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.

Engineering Engineering Deinococcus radiodurans Deinococcus radiodurans forfor

metal remediation in radioactive metal remediation in radioactive mixedmixed

waste environmentswaste environments

William ChenWilliam ChenKeng TeoKeng Teo

OverviewOverview

BackgroundBackground

Deinococcus radioduransDeinococcus radiodurans

Engineering Engineering Deinococcus radioduransDeinococcus radiodurans

Experimental results and analysisExperimental results and analysis

Discussion & ConclusionsDiscussion & Conclusions

Future DevelopmentFuture Development

BackgroundBackground

In U.S. alone, In U.S. alone, 1000 1000 waste sites with waste sites with radiation level radiation level 10 mCi/L 10 mCi/L

7.5 x107.5 x107 7 mm33 soil & 2 x10 soil & 2 x1012 12 ddmm3 3 ground water ground water contaminated by 3 x10contaminated by 3 x106 6 mm33 leaking waste leaking waste

Clean-up cost > $265 billionClean-up cost > $265 billion

Potential targets for less expensive and Potential targets for less expensive and more effective bioremediation methodsmore effective bioremediation methods

What is bioremediation?What is bioremediation? Definition:Definition: Use of biological mechanisms to destroy, Use of biological mechanisms to destroy,

transform or immobilize environmental transform or immobilize environmental contaminants to protect potential sensitive contaminants to protect potential sensitive receptors receptors

Applications:Applications:

Agricultural chemicals, gasoline contamination & radioactive Agricultural chemicals, gasoline contamination & radioactive wastes….etc.wastes….etc.

Examples:Examples: 1) Xenobiotics by 1) Xenobiotics by Pseudomonas sp.Pseudomonas sp.

2) n2) n-alkane metabolism by -alkane metabolism by Desulfobacterium cetonicum &Desulfobacterium cetonicum & Pseudomonas sp.Pseudomonas sp.

Denococcus radioduransDenococcus radiodurans

Physiology:Physiology: Gram(+), Non-pathogenicGram(+), Non-pathogenic

Red-pigmentedRed-pigmented

Radiation-resistantRadiation-resistant:: UV & Ionic UV & Ionic

Selective solvent tolerant Selective solvent tolerant

Non-motile Non-motile

Soil bacteriumSoil bacterium

Denococcus radioduransDenococcus radiodurans

R1 strain genome:R1 strain genome: 2 chromosomes (2.65 Mbp & 412 kbp), with 4-2 chromosomes (2.65 Mbp & 412 kbp), with 4-

10 genome equivalents/copies in growing cells10 genome equivalents/copies in growing cells 1 megaplasmid (177 kbp)1 megaplasmid (177 kbp) 1 small plasmid (46 kbp)1 small plasmid (46 kbp) Able to withstand high energy radiation due to:Able to withstand high energy radiation due to:

1. Strong DNA repair1. Strong DNA repair

2. DNA damage prevention2. DNA damage prevention

3. Genetic redundancy3. Genetic redundancy

Objectives of EngineeringObjectives of EngineeringD. radioduransD. radiodurans

Confer resistance to toxic metallic Confer resistance to toxic metallic waste constituents waste constituents

Transform toxic metals to less Transform toxic metals to less toxic and less soluble chemical toxic and less soluble chemical formsforms

For example: For example:

Bacterial mercuric reductase gene Bacterial mercuric reductase gene merAmerA, , encoding mercuric ion reductase MerA that encoding mercuric ion reductase MerA that reduces reduces toxic Hg(II) to inert Hg(0)toxic Hg(II) to inert Hg(0)

Construction of Metal-Remediating Construction of Metal-Remediating D. radiodurans D. radiodurans StrainsStrains

Clone Clone merAmerA locus locus from from E. coliE. coli BL308 into BL308 into D. D. radiodurans radiodurans R1 strainR1 strain

Combining organic degrading function into Combining organic degrading function into Hg(ll)Hg(ll)RR-D-D. radiodurans. radiodurans

Five different Constructs: Five different Constructs: - - Hg(ll)-resistant:Hg(ll)-resistant: MD 735, MD 736, MD 737, MD 767MD 735, MD 736, MD 737, MD 767 - - Hg(ll)-resistant & toluene metabolizing:Hg(ll)-resistant & toluene metabolizing: MD 764MD 764

