The document discusses the development of a "superbug" bacterium for oil spill cleanup. It describes how researchers genetically engineered Pseudomonas putida by transferring plasmids containing genes for degrading various hydrocarbons. This created a strain that could break down compounds like camphor, octane, xylene and naphthalene. The superbug was the first genetically engineered microorganism to be patented. While genetically engineered microbes show promise for bioremediation, they also risk disturbing ecosystems if released.

1. DEGRADATIVE PLASMIDS &
SUPERBUG FOR OIL SPILLS
DR. ANU P. ABHIMANNUE
DEPARTMENT OF BIOTECHNOLOGY
ST. MARY’S COLLEGE, THRISSUR

2. Introduction
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 2
Degradative plasmids helps digest unusual substances

3. Introduction
Bacterial plasmids carry genes which help the host to degrade recalcitrant
organic compounds
eg: plasmid of pseudomonas putida reported to have genes that enable
degradation of Xylene, octane, naphthalene and camphor
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 3

4. Degradative pathway
Pathway for degradation of hydrocarbons comprises 2 parts
1. Upper/peripheral pathway: aromatic compounds are converted to catechol
or its derivatives
2. Lower/central pathway: catechol or its derivatives are converted to common
intermediates
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 4

5. Degradative plasmids - Types
TOL/NAH/SAL plasmids
Degrade via meta cleavage pathway
Mainly toluene, naphthalene,
salicylate and biphenyls are degraded
via this pathway
2,4-D plasmids
Degrade via ortho cleavage pathway
Mainly 2,4-D, 3-CBA and MCPA are
degraded via this pathway
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 5

6. ORTHO & META CLEAVAGE PATHWAY
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 6

7. TOL/NAH/SAL plasmids
Sl no: Plasmid Size (kb) Organism Organic compounds degraded
1 pWWO 117 Pseudomonas putida XYL, TOL
2 SAL 68 Pseudomonas aeruginosa SAL
3 pNL1 184 Sphingomonas aromaticivorans XYL, NAP, Biphenyls
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 7

8. 2,4-D plasmids
Sl no: Plasmid Size (kb) Organism Organic compounds degraded
1 pJP4 80 Ralstonia eutrophus 2,4-D, 3-CBA and MCPA
2 pMAB1 90 Alcaligens sp. 2,4-D and MCPA
3 pRC 45 Flavobacterium sp. 2,4-D, 3-CBA and MCPA
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 8

9. Genetic Manipulation by Transfer of
Plasmids
Degradative plasmids are equipped with genes that synthesize bio-degradative
enzymes.
Hence, genetic manipulations of plasmids leads to new bacterial strains with
better biodegradation potential.
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 9

10. Super bug for oil spills – P. putida
Pioneering work in genetic manipulation of plasmid was by Chakrabarty and his
co-workers in 1970s.
They developed a new strain of bacterium (Pseudomonas)
They incorporated different plasmids and constructed a new bacterium called
superbug
Superbug could degrade a variety of hydrocarbons.
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 10

11. Patent
United States granted patent to this superbug in 1981 (as per the directive of
American Supreme Court).
Thus, superbug became the first genetically engineered microorganism to be
patented.
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 11

12. Method
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 12

13. 1
• Bacterium with CAM (camphor- degrading) plasmid was conjugated with another
containing OCT (octane-degrading) plasmid
• Due to the presence of homologous DNA regions of DNA, recombination occurred and
resulted in a single CAM-OCT plasmid.
2
• Bacterium with XYL (xylene-degrading) plasmid conjugated with NAH (naphthalene-
degrading) plasmid containing bacterium
• XYL and NAH plasmids are compatible and therefore coexist in the same bacterium
3
• Final step is the conjugation of bacterium containing CAM-OCT plasmid with the other
bacterium containing XYL and NAH plasmids
• This genetically engineered superbug can degrade camphor, octane, XYL and NAH
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 13

14. Bio-surfactant Producing Genetically
Engireed Microbes (GEM)
 Chakarabarty and his group created a genetically engineered Pseudomonas
aeruginosa
This modified strain produce a glycolipid emulsifier (a bio-surfactant) which can
reduce the surface tension at oil water interface.
The reduced interfacial tension promotes biodegradation of oils.
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 14

15. Risks associated with GEM
Once introduced to ecosystem, they may disturb ecological balance
GEM could cause harm to the habitat
New strain may turn virulant thereby causing great harm to humankind
Hence, GEM are confined to the laboratories, and being utilized for controlled
processes of biodegradation
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 15

16. References
https://pt.slideshare.net/15swarka/biology-powerpoint-13029002/2
http://www.biologydiscussion.com/biotechnology/biodegradation/biodegradation-and-
bioremediation-with-diagram/11043
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 16

17. Thank you
DR. ANU P. ABHIMANNUE, ST. MARY'S COLLEGE, THRISSUR. 17