Radiation resistant bacterium/E. coli shuttle vector

Details Radiation resistant bacterium/E. coli shuttle vector Radiation resistant bacterium/E. coli shuttle vector

2002-11-26

2004-08-03

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C435S471000, C536S023100

Reexamination Certificate

active

06770476

ABSTRACT:

BACKGROUND OF THE INVENTION
This application is based upon and claims the benefit of priority from Japanese Patent Application No. 2002-46377, filed Feb. 22, 2002, the entire contents of this application are incorporated herein by reference.
This invention relates to a novel vector capable of replication in cells of bacteria of the genus Deinococcus, a novel shuttle vector capable of replication in cells of both a bacterium of the genus Deinococcus and
E. coli
, a shuttle vector incorporating the novel shuttle vector, and transformants obtained by introducing those shuttle vectors into bacteria of the genus Deinococcus.
Although the radioresistance of organisms varies greatly with their species, a group of microorganisms having resistance to radiation are known to exist and they are collectively referred to as radioresistant bacteria. Bacteria of the genus Deinococcus are known as representative radioresistant bacteria and to date seven species have been identified (
D. radiodurans, D. radiopugnans, D. radiophilus, D. grandis, D. proteolyticus, D. geothermalis
and
D. murrayi
) (Ferreira et al., Int. J. Syst. Bacteriol., 47:939-947, 1997). It is also known that the radioresistance of those microorganisms is at least about 100 times the resistance of
E. coli
and at least 1000 times that of human cells. If foreign genes that are used to remove difficult-to-decompose substances, toxic substances, etc. could be introduced into these radioresistant bacteria by genetic engineering techniques, it would be possible to use the transformed bacteria to remove difficult-to-decompose substances, toxic substances, etc. that are contained in wastes contaminated by radioactive substances.
Two cases have heretofore been known as examples of incorporating a foreign gene into a bacterium of the genus Deinococcus and expressing a heterologous protein; in one case, toluene dioxygenase was expressed in
D. radiodurans
and difficult-to-dissolve toluene was converted to a substance that was less difficult to dissolve (Lange et al., Nature Biotechnol., 16: 929-933, 1998); in the other case, mercuric(II) ion reductase was expressed in
D. radiodurans
and mercuric ion, Hg(II) ion was reduced to less toxic volatile metallic mercury (Brim et al., Nature Biotechnol., 18: 85-90, 2000). However, in no case have bacteria of the genus Deinococcus other than
D. radiodurans
been transformed with a plasmid vector containing a foreign gene. Furthermore, in the reported cases of transformant preparation, integration-type plasmids which cannot autonomously replicate in bacteria of the genus Deinococcus are used, so there have been several defects such as the inability to control copy number, the small amount of a protein that can be expressed and the need to add an antibiotic to the culture media.
With a view to overcoming these defects, pRAD1, a
D. radiodurans/E. coli
shuttle vector, was developed using a replicon of plasmid pUE10 from
D. radiodurans
Sark strain and an
E. coli
vector pMTL23 (Meima and Lidstrom, Appl. Environ. Microbiol., 66: 3856-3867, 2000). However, there has been no report of using pRAD1 to transform other bacteria of the genus Deinococcus than
D. radiodurans
. In addition, pRAD1 which has low segregational stability can be stably replicated in a selective medium containing an antibiotic chloramphenicol but in a non-selective medium that does not contain chloramphenicol, the frequency of appearance of a pRAD1 containing bacterium cannot be maintained at high level and hence such bacterium cannot exist stably. This defect is particularly significant when a radioresistant bacterium into which a foreign gene has been introduced is released into an outside open-air system. For it is extremely difficult to maintain a constant antibiotic level in environments such as soil or liquid wastes contaminated with radioactive substances in order to ensure that the frequency of appearance of the pRAD1 containing bacterium is maintained at high level; what is more, the release of antibiotics into the open-air might increase the possibility of the appearance of an unwanted antibiotic-resistant bacterium, thus eventually polluting the environment.
SUMMARY OF THE INVENTION
An object, therefore, of the invention is to provide a novel vector that is useful in transforming bacteria of the genus Deinococcus and which can stably exist in bacteria of the genus Deinococcus under conditions that have no selective pressure such as antibiotics. Another object of the invention is to provide a novel shuttle vector that can replicate in both a bacterium of the genus Deinococcus and
E. coli
and which can stably exist under conditions that have no selective pressure such as antibiotics.
The present inventors made intensive studies with a view to attaining the stated objects and found that the first object could be attained by using pUE30, an endogenous plasmid from radioresistant bacterium
D. radiopugnans
, or a derivative thereof.
Thus, the first object of the invention can be attained by providing a plasmid capable of autonomous replication in bacteria of the genus Deinococcus, which is the endogenous plasmid pUE30 from
D. radiopugnans
strain ATCC19172 or a derivative thereof.
The present invention attains its second object by providing a shuttle vector that contains both the endogenous plasmid pUE30 from
D. radiopugnans
strain ATCC19172 or a derivative thereof and a plasmid capable of autonomous replication in
E. coli
or a derivative thereof and which can replicate in both a bacterium of the genus Deinococcus and
E. coli.


REFERENCES:
Mackay et al. The plasmids of Deinococus spp. and the cloning and restriction mapping of plasmid pUE1 Archives of Microbiology vol. 141 pp91-94 1985 (IDS).*
Lange et al., “Engineering a Recombinant Deinococcus radiodurans For Organopollutant Degradation in Radioactive Mixed Waste Environments”, Nature Biotechnology, vol. 16, pp. 929-933 (Oct. 1998).
Brim et al., “Engineering Deinococcus radiodurans For Metal Remediation In Radioactive Mixed Waste Environments”, Nature Biotechnology, vol. 18, pp. 85-90 (Jan. 2000).
Rob Meime et al., “Characterization of the Minimal Replicon of a Cryptic Deinococcus radiodurans SARK Plasmid and Development of Versatile Escherichia coli-D, radiodurans Shuttle Vectors” Applied and Environmental Microbiology,pp. 3856-3867, vol. 66, No. 9, (Sep. 2000).
Martin W. Mackay, et al., “The Plasmids of Deinococcus ssp. and the Cloning and Restriction Mapping of the D. Radiophilus Plasmid pUE1”,Archives of Microbiology, vol. 141, pp. 91-94 (1985).

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