MD 735MD 735

Starting material:merA operon from the E. coli strain BL308D. radiodurans autonomously replicating plasmid pMD66D. radiodurans wild-type strain R1

KanR: kanamycinresistance gene, aphA

P1 & P2: D. radiodurans Constitutive Promoter

MD 736MD 736

MD 737MD 737

MD 767MD 767

CmR: chloramphenicol resistance gene, cat

tod Operon cloned from Pseudomonas putida

MD 764MD 764

Summary of Hg(ll)-Resistant Summary of Hg(ll)-Resistant ConstructsConstructs

ConstructConstruct Type of IntegrationType of Integration D.radioduransD.radiodurans Constitutive PromoterConstitutive Promoter

MD 735MD 735 Plasmid (No Integration)Plasmid (No Integration) YesYes

MD 736MD 736 Tandem DuplicationTandem Duplication YesYes

MD 767MD 767 Direct InsertionDirect Insertion YesYes

MD 737MD 737 Amplification VectorAmplification Vector NoNo

MD 764MD 764 Direct InsertionDirect Insertion NoNo

Experimental Results & AnalysisExperimental Results & Analysis

merAmerA copy number copy number

Resistance to Hg(II)Resistance to Hg(II)

Effect of Effect of γγ-radiation-radiation

Reduction of Hg(II) to Hg(0) Reduction of Hg(II) to Hg(0)

Assess toluene-metabolizing potential of Assess toluene-metabolizing potential of MD 764MD 764

merA merA copy numbercopy 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

ConstructConstruct merA Operon merA Operon

Copy NumberCopy Number

Type of Type of IntegrationIntegration

D.radioduransD.radiodurans Constitutive PromoterConstitutive Promoter

MD 735MD 735 11 Plasmid (No Plasmid (No Integration)Integration)

YesYes

MD 736MD 736 1010 Tandem Tandem DuplicationDuplication

YesYes

MD 767MD 767 1010 Direct Direct InsertionInsertion

YesYes

MD 737MD 737 150150 Amplification Amplification VectorVector

NoNo

MD 764MD 764 115050

+ + todtod operons operonsDirect Direct InsertionInsertion

NoNo

E. ColiE. Coli 20-3020-30 Plasmid (No Plasmid (No Integration)Integration)

No (No (E. ColiE. Coli Promoter) Promoter)

Summary of Hg(ll)-Resistant Summary of Hg(ll)-Resistant ConstructsConstructs

Resistance to Hg(II)Resistance to Hg(II)

• Inoculate 5 x 106 cells into growth medium• Order of resistance:

BL308 > (MD737,MD736) > MD735 > MD767 > R1

Effect of Effect of γγ-radiation-radiation

Hg(-)Hg(-)

γγ(-)(-)

Hg(-)Hg(-)

γγ(+)(+)

Hg(+)Hg(+)

γγ(-)(-)

Hg(+)Hg(+)

γγ(+)(+)

Reduction of Hg(II) to Hg(0)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)

Assess toluene-metabolizing Assess toluene-metabolizing potential of MD 764potential 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) MD737 (20 h)

MD764 (20h)(40h)

Pure

tod Operon

2-hydroxypenta-2,4-dienoate + acetate

Summary of Experimental ResultsSummary of Experimental ResultsConstructConstruct merA merA

copy No.copy No.Hg(II)Hg(II)RR γγ-ray-rayRR HgHg(II)(II)HgHg(0)(0)

ReductionReduction

Metabolizing Metabolizing

TolueneToluene

MD 735MD 735 11 ++++ ++ ++ N/AN/A

MD 736MD 736 1010 ++++++ ++ ++++++ N/AN/A

MD 767MD 767 1010 ++ ++ ++++ N/AN/A

MD 737MD 737 150150 ++++++ ++ ++++++ – –

MD 764MD 764 1150 50

+ + todtod ++++++ ++ ++++++ ++

Discussion & ConclusionsDiscussion & Conclusions D. Radiodurans wereD. Radiodurans were

1)1) Resistant to bacteriacidal effects of Hg(II)Resistant to bacteriacidal effects of Hg(II)

2)2) Able to reduce Hg(II) to Hg(0)Able to reduce Hg(II) to Hg(0)

3)3) Resistant to Hg(II) in irradiating environmentsResistant to Hg(II) in irradiating environments

