2. In this paper, the authors had cloned two different functional loci from bacteria into Deinococcus radiodurans for the purpose of bioremediation. What are those two functional loci? Please specify their names and main functions respectively.

3. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments William Chen Keng Teo

4. Overview  Background  Deinococcus radiodurans  Engineering Deinococcus radiodurans  Experimental results and analysis  Discussion & Conclusions  Future Development

5. Background  In U.S. alone, 1000 waste sites with radiation level  10 mCi/L  7.5 x10 7 m 3 soil & 2 x10 12 dm 3 ground water contaminated by 3 x10 6 m 3 leaking waste  Clean-up cost > $265 billion  Potential targets for less expensive and more effective bioremediation methods

6. What is bioremediation?  Definition: Use of biological mechanisms to destroy, transform or immobilize environmental contaminants to protect potential sensitive receptors Use of biological mechanisms to destroy, transform or immobilize environmental contaminants to protect potential sensitive receptors  Applications: Agricultural chemicals, gasoline contamination & radioactive wastes….etc. Agricultural chemicals, gasoline contamination & radioactive wastes….etc.  Examples: 1) Xenobiotics by Pseudomonas sp. 1) Xenobiotics by Pseudomonas sp. 2) n-alkane metabolism by Desulfobacterium cetonicum & 2) n-alkane metabolism by Desulfobacterium cetonicum & Pseudomonas sp. Pseudomonas sp.

7. Denococcus radiodurans Physiology:  Gram(+), Non-pathogenic  Red-pigmented Red-pigmented  Radiation-resistant: UV & Ionic  Selective solvent tolerant  Non-motile  Soil bacterium

8. Denococcus radiodurans R1 strain genome:  2 chromosomes (2.65 Mbp & 412 kbp), with 4- 10 genome equivalents/copies in growing cells  1 megaplasmid (177 kbp)  1 small plasmid (46 kbp)  Able to withstand high energy radiation due to: 1. Strong DNA repair 1. Strong DNA repair 2. DNA damage prevention 2. DNA damage prevention 3. Genetic redundancy 3. Genetic redundancy

9. Objectives of Engineering D. radiodurans  Confer resistance to toxic metallic waste constituents  Transform toxic metals to less toxic and less soluble chemical forms  For example: Bacterial mercuric reductase gene merA, encoding mercuric ion reductase MerA that reduces toxic Hg(II) to inert Hg(0) Bacterial mercuric reductase gene merA, encoding mercuric ion reductase MerA that reduces toxic Hg(II) to inert Hg(0)toxic Hg(II) to inert Hg(0)toxic Hg(II) to inert Hg(0)

10. Construction of Metal-Remediating D. radiodurans Strains  Clone merA locus from E. coli BL308 into D. radiodurans R1 strain merA locus merA locus  Combining organic degrading function into Hg(ll) R -D. radiodurans  Five different Constructs: - Hg(ll)-resistant: - Hg(ll)-resistant: MD 735, MD 736, MD 737, MD 767 MD 735, MD 736, MD 737, MD 767 - Hg(ll)-resistant & toluene metabolizing: - Hg(ll)-resistant & toluene metabolizing: MD 764 MD 764

11. MD 735 Starting material: merA operon from the E. coli strain BL308 D. radiodurans autonomously replicating plasmid pMD66 D. radiodurans wild-type strain R1 Kan R : kanamycinresistance gene, aphA P1 & P2: D. radiodurans Constitutive Promoter

12. MD 736

13. MD 737 MD 737

14. MD 767 Cm R : chloramphenicol resistance gene, cat

15. tod Operon cloned from Pseudomonas putida MD 764

16. Summary of Hg(ll)-Resistant Constructs Construct Type of Integration D.radiodurans Constitutive Promoter MD 735 Plasmid (No Integration) Yes Yes MD 736 Tandem Duplication Yes Yes MD 767 Direct Insertion Yes Yes MD 737 Amplification Vector No No MD 764 Direct Insertion No No

17. Experimental Results & Analysis  merA copy number  Resistance to Hg(II)  Effect of γ-radiation  Reduction of Hg(II) to Hg(0)  Assess toluene-metabolizing potential of MD 764

18. merA copy number A shows electrophoresis of genomic DNA of different strains B examines the intensity of merA bands after hybridization C examines change in copy number after induction in MD767 & MD736

19. Construct merA Operon Copy Number Type of Integration D.radiodurans Constitutive Promoter MD 735 1 Plasmid (No Integration) Yes MD 736 10 Tandem Duplication Yes MD 767 10 Direct Insertion Yes MD 737 150 Amplification Vector No MD 764 150 + tod operons Direct Insertion No E. Coli 20-30 Plasmid (No Integration) No (E. Coli Promoter) Summary of Hg(ll)-Resistant Constructs

