Chemistry: molecular biology and microbiology – Vector – per se
Patent
1994-12-27
2000-02-15
Degen, Nancy
Chemistry: molecular biology and microbiology
Vector, per se
435 691, 4351723, 4352523, 43525231, 43525233, C12N 1563, C12N 1570, C12N 1574, C12N 121
Patent
active
060251905
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a plasmid which is usable for the genetic modification of bacteria displaying positive Gram staining, especially lactic bacteria of industrial or medical importance.
It also relates to bacteria containing such a plasmid. Lastly, it relates to genetic modification methods employing such a plasmid, either to inactivate a gene normally present in the bacterial chromosome, or to introduce and express a gene of interest.
BACKGROUND OF THE INVENTION
Many Gram bacteria displaying positive Gram staining are subjects of study as a biological model (for example bacteria of the genus Bacillus), as a fermentation strain of industrial importance (lactic acid bacteria) or as a pathogen (for example Clostridia, Listeria, Staphylococcus, Streptococcus). Many of these strains are characterized from a physiological standpoint, but few have been studied or modified genetically. The study or modification of the strains may be facilitated by the use of vectors permitting directed or non-specific insertions into the bacterial chromosome. Delivery systems which are based on the non-replicative vectors are limited to bacteria which can be transformed with a high frequency, and those utilizing replicons which are active only under certain conditions are often limited to their host range. Thus, the construction of recombinant strains requires considerable effort, and can be applied efficaciously only to certain specific micro-organisms.
The addition, loss or modification of genes can transform the role of an organism in an industrial process such as fermentation.
Biotechnology seeks to facilitate the industrial use of microorganisms. For example, lactic bacteria are used in agri-foodstuffs, predominantly for the manufacture of fermented dairy products, but also outside the milk industry for the manufacture of wine, cider, cooked meats and silage.
It is hence especially desirable to have effective means available for introducing or modifying specifically and permanently certain genes in these organisms.
At the present time, modification of the chromosome in lactic bacteria is performed via a system by transformation of a non-replicative plasmid. In a single step, it is necessary to have two low-frequency events, transformation with a plasmid and recombination in the chromosome. The probability of obtaining these two events in a single step is the product of the probabilities of each; there is hence a very small chance of obtaining the modification.
Plasmid pWV01 is a cryptic plasmid initially isolated in Lactococcus lactis subsp. cremoris; it is a broad-host-range plasmid which is replicative in both Gram-positive and Gram-negative bacteria, in particular in E. coli, Bacillus subtilis, Lactococcus lactis Streptococcus and Lactobacillus. It has been characterized, and its nucleotide sequence has been published by Leenhouts et al. (1991).
In Application WO 85/03495, large fragments of this plasmid are used to construct a recombinant plasmid pGK12 marked with the gene for resistance to erythromycin and/or the gene for resistance to chloramphenicol (chloramphenicol acetyltransferase (CAT)). This plasmid pGK12 cannot be used to make integrations in the bacterial chromosome.
The non-replicative plasmids used hitherto enable this problem to be alleviated, but this system requires high degrees of transformation to permit the detection of low-frequency events such as transposition or recombination in the chromosome; now, most lactic bacteria are weakly transformable.
It would be possible to overcome all these difficulties by obtaining a temperature-sensitive replicon which could be used as a delivery vector in lactic or other bacteria.
Plasmids pE194 and pSH71 have been described as naturally temperature-sensitive, above a temperature of 51.degree. C. (J. Bacteriol., 1990, 172, 4543-4548).
SUMMARY OF THE INVENTION
Thus, the subject of the present invention is a bacterial vector plasmid of the type containing an origin of replication which is effective in Gram+ bacteria, character
REFERENCES:
patent: 4711845 (1987-12-01), Gelfand et al.
Applied and Enviromental Microbiology; vol. 57, No. 2, 1991, Washington DC, pp. 539-548. Feiertag J M; Petzel J P; Pasalodos E; Baldwin K A; McKay L L "Thermosensitive Plasmid Replication Temperature-Sensitive Host Growth and Chromosomal Plasmid Integration Conferree by Lactococcus-Lactis-SSP-Cremoris Lactose Plasmids in Lactococcus-Lactis-SSP-Lactis" see the whole document. Relevant to Claims 1-4, 19-24.
Journal of Bacteriology; vol. 172, No. 8, 1990, Baltimore, U.S., pp. 4543-4584. Sozhammannan S; Dabert P; Moretto V; Ehrlich S D; Gruss A "Plus-Origin Mapping of Single-Stranded DNA Plasmid P-E-194 and Nick Site Homologies With Other Plasmids" cited in the application. See the whole document. Relevant to Claims 1,2,19-24.
Biological Abstracts; vol. 87 Philadelphia, PA, U.S., abstracts No. 047423. Alonso J C; Stiege C A; Tailor R H; Viret J-F "Functional Analysis of the DNA-TS Mutants of Bacillus-Subtilis Plasmid PUB110 Replication As A Model System"; see abstract & Mol Gen Genet 214 (3). 1988. 482-489. Relevant to Claims 1,9-11.
Biological Abstracts; vol. 92, Philadelphia, PA, U.S., abstract No. 075453. Leenhouts K J; Kok J; Venema G "Replacement Recombination In Lactococcus-Lactis"; see abstract & J. Bacteriol 173 (15). 1991. 4794-4798. Relevant to Claims 5, 8.
Biological Abstracts; vol. 88, Philadelphia, PA, U.S., abstract No. 107483. Priebe S D; Lacks S A "Region Of The Streptococcal Plasmid PMV158 Required For Conjugative Mobilization"; see abstract & J. Bacteriol 171 (9). 1989. 4778-4784. Relevant to Claim 15.
Journal of Bacteriology; vol. 174, No. 17, 1992, Baltimore U.S., pp. 5633-5638. Maguin E; Duwat P; Hege T; Ehrlich D; Gruss A "New Thermosensitive Plasmid For A Gram-Positive Bacteria"; see the whole document. Relevant to Claims 1-24.
Alonso et al. "Functional Analysis of the dna (Ts) Mutants of Bacillus subtilis" Mol Gen Genet 214 482-489 1988.
Danilevich et al. "Isolatin & Characterization of a Temperature-Sensitive Plasmid . . . " Mol Biol 18(4) 1111-1120 1984. Abstract only.
Urlapova et al. "Temperature Sensitive Mutants of the Plasmid RP-1" Genetika 15(3) 1979 433-443. Abstract only.
Leenhouts et al. "Nucleotide Sequence & Charcterization of the Broad Host Range Lactococcal Plasmid pWVO1" Plasmid 26 55-66 1991.
Gruss Alexandra
Maguin Emmanuelle
Degen Nancy
Institut National de la Recherche Agronomique
LandOfFree
Temperature-sensitive plasmid does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Temperature-sensitive plasmid, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Temperature-sensitive plasmid will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1905046