FR2639959A1 - Recombinant bacteria which are toxic towards Diptera larvae and their use for the production of larvicidal compositions. - Google Patents

Abstract

The invention relates to recombinant bacteria such as those obtained by modification of wild-type bacteria belonging to the family of Bacillaceae, which are different from B. sphaericus, having a toxic activity towards Diptera larvae especially of the mosquito or simuliid family. The invention also relates to recombinant vectors containing a DNA sequence which is foreign to the genome of the above wild-type bacterium and which is capable of producing a protein or an active protein portion having the toxic activity defined above. The invention also relates to the use of the recombinant bacteria in larvicidal compositions.

Description

Recombinant bacteria toxic to larvae of
Diptera and their use for the elaboration of larvicidal compositions.

 The invention relates to recombinant bacteria that are toxic to Diptera larvae and their use for the preparation of larvicidal compositions.

 Diptera are known to cause nuisance and / or transmit diseases.

For example, in Mosquitoes, which for the most part belong to 3 genera, namely
Aedes, Culex, AnoPhelesw Aedes aeavnti is responsible for yellow fever, some Culex species transmit arboviruses, filiaires and Anopheles sambiae transmits including malaria.

Larvicidal activities vis-à-vis
Diptera have been found in some bacteria, the latter were used for the development of larvicidal compositions. It is in particular bacteria of the genus Bacillus thurinaiensis or
Bacillus sphaericus. In particular it is known that Bacillus thurinaiensis israelensis (Bti) has a toxic activity on mosquito larvae belonging to several genera, namely on the larvae of Culicinae (in particular the Aedes and Culex larvae) and on the larvae of AnoDhelinae (AnoDheles). . Bti's activity is apparently more pronounced with regard to the larvae
Aedes and Culex. In addition, Bti activity on Simulium larvae was demonstrated. Similar characteristics to the previous ones in terms of toxic activity and specificity were found in B.thurinviensis morrisoni strain.

 The activity of B.thurinaiensis and in particular of Bti on mosquito larvae results from the production, during sporulation, of a protein crystal whose entomopathogenic activity is manifested in the cells of the digestive tract of the larvae ingesting this crystal.

 The toxic protein crystal of Bti is composed of several proteins, toxins or protoxins, in particular the proteins of 28, 68, 125 and 135 kDalton (kDa) solubilized and activated in the digestive tract of the larvae by the combined action of an alkaline pH and intestinal proteases. The formulations of Bti currently used (especially in the fight against onchocerciasis) consist of sporulated bacteria and crystals. New formulations have been developed from an asporogenous crystal producing strain. However, the effectiveness of these formulations proves to be insufficient, in particular because of a weakness of the persistence of the larvicidal activity. It is found that the persistence of the activity of Bti is reduced and that its activity in waters loaded with organic compounds is limited.

 Moreover, the bacterial property of Bacillus sPhaericus is known to have entomopathogenic activity on mosquito larvae.

As in Bti this activity is due to one or more proteins or toxins synthesized during sporulation and most often assembled in crystals.

B.sPhaericus activity against mosquito larvae also varies according to the mosquito genera involved, within this family. Unlike Bti, most strains of B.sPhaericus are inactive on Aedes larvae and very active on larvae of Culex, AnoDhèles and Mansonia.
It has been found, however, that the action of B.sphaericus on mosquito larvae is slower than that of Bti in relation to the same insects.

B.SPhaericus also showed a loss of spore activity after exposure to sunlight. In addition, B.sPhaericus has several constraints hindering its use in larvicidal compositions This bacteria does not develop for example on media whose carbon source is constituted by sugars, which limits the use of inexpensive media for the preparation of larvicidal compositions.

The many limitations mentioned above, found in bacteria with a certain larvicidal activity vis-à-vis Diptera larvae including the family of
Mosquitoes or Simulia constitute a disadvantage for the extension of their employment. The inventors have sought new ways to provide improved larvicide compositions with a higher cost-effectiveness ratio.

 By applying genetic engineering techniques, recombinant bacteria possessing larvicidal properties of great interest have been developed.

The object of the invention is therefore to provide recombinant bacteria as obtained by modification of wild-type bacteria belonging to the Bacillaceae family, different from B sPhaericus, having a toxic activity with respect to the larvae of
Diptera, in particular from the family of Mosquitos or
Blackflies.