20. Resistance to Hg(II) Inoculate 5 x 10 6 cells into growth medium Order of resistance: BL308 > (MD737,MD736) > MD735 > MD767 > R1

21. Effect of γ-radiation Hg(-) γ(-) γ(-)Hg(-) γ(+) γ(+)Hg(+) γ(-) γ(-)Hg(+) γ(+) γ(+)

22. Reduction of Hg(II) to Hg(0) Oxidized Hg(II)-dependant NADPH Decrease in absorbance is a decrease in NADPH Using X-ray film to measure production of volatile Hg(0)

24. Assess toluene-metabolizing potential of MD 764 Fig.B Genomic DNA of MD764 Fig.C Growth of MD764(Merbromin &  -ray) Fig.D Thin layer chromatography(TLC) Lane1: Pure cis-toluene dihydrodiol(marker) Lane3: 3-methylcatechol(40 h) MD73 7 (20 h) MD764 (20h)(40h) Pure

25. tod Operon

26. 2-hydroxypenta-2,4-dienoate + acetate

27. Summary of Experimental Results Construct merA copy No. merA copy No. Hg(II) R γ-ray R Hg (II)  Hg (0) ReductionMetabolizing Toluene Toluene MD 735 1 ++ ++ + + N/A N/A MD 736 10 10 +++ +++ + N/A N/A MD 767 10 10 + + ++ ++ N/A N/A MD 737 150 150 +++ +++ + – MD 764 150 150 + tod + tod +++ +++ + +

28. Discussion & Conclusions  D. Radiodurans were 1)Resistant to bacteriacidal effects of Hg(II) 2)Able to reduce Hg(II) to Hg(0) 3)Resistant to Hg(II) in irradiating environments 4)Other metal-resistance genes as well  Modulating gene expression in D. radiodurans 1) By varying gene dosage between 1 – 150 copies per cell 1) By varying gene dosage between 1 – 150 copies per cell 2) Good correlation between merA copy number and 2) Good correlation between merA copy number and resistance/reduction of Hg (II) resistance/reduction of Hg (II) 3) By Deinococcal constitutive promoter upstream of merA 3) By Deinococcal constitutive promoter upstream of merA 4) Tandem duplications better than amplification vectors: 4) Tandem duplications better than amplification vectors: better adaptation + less a burden better adaptation + less a burden

29.  New selection system 1) Kan R /Cm R can be removed, using metal for selection 1) Kan R /Cm R can be removed, using metal for selection 2) More efficient, More stable & More genes 2) More efficient, More stable & More genes  Great genome plasticity of D. radiodurans 1) MD737: 150 copies of 20-kb vector = ~3 Mbp more DNA 1) MD737: 150 copies of 20-kb vector = ~3 Mbp more DNA 2) MD764: even more because of tod cassette 2) MD764: even more because of tod cassette 3) Able to maintain, replicate, and express extremely large 3) Able to maintain, replicate, and express extremely large foreign DNA foreign DNA 4) Accommodating more gene cassettes for remediating 4) Accommodating more gene cassettes for remediating complex mixtures complex mixtures Discussion & Conclusions

30. Future developments  Incorporating different gene clusters into a single promising host, for example, Pseudomonas sp.& D. radiodurans…etc.  A long way to go before real field bioremediation: 1.Identification of a promising host from its genome 2.Test its ability in the lab 3.Clone multiple genes into it to deal with “real waste” 4.Prove it has no danger to human and environment 5.Field study 6.Practical application

31. References 1.Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments, Hassan Brim et al. NATURE BIOTECHNOLOGY VOL 18 JANUARY 2000 2.Genome Sequence of the Radioresistant Bacterium Deinococcus radiodurans R1, Owen White et al. SCIENCE VOL 286 19 NOVEMBER 1999 3.Engineering radiation-resistant bacteria for environmental Biotechnology, Michael J Daly CURRENT OPINION IN BIOTECHNOLOGY 2000, 11:280–285 4.Bacterial mercury resistance from atoms to ecosystems, Tamar Barkay et al. FEMS MICROBIOLOGY REVIEWS 27 (2003) 355-384 5.Toluene Degradation by Pseudomonas putida F1, Gerben J. Zylstra and David T. Gibson THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264 No. 25 1989 14940-14946 6.Molecular Biotechnology third edition, Glick and Pasternak, ASM Press

32. Appendix

33. The Mercury Crisis

34. 4.2-kb mer operon of pBD724 encodes six proteins: MerR: activation/repression of the mer operon MerT: mercuric ion transport protein MerP: periplasmic mercuric ion binding protein MerC: transmembrane protein MerA: mercuric reductase MerD: putative secondary regulatory protein OP: operator/ promoter sequence

35. G-S-S-G 2 G-SH NADPH + H + NADP + FADGlutathione reductase Glutathione Redox: