Transcript Engineering Deinococcus radiodurans for metal remediation

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 Deinococcus radiodurans for
metal remediation in radioactive mixed
waste environments
William Chen
Keng Teo
Overview
 Background
 Deinococcus radiodurans
 Engineering Deinococcus radiodurans
 Experimental results and analysis
 Discussion & Conclusions
 Future Development
Background
 In U.S. alone, 1000 waste sites with
radiation level  10 mCi/L
 7.5 x107 m3 soil & 2 x1012 dm3 ground
water contaminated by 3 x106 m3 leaking
waste
 Clean-up cost > $265 billion
 Potential targets for less expensive and
more effective bioremediation methods
What is bioremediation?

Definition:
Use of biological mechanisms to destroy, transform
or immobilize environmental contaminants to
protect potential sensitive receptors

Applications:
Agricultural chemicals, gasoline contamination &
radioactive wastes….etc.

Examples:
1) Xenobiotics by Pseudomonas sp.
2) n-alkane metabolism by Desulfobacterium cetonicum &
Pseudomonas sp.
Denococcus radiodurans
Physiology:
 Gram(+), Non-pathogenic
 Red-pigmented
 Radiation-resistant: UV & Ionic
 Selective solvent tolerant
 Non-motile
 Soil bacterium
Denococcus radiodurans
R1 strain genome:
 2 chromosomes (2.65 Mbp & 412 kbp), with 410 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
2. DNA damage prevention
3. Genetic redundancy
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)
Construction of Metal-Remediating
D. radiodurans Strains

Clone merA locus from E. coli BL308 into D.
radiodurans R1 strain

Combining organic degrading function into
Hg(ll)R-D. radiodurans
Five different Constructs:
- Hg(ll)-resistant:
MD 735, MD 736, MD 737, MD 767
- Hg(ll)-resistant & toluene metabolizing:
MD 764

MD 735
P1 & P2: D. radiodurans Constitutive Promoter
KanR: kanamycinresistance gene, aphA
Starting material:
merA operon from the E. coli strain BL308
D. radiodurans autonomously replicating plasmid pMD66
D. radiodurans wild-type strain R1
MD 736
MD 737
MD 767
CmR: chloramphenicol resistance gene, cat
MD 764
tod Operon cloned from Pseudomonas putida
Summary of Hg(ll)-Resistant
Constructs
Construct
Type of Integration
D.radiodurans
Constitutive Promoter
MD 735
Plasmid (No Integration)
Yes
MD 736
Tandem Duplication
Yes
MD 767
Direct Insertion
Yes
MD 737
Amplification Vector
No
MD 764
Direct Insertion
No
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
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
Summary of Hg(ll)-Resistant
Constructs
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)
Resistance to Hg(II)
• Inoculate 5 x 106 cells into growth medium
• Order of resistance:
BL308 > (MD737,MD736) > MD735 > MD767 > R1
Effect of γ-radiation
Hg(-)
γ(-)
Hg(-)
γ(+)
Hg(+)
γ(-)
Hg(+)
γ(+)
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
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)
MD764 MD73
7
(20h)(40h) (20 h)
Pure
tod Operon
2-hydroxypenta-2,4-dienoate + acetate
Summary of Experimental Results
Construct
merA
Hg(II)R γ-rayR
copy No.
Hg(II)Hg(0) Metabolizing
Reduction
Toluene
MD 735
1
++
+
+
N/A
MD 736
10
+++
+
+++
N/A
MD 767
10
+
+
++
N/A
MD 737
150
+++
+
+++
–
MD 764
150
+ tod
+++
+
+++
+
Discussion & Conclusions

D. Radiodurans were
1)
2)
3)
4)

Resistant to bacteriacidal effects of Hg(II)
Able to reduce Hg(II) to Hg(0)
Resistant to Hg(II) in irradiating environments
Other metal-resistance genes as well
Modulating gene expression in D. radiodurans
1) By varying gene dosage between 1 – 150 copies per cell
2) Good correlation between merA copy number and
resistance/reduction of Hg (II)
3) By Deinococcal constitutive promoter upstream of merA
4) Tandem duplications better than amplification vectors:
better adaptation + less a burden
Discussion & Conclusions

New selection system
1) KanR/CmR can be removed, using metal for selection
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
2) MD764: even more because of tod cassette
3) Able to maintain, replicate, and express extremely large
foreign DNA
4) Accommodating more gene cassettes for remediating
complex mixtures
Future developments

Incorporating different gene clusters into a single
promising host, for example, Pseudomonas sp.&
D. radiodurans…etc.

1.
2.
3.
4.
5.
6.
A long way to go before real field bioremediation:
Identification of a promising host from its genome
Test its ability in the lab
Clone multiple genes into it to deal with “real waste”
Prove it has no danger to human and environment
Field study
Practical application
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
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 protein
MerP: periplasmic mercuric ion binding
protein MerC: transmembrane protein
MerA: mercuric reductase
MerD: putative secondary regulatory protein
OP: operator/ promoter sequence
Glutathione
Redox:
NADPH + H+
G-S-S-G
FAD Glutathione
reductase
+
NADP
2 G-SH