 Wild bacteria meeting the above criteria (also referred to as "host bacteria") are either sporulating bacteria, at least able to perform the early stages of sporulation as will be defined later, or bacteria in phase vegetative growth, able under certain conditions defined below, to behave in terms of the desired toxicity, such as sporulating bacteria.

 By ecologically acceptable bacterium is meant a bacterium that does not produce a pollutant or toxic effect in the environment in which it is administered with the exception of the desired larvicidal effect discussed above.

 The invention also aims at providing recombinant vectors containing a DNA sequence foreign to the genome of the above wild-type bacterium and capable of producing a protein or a protein active part producing or inducing the toxic activity defined above or still participating in this toxicity when it is in synergy with a protein or part of a toxic or potentially toxic protein.

Such recombinant vectors are capable of being introduced and expressed in a wild-type bacterium meeting the above criteria.

The invention also aims to provide larvicidal compositions for the control of Diptera larvae, in particular against larvae of
Mosquitoes and larvae of Simulia.

The bacteria according to the invention are recombinant bacteria, as obtained by genetic modification of a strain of wild bacteria belonging to the Bacillaceae family and are endowed with a larvicidal activity with respect to the larvae of the Bacillaceae.
Diptera, especially against larvae of the family Mosquitoes and Simulia.

 These recombinant bacteria are characterized in that - they are different from B.sphaericus, - they comprise, possibly under the control of an exogenous promoter, a DNA sequence foreign to the genome of the wild-type bacterium, coding for at least the part active ingredient of at least one protein as produced by B.sPhaericus, having a toxic activity vis-à-vis Diptera larvae or capable of inducing this activity, - they are capable of expressing both the sequence of Foreign DNA above and the gene (s) specific to the wild-type bacterium responsible for its larvicidal activity vis-à-vis the Diptera larvae, either during the process of sporulation of the recombinant bacterium formed, or during the vegetative phase of growth of this recombinant bacterium, when the genes specific to the wild-type bacterium and the foreign DNA sequence are placed under the control of functional promoters in the vegetative phase c hez the host bacterium, so as to obtain an expression of toxicity, comparable to that observed during the course of sporulation.

For convenience of language, the characteristic defined by "having a toxic activity with respect to the larvae of
Diptera or capable of inducing or participating in this activity in combination with another protein "by the expression" involved in larvicidal activity with respect to Diptera.

 Advantageously, the recombinant bacteria according to the invention are capable of synthesizing in the same organism, the toxins specific to the wild-type bacterium and the products resulting from the expression of the foreign DNA sequence.

 In particular, the recombinant bacteria of the invention are capable of expressing simultaneously, the larvicidal activity of the wild-type bacterium above and that of the expression product of the foreign DNA sequence incorporated in this wild-type bacterium, which makes them confers increased toxic activity.

 Preferably, the expression product of the foreign DNA sequence comprises one or more proteins or active parts of proteins, producing or inducing the specific desired larvicidal activity, or participating in combination with a protein or part of a protein with said activity. larvicide. This product is preferably in the form of crystalline inclusions occurring either during the sporulation of the recombinant bacterium, or during the vegetative phase when the conditions determined above, to achieve the expression of said crystalline inclusions are present.

 These toxic proteins or polypeptides can be located in separate crystals depending on whether they result from the expression of the genes of the host bacterium or the expression of the foreign DNA sequence.

 These crystals can also be assembled in the same entity to form a new crystalline mass.

 Advantageously, a recombinant bacterium of the invention allows the synthesis of specific combinations of the various proteins and toxins sought. Depending on the desired spectrum of action, it is indeed possible to limit the expression of the genes of the wild-type bacterium, especially those which code for the above toxic proteins, to certain specific toxins.

 As indicated above in a preferred embodiment of the invention, a recombinant bacterium of the invention retains its ability to carry out the sporulation process, at least the ability to perform the sporulation phases necessary to produce sporulation. crystals containing the toxins of the host bacterium and crystals containing the proteins resulting from the expression of the foreign DNA sequence, when the natural external conditions of this production are met.

 In one embodiment of the invention involving a spore-forming bacterium, it is particularly advantageous for the recombinant bacterium to be capable of carrying out sporulation at least until the course of stage 2. In the following expression "sporulation" will have to be understood in this sense, and will not be limited to the achievement of all stages of the sporulation process that is to say to obtain spores.

