Microorganisms having multiple compatible degradative energy-generating plasmids and preparation thereof
Abstract
Unique microorganisms have been developed by the application of genetic engineering techniques. These microorganisms contain at least two stable (compatible) energy-generating plasmids, these plasmids specifying separate degradative pathways. The techniques for preparing such multi-plasmid strains from bacteria of the genus Pseudomonas are described. Living cultures of two strains of Pseudomonas (P. aeruginosa [NRRL B-5472] and P. putida [NRRL B-5473]) have been deposited with the United States Department of Agriculture, Agricultural Research Service, Northern Marketing and Nutrient Research Division, Peoria, Ill. The P. aeruginosa NRRL B-5472 was derived from Pseudomonas aeruginosa strain 1c by the genetic transfer thereto, and containment therein, of camphor, octane, salicylate and naphthalene degradative pathways in the form of plasmids. The P. putida NRRL B-5473 was derived from Pseudomonas putida strain PpG1 by genetic transfer thereto, and containment therein, of camphor, salicylate and naphthalene degradative pathways and drug resistance factor RP-1, all in the form of plasmids.
Claims
1. A bacterium from the genus Pseudomonas containing therein at least two stable energy-generating plasmids, each of said plasmids providing a separate hydrocarbon degradative pathway.
2. The Pseudomonas bacterium of claim 1 wherein the hydrocarbon degradative pathways are selected from the group consisting of linear aliphatic, cyclic aliphatic, aromatic and polynuclear aromatic.
3. The Pseudomonas bacterium of claim 1, said bacterium being of the specie P. aeruginosa.
4. The P. aeruginosa bacterium of claim 3 wherein the bacterium contains CAM, OCT, SAL and NPL plasmids.
5. The Pseudomonas bacterium of claim 1, said bacterium being of the specie P. putida.
6. The P. putida bacterium of claim 5 wherein the bacterium contains CAM, SAL, NPL and RP-1 plasmids.
7. An inoculum for the degradation of a preselected substrate comprising a complex or mixture of hydrocarbons, said inoculum consisting essentially of bacteria of the genus Pseudomonas at least some of which contain at least two stable energy-generating plasmids, each of said plasmids providing a separate hydrocarbon degradative pathway.
8. The inoculum of claim 7 wherein the hydrocarbon degradative pathways are selected from the group consisting of linear aliphatic, cyclic aliphatic, aromatic and polynuclear aromatic.
9. The inoculum of claim 8 wherein the bacteria having multiple energy-generating plasmids are of the specie P. aeruginosa.
10. The inoculum of claim 8 wherein the bacteria having multiple energy-generating plasmids are of the specie P. putida.
11. In the process in which a first energy-generating plasmid specifying a degradative pathway is transferred by conjugation from a donor Pseudomonas bacterium to a recipient Pseudomonas bacterium containing at least one energy-generating plasmid that is incompatible with said first plasmid, said transfer occurring in the quiescent state after the mixing of substantially equal volumes of cultures of said donor and said recipient, each culture presenting the respective organisms in a complex nutrient liquid medium at a population density of at least about 1,000,000 cells/ml, the improvement wherein after conjugation has occurred, the multi-plasmid conjugatant bacteria are subjected to DNA-cleaving radiation in a dosage sufficient to fuse the first plasmid and the plasmid incompatible therewith located in the same cell.
12. The improvement of claim 11 wherein the DNA-cleaving radiation is UV radiation.
13. The improvement of claim 12 wherein the first plasmid provides the degradative pathway for camphor and the recipient Pseudomonas contains the degradative pathway for n-octane.
14. An inoculated medium for the degradation of liquid hydrocarbon substrate material floating on water, said inoculated medium comprising a carrier material able to float on water and bacteria from the genus Pseudomonas carried thereby, at least some of said bacteria each containing at least two stable energy-generating plasmids, each of said plasmids providing a separate hydrocarbon degradative pathway and said carrier material being able to absorb said hydrocarbon material.
15. The inoculated medium of claim 14 wherein the carrier material is straw.
16. The inoculated medium of claim 14 wherein the hydrocarbon degradative pathways are selected from the group consisting of linear aliphatic, cyclic aliphatic, aromatic and polynuclear aromatic.
17. The inoculated medium of claim 14 wherein the bacteria are of the specie P. aeruginosa.
18. The inoculated medium of claim 14 wherein the bacteria are of the specie P. putida.
Cabinet Chaillot