4)4) Other metal-resistance genes as wellOther metal-resistance genes as well

Modulating gene expression in Modulating gene expression in D. radioduransD. radiodurans

1) By varying gene dosage between 1 1) By varying gene dosage between 1 –– 150 copies per cell 150 copies per cell

2) Good correlation between 2) Good correlation between merA merA copy number and copy number and resistance/reduction of Hg (II)resistance/reduction of Hg (II)

3) By 3) By DeinococcalDeinococcal constitutive promoter upstream of constitutive promoter upstream of merAmerA

4) 4) Tandem duplications better than amplification vectors: Tandem duplications better than amplification vectors: better adaptation + less a burdenbetter adaptation + less a burden

New selection systemNew selection system

1) Kan1) KanRR/Cm/CmRR can be removed, using metal for selection can be removed, using metal for selection

2) More efficient, More stable & More genes2) More efficient, More stable & More genes

Great genome plasticity of Great genome plasticity of D. radioduransD. 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 2) MD764: even more because of tod tod cassette cassette

3) Able to maintain, replicate, and express extremely large 3) Able to maintain, replicate, and express extremely large foreign DNAforeign DNA

4) Accommodating more gene cassettes for remediating 4) Accommodating more gene cassettes for remediating complex mixturescomplex mixtures

Discussion & ConclusionsDiscussion & Conclusions

Future developmentsFuture developments

Incorporating different gene clusters into a single Incorporating different gene clusters into a single promising host, for example, promising host, for example, Pseudomonas sp.& Pseudomonas sp.& D. radiodurans…etc. D. radiodurans…etc.

A long way to go before real field bioremediationA long way to go before real field bioremediation: : 1.1. Identification of a promising host from its genomeIdentification of a promising host from its genome2.2. Test its ability in the labTest its ability in the lab3.3. Clone multiple genes into it to deal with “real Clone multiple genes into it to deal with “real

waste”waste”4.4. Prove it has no danger to human and environmentProve it has no danger to human and environment5.5. Field studyField study6.6. Practical application Practical application

ReferencesReferences1.1. Engineering Engineering Deinococcus radiodurans Deinococcus radiodurans for metal remediation in radioactive for metal remediation in radioactive

mixed waste environments, Hassan Brim et al.mixed waste environments, Hassan Brim et al. NATURE NATURE BIOTECHNOLOGY VOL 18 JANUARY 2000BIOTECHNOLOGY VOL 18 JANUARY 2000

2.2. Genome Sequence of the Radioresistant Bacterium Genome Sequence of the Radioresistant Bacterium Deinococcus Deinococcus radiodurans radiodurans R1, Owen White et al.R1, Owen White et al. SCIENCE SCIENCE VOL 286 19 NOVEMBER 1999VOL 286 19 NOVEMBER 1999

3.3. Engineering radiation-resistant bacteria for environmental Biotechnology, Engineering radiation-resistant bacteria for environmental Biotechnology, Michael J DalyMichael J Daly CURRENT OPINION IN CURRENT OPINION IN BIOTECHNOLOGY 2000, 11:280–285BIOTECHNOLOGY 2000, 11:280–285

4.4. Bacterial mercury resistance from atoms to ecosystems, Bacterial mercury resistance from atoms to ecosystems, Tamar Barkay et Tamar Barkay et al. al. FEMS MICROBIOLOGY REVIEWS 27 (2003) 355-384FEMS MICROBIOLOGY REVIEWS 27 (2003) 355-384

5.5. Toluene Degradation by Toluene Degradation by Pseudomonas putida Pseudomonas putida F1, Gerben J. Zylstra and F1, Gerben J. Zylstra and David T. GibsonDavid T. Gibson THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264 No. 25 1989 14940-14946THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 264 No. 25 1989 14940-14946

6.6. Molecular Biotechnology third edition, Glick and Pasternak, ASM PressMolecular Biotechnology third edition, Glick and Pasternak, ASM Press

Appendix

The Mercury Crisis

4.2-kb mer operon of pBD724 encodes six proteins:

MerR: activation/repression of the mer operon MerT: mercuric ion transport proteinMerP: periplasmic mercuric ion binding protein MerC: transmembrane proteinMerA: mercuric reductaseMerD: putative secondary regulatory proteinOP: operator/ promoter sequence

G-S-S-G

2 G-SH

NADPH + H+

NADP+

FAD Glutathione reductase

Glutathione Redox: