Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Patent
1996-07-05
1998-11-03
Degen, Nancy
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
4352523, 4353201, 435419, 514 2, 530300, 536 237, C12N 1531, C12N 121, C07K 1433, C07H 2104
Patent
active
058307222
DESCRIPTION:
BRIEF SUMMARY
The invention relates to new toxins isolated from a strain of anaerobic bacterium of the species Clostridium bifermentans. The strain CH18 of Clostridium bifermentans serovar malaysia (Cbm) has been described by H. de Barjac and M. Sebald, in C.R. Acad. Sci. Paris, vol. 310, series III, p. 383-387, 1990.
It exhibits a toxic activity during the sporulation phase via toxins having a larvicidal power against the larvae of arthropods, in particular of insects and especially against the larvae of Diptera, for example the larvae of mosquitoes or of similis. These particular Diptera are vectors of diseases or cause harmful effects.
Until now, toxins specific to bacteria of the species Bacillus thuringiensis or of the species Bacillus sphaericus were known for their entomopathogenic activity and especially their toxic activity against mosquito larvae (Nicolas (1992). Pes. agropec. bras., Brasilia, abr. 1992, 27, 37-46). In spite of the obvious benefit of these Bacillus toxins for controlling insects and especially insect larvae, the search for new toxins active against certain insects proved to be important especially with the aim of offering means for controlling the risk of development of resistance against these products with larvicidal activity. In this context, the inventors have detected the existence of a larvicidal activity in the bacterium of the species Clostridium bifermentans. In this regard, the invention proposes, for the first time, toxins present in anaerobic bacteria, which are capable of having a toxic activity and in particular a larvicidal activity against arthropods, and in particular insects of the Diptera type. The strain of Clostridium bifermentans serovariety malaysia used is harmless against the mammals and fish tested as well as against all aquatic organisms outside the natural target for the larvicidal activity of Cbm (Thiery et al. (1992) J. econ. Entomol. 85(5), 1618-1623). The existence of the larvicidal activity of Cbm has already been observed and associated, in the prior art, with sporulating cells (Charles et al. (1990) Res. Microbiol. 141, 721d-733). It has also been noted that this activity decreases very substantially after cell lysis because of inactivation by cellular proteases (Nicolas et al (1990). Appl. Microbiol. Biotechnol. 34, 36-41).
The subject of the invention is therefore nucleotide sequences encoding polypeptides capable of taking part in the toxic activity against the larvae of arthropods and in particular of insects, especially of Diptera such as mosquitoes and simuliids.
The invention also relates to these polypeptides as well as recombinant cells containing the nucleotide sequences and the nucleotide fragments of the invention, under conditions allowing their expression.
Also entering into the framework of the present application are antibodies which recognize the polypeptides of the invention, as well as compositions with larvicidal activity.
A nucleotide sequence of the invention is characterized by the following properties: 4A, as obtained from the plasmid pCBM1 deposited at CNCM on 15 Jun. 1993 under the No. I-1317; AAT TGT GA) and/or with the oligonucleotide probe 16A (SEQ ID NO:2) (TTT CAT ATI GAA GCI GTI AAT GAA GG) and/or with at least one of the probes 66A (SEQ ID NO:3) (ATG AAT ACI AAT ATI TTT TCI ACI AA) or 66B (SEQ ID NO:4) (TC IGG TTC ICC ATA IAT CCA TTC ATC) under stringent conditions described in the experimental part; take part in the toxic activity of the products of expression of the 7 kb XbaI fragment against the larvae of Diptera and in particular the larvae of mosquitoes or of simuliids.
The toxic activity is defined within the frame-work of the invention in relation to the "target" on which it is desired to obtain this activity. This "target" is generally an arthropod and more particularly an insect, especially of the Diptera family and for example, a mosquito or simuliid larva.
It will be considered within the framework of the invention that a nucleotide sequence encodes a protein or a polypeptide having the capacity to take part in the
REFERENCES:
Porter, Mosquitocidal toxins, genes and bacteria: the hit squad. Parasitology Today. vol. 12(5):175-179, Apr. 25, 1996.
Barloy Frederique
Charles Jean-Francois
Delecluse Armelle
Nicolas Luc
Degen Nancy
Institut Pasteur
Sandals William
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