008) Uranium precipitation (% of input) Uranium bioprecipitation at pH 9.0 using PhoK 0.5 mM UC, 5 mM BGP 100 80 BSAR1 KN20 EK4 60 40 20 0 0 1 2 3 4 5 6

7 8 24 Time (h) (Nilgiriwala, Alahari, Rao and Apte, Appl. Env. Microbiol. 74: 5516-5523, 2008) 30 20 40.81 41.79 15.99 17.93 20.47 In ten sity 40 30.25 32.23 50 23.49 25.47 27.29 Precipitate identified as H2(UO2)2(PO4)2.8H2O, metaautunite or

chernikovite by Powder-XRD analysis 10 0 10 15 20 25 30 35 40 45 50 2 (Nilgiriwala, Alahari, Rao and Apte, Appl. Env. Microbiol. 74: 5516-5523, 2008) Uranium precipitation at pH 9.0 using PhoK alkaline phosphatase BSAR-1 White light KN20 pET29b

EK4 - cells - GP UV light Seeing is believing . TEM images of Deino-PhoK cells 500 nm Deino-PhoK without uranium treatment 500 nm Deino-PhoK with uranium treatment Needle shaped crystals of uranyl phosphate seen in uranium treated samples Easy recovery of precipitated uranyl phosphate through beads A C B Empty beads D Deino-PhoK (-U) E Deino-PhoK beads Deino-pRAD1 beads

100 Deino-PhoK (+U) Deino-PhoK beads supernatant Deino-pRAD1 beads supernatant 120 Fluroscence intensity (AU) % U precipitated 80 60 40 20 0 0.5 1.0 1.5 2.0

2.5 Time (h) 3.0 3.5 4.0 4.5 100 80 60 40 20 0 1 2 Time (h) 3 4 Comparison of Deino-PhoN and Deino-PhoK strains Recombinant Deinococcus strains on PDP-MG plate

Phosphatase activity of recombinant strains Specific Activity Clones (nmoles of p-NP liberated/min/mg of total cellular protein) Deino- pRAD1 18 + 5 Deino-PhoK 7000 + 1000 Deino-PhoN 200 + 10 Deino-PhoK Deino-PhoN Deino-control 100 1 Deino-pRAD1 2 Deino-PhoK 3 Deino-PhoN %U precipitated 80 60

40 20 0 1 2 Time in hours 3 4 Maximum loading possible with Deino-PhoN Maximum loading possible with Deino-PhoK 10mM 10 % U precipitated 80 9 Deino-PhoK 8 1 mM U + 5mM GP 60 2 mM U + 5mM GP

7 5 mM U + 10mM GP 6 10 mM U + 20mM GP 40 5mM controls (Deino-pRAD1) 20 0 11 10 mMU + 20mM GP 4 10 mM U + 20mM GP 3 ( without cells) 0.0 0.5

1.0 5 1.5 Time(h) 2.0 2mM 2 1mM 1 2.5 0 U g/g of dry weight of Deino-PhoK cells 12 100 SUMMARY Metal precipitation using phosphatases is an old story. Novelty of the present work : Use of heavy metal tolerant enzymes Cloning/characterization of a very active alkaline phosphatase (PhoK)

Extension of metal bioremediation to alkaline solutions Recombinant radioresistant microbes to biorecover uranium from acidic/alkaline solutions in high radiation environments. Lyophilization to extend shelf-life while retaining precipitation ability Volume reduction, high U loading, easy recovery Related Publications Appukuttan, D, Rao, A. S. and Apte, S. K. (2006) Appl. Env. Microbiol. 72 : 7873-7878. Nilgiriwala, K., Alahari, A., Rao, A. S. and Apte, S. K. (2008) Appl. Env. Microbiol. 1784 : 1256-1264. Seetharam, C., Soundarajan, S., Udas, A. C., Rao, A. S. and Apte, S. K. (2009) Proc. Biochem. 44 : 246250. Nilgiriwala, K., Bihani, S C., Das, A., Prashar, V., Kumar, M., Ferrer, J-L, Apte, S. K. and Hosur, M. V. (2009) Acta Cryst. F65 : 917-919. Ujaoney, A. K., Potnis, A., Mukhopadhyay, R. and Apte, S. K. (2010) J. Bacteriol. 192 : 5637-5644. Bihani, S., Das, A., Nilgiriwala, K., Prashar, V., Pirocchi, M., Apte, S. K., Ferrer, J. and Hosur, M. V. (2011) PLoS ONE 6 : e22767.

Appukuttan, D., Seetharam, C., Padma, N., Rao, A. S. and Apte, S. K. (2011) J. Biotechnol. 154 : 285290. Seetharam-Misra C., Appukuttan D., Kantamreddi V. S. S., Rao A. S. and Apte S. K. (2012) Bioengineered Bugs 3 : 44-48. Kulkarni, S., Ballal, A. and Apte, S. K. (2013) J. Hazard. Metals 262 : 853-861. Misra, C.S., Mukhopadhyaya, R. and Apte, S. K. (2014) J. Biotechnol. 189 : 8893. ACKNOWLEDGEMENTS M. Daly & K. Minton Deinococcus radiodurans strain R1 Mary Lidstrom Useful Vectors PhoN for U/Cd bioprecipitation Seetharam Deepti Appukuttan, Chitra

& A.S. Rao PhoK for U bioprecipitatio Kayzad Nilgiriwala, Anuradha Alahari & A. S. Rao, Sayali Kulkarni, SEM-EDX Shovit Bhattacharya & N. Padma (TPPED, BARC) TEM ICP-MS BARC) Anand Ballal, Alka Gupta Sanjukta A. Kumar (ACD, Recombinant strain functions well in high radiation environment A B Deino-pRAD1 Deino-PhoK 8 10

Unirradiated cells Irradiated cells 100 80 7 % U precipitated CFU/ml 10 6 10 60 40 20 5 10 D10=15.63kGy D10=15.5kGy 0 3

6 9 12 Dose (kGy) 15 18 21 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time (h) Addition of PhoK does not compromise or alter radioresistance Irradiation (6 kGy, 60Co -rays) does not influence bioprecipitation

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