 The integrated bacterial DNA sequence encodes at least the active portion of one or more proteins exhibiting larvicidal or larvicidal toxic activity such as larvicidal activity directly or indirectly related to B protein P43. .sPhaericus. This DNA sequence may also encode at least the active portion of a protein such as B. sDhaericus P56 protein as long as the protein or portion of encoded protein is involved in the desired activity.

 In a particularly advantageous embodiment, the DNA sequence integrated into the host bacterium encodes at least one. active part of proteins such as B.sphaericus P43 and P56 together. The genes coding for each of these P43 and P56 proteins whose apparent molecular weights in polyacrylamide gel electrophoresis are 43 kDa and 56 kDa, were sequenced and described by BAUMANN et al (1).

A foreign DNA sequence suitable for carrying out the invention is the DNA sequence coding for at least the active part of the P43 protein defined above. Another DNA sequence encodes the P56 protein. Another preferred sequence codes for these two B.sPhaericus proteins together, since in each case the desired larvicidal activity is obtained. The invention also relates to a sequence which preserves with one of the above sequences, which are characteristic of B. shaericus, a homology sufficient for the protein or the active portion of protein encoded by this foreign DNA sequence to be capable of exhibiting a toxic activity of the same qualitative and quantitative order, or of inducing or participating in the production of a similar toxicity with respect to Diptera larvae, especially larvae of the family of
Simulias or Mosquitoes and preferably vis-à-vis larvae of the genus Aedes, Anopheles and Culex.

For example, it is possible to use, in a particular embodiment of the invention, a protein derived from P43, namely the P38 protein (having a molecular weight of approximately 38-40 kDa), which is an activated form of
P43 in the presence of the intestinal proteases of Mosquito.

 Advantageously, to allow the expression in the recombinant bacterium of proteins, parts of these proteins, or polypeptides, involved in the desired larvicidal activity, the foreign DNA sequence is incorporated under the control of a specific promoter.

 Different types of promoters may be used, among which are strong promoters and advantageously strong promoters subject to regulation. By way of example, mention is made of the spac promoter as described in (2) or the promoter of the B. subtilis sac B gene, as described in (3).

 These promoters are particularly suitable when it is desired to obtain the above larvicidal activity in the vegetative phase of growth of the bacterium.

 In this case, promoters must also be placed upstream of the genes specific to the host bacterium in order to obtain their expression prematurely.

Regarding the genes specific to the host bacterium, a prior cloning step can be performed to place them under the control of the vegetative promoter. Thus, in this case, the expression of the genes specific to the host bacterium and of the foreign DNA sequence coding for at least one part of protein involved in the larvicidal activity is carried out under the control of a vegetative promoter capable of induce the production of the proteins with the desired activity, to obtain a level of toxicity comparable to that obtained during the sporulation phase.

 Other promoters of interest for controlling the expression of the foreign DNA sequence, when the host bacterium is a spore-forming bacterium, are so-called sporulation promoters that function with a sporulation-specific RNA polymerase. A sporulation promoter may be derived from the genome of B.sPhaericus or may be any exogenous promoter compatible with the expression of the B.sPhaericus DNA fragment above, in the wild-type bacterium.

 In a preferred embodiment of the invention, the starting wild-type bacterium subsequently modified with the foreign DNA sequence defined above is a bacterium of the strain B.thurinaiensis (Bt). In a particularly preferred manner, the wild-type bacterium belongs to the Bti variety or to the B.thurinaiensis morrisoni variety, such as the PG14 strain isolated by PADUA and (4).

The invention also provides a recombinant expression and cloning vector containing said foreign DNA sequence. This vector is characterized by the following properties
it contains an optionally exogenous promoter, allowing the expression of the above-defined DNA sequence defined in a host bacterium chosen from Bacillaceae different from B.sPhaericus,
it is compatible with the production of at least the active part of at least one protein having the larvicide activity specific to the host bacterium and the larvicidal activity obtained by expression of the foreign DNA sequence.

 Particularly advantageous vectors for carrying out the invention are bifunctional vectors, capable of replicating in several types of microorganisms, for example E. coli, B. subtilis and recombinant bacteria according to the invention.

In an advantageous embodiment, the recombinant vector contains the DNA sequence coding for at least the active part of the P43 proteins and / or
P56 of B.sphaericus and is compatible with their expression in Bt preferably in Bti in conjunction with the expression of toxicity genes specific to Bt.

 When the host bacterium is sporulant, several types of vectors can be used to obtain the recombinant vector above, as long as they do not inhibit the sporulation of the recombinant bacterium. For example, it is possible to use plasmid vectors which do not cause the inhibition of bacterial sporulation and therefore do not cause any limitation in the expression of the toxins of the modified bacterium.

 When the expression of the toxicity takes place in a bacterium in the vegetative phase of growth, the vector carrying the foreign DNA sequence is provided with a strong vegetative promoter of the type of those described above. In this case, the same promoter may be placed upstream of the toxicity genes of the host bacterium.

 To realize the recombinant vectors according to the invention, one skilled in the art can easily take advantage of the teaching of the articles of Sullivan et al (5) and MITEVA et al (6) whose content is part of this description. In general, the insertion of the foreign DNA sequence into the vector is carried out at a site such that the replication properties of the vector are not altered.

 Such a plasmid vector which after modification by the foreign DNA sequence is adapted for insertion into a sporulating host bacterium is the replicable plasmid pBU4, obtained by melting the vectors pUCI9 and pBC16.1. These two vectors have been described in (7) and (8).

The invention thus relates in particular to recombinant vectors resulting from incorporation into the pBU4 vector of the foreign DNA sequence. It aims particularly advantageously the recombinant plasmids pGSP10 and pGSP11. These two vectors described in FIGS. 1 and 2, formed from pBU4 contain the B.sDhaericus DNA sequence comprising the sequences coding for the P43 and P56 proteins. This DNA fragment of interest in the context of the invention is further characterized by the following restriction sites
Hind III, Bgl II, Sph I, EcoR I, EcoR V, Sca I, EcoR V,
Sca I, Hind III.

 It is located on a 3.6 kb Hind III DNA fragment.

 To make the recombinant plasmids pGSP10 and pGSP11, the above DNA sequence of B.sDhaericus is cloned at the Hind III site of the multiple cloning site (polylinker) of the pBU4 vector. Both plasmids pGSP10 and pGSP11 differ from each other by the orientation of the B.sPhaericus DNA sequence to the lacZ gene promoter (placZ).

 Another vector suitable for producing the above recombinant vector is the vector pBU5 constructed from the same plasmids pUC19 and pBC16.1 and in which the orientation of pBC16.1 has been reversed with respect to the orientation of this plasmid. in pBU4.

The plasmid pBU4 transformed as indicated above does not inhibit the sporulation of the transformed bacteria. It also has the property of being bifunctional, which gives it the ability to replicate in several types of microorganisms, for example E.coli or B.subtilis or bacteria of the Bacillaceae family. As a result, cloning of the DNA encoding the proteins or active parts of larvicide-active proteins described above can be performed in
E.coli. The clone thus produced makes it easy to purify the recombinant vector formed between pBU4 and the foreign DNA sequence. This recombinant vector can then be introduced into Bti to obtain the final recombinant bacterium.

 Other recombinant vectors can be envisioned for preparing recombinant Bti bacteria.

By way of example, the pMK3 vectors obtained by fusion between plasmids pUC9 and pUB110 and pMK4 obtained by fusion of plasmids pUC9 and pC194 are cited. These two plasmids pMK3 and pMK4 have been described by Sullivan et al. in (5).

 Recombinant bacteria formed from a Bti wild-type bacterium transformed with plasmids pGSP10 and pGSP11 are also within the scope of the invention.

 Particularly advantageous clones are the clones 4Q2-72 / pGSP10 and 4Q2-72ipGSP11 which express both toxins specific to Bti and toxins specific to B sPhaericus. These clones result from the transformation of a "crystal +" strain of Bti (strain 4Q2-72) by the previously described recombinant plasmids.

 The introduction of the foreign DNA sequence into the wild-type bacterium can be carried out according to conventional techniques, for example by transformation, conjugation, electroporation using techniques such as those described by LUCHANSRY et al (9) or transduction. For this purpose, it is possible to use the previously defined recombinant vectors.

 These recombinant vectors are then brought into contact with wild bacteria defined above, under conditions allowing their modification by the recombinant vector. According to an alternative embodiment, said recombinant vector is used to transform a bacterium of the E.coli or B.subtilis type. This step allows the cloning and increased production of said vector which is then inserted by transformation into the sporulating wild-type bacterium.

 The recombinant bacteria obtained are selected by any appropriate physical or chemical means.

 In a particular embodiment of the above method, the recombinant bacteria result from the transformation of Bt, preferably Bti, by the previously described B.sPhaericus DNA sequence.

 The implementation of the transformation process may in particular be controlled by detecting the production of crystals with larvicidal activity corresponding to those naturally produced by each of the original bacteria involved in obtaining the recombinant bacteria.

The introduction of the foreign DNA sequence by conjugation can be carried out using, as a donor strain, a bacterium such as B. subtilis or
E. coli following techniques such as those described in (10) or (11).

The elaboration of the recombinant bacteria according to the invention has allowed the inventors to propose new compositions for controlling Diptera larvae, in particular larvae of the family of
Mosquitoes.

The invention therefore relates to a larvicidal composition comprising recombinant bacteria as defined above, in an amount sufficient to exhibit an activity on larvae of Diptera of the family Simulia or Mosquitoes, preferably on all the larvae of the genus Aedes, Culex and
Anopheles.

 Such a larvicidal composition can be presented in various forms depending on the desired location and mode of administration. Thus, the larvicidal compositions may be prepared in the form of powder, oily suspension, or briquettes. Such compositions will advantageously be prepared from recombinant bacteria allowing a significant quantitative expression of the different toxins active on mosquito larvae and / or Simulia.

Advantageously, a composition that can be used for its larvicidal activity vis-à-vis Diptera larvae, particularly from the family of
Mosquitoes and / or Simulia, includes B.thurinctiensis bacteria and in particular Bti modified according to the invention by a foreign DNA sequence.

 A particularly suitable larvicidal composition comprises Bti bacteria modified with B.shaericus DNA sequences expressing at least the active part of at least one protein involved in larvicidal activity with respect to Diptera larvae, especially the family of Mosquitos.

Other features and benefits of
The invention appears in the following examples and in the figures which represent - Figure 1: Construction of plasmids pBU4 and pBU5, - Figure 2: representation of the restriction maps of plasmids pBU4, pGSP10 and pGSP11. The DNA fragment
Hind III containing the 41, 9 and 51.4 kDa protein genes of B. sPhaericus was inserted into pBU4 at the Hind III site of a multiple cloning site (MCS) originating from the pUC vector. Tet and Amp indicate the positions of the markers of resistance to tetracycline and ampicillin. The names of the restriction enzymes in parenthesis correspond to the cloning sites used to construct pBU4 and are no longer usable.

FIG. 3: restriction map of the DNA fragments coding for the p43 and p56 proteins; FIG. 4: analysis of the proteins of the transformants of
B. thurinqiensis expressing crystal proteins
B. sPhaericus. Bacterial extracts were deposited on a 10% polyacrylamide gel containing SDS and stained with Coomassie blue. Lanes 1,2,3: strain 4Q2-72 containing pGSP11, pSGSP10 and pBU4 respectively; lanes 4, 5, 6: strain-402-81 containing pGSP11, pGSP10 and pBU4 respectively; line 7: purified crystals of Bti; Lane 8: purified crystals of B. sPhaericus; S: markers. The size of the markers is indicated in the right margin. In the left margin, the small numbers indicate the size of the Bti crystal proteins, the large numbers indicate the size of the proteins of the crystals of Bti.
B. sPhaericus.

FIGS. 5 and 6: immunodetection (WESTERN-BLOT) of the transformant proteins. Samples were numbered in the same way as in Figure 4.

 Figure 5: Detection of B. crystal proteins

sphaericus.

 Figure 6: Detection of Bti proteins.

FIG. 7: observation under the electron microscope of the strains Bti4Q2-81, a: 4Q2-81 / pBU4, b and c: 4Q2-81 / pGSP10 producing the proteins of the crystal of
B. sPhaericus.

FIG. 8: observation under the electron microscope of
Bti4Q2-72, a: 4Q2-72 / pBU4, b and c: 4Q2-72 / pGSP10 producing large (L) and small (S) crystals probably corresponding to Bti proteins and B. sohaericus crystal proteins, respectively .

EXAMPLE 1
Transformation of B.thurinqiensis israelensis By the ozens of B.sphaericus toxins.

The transformation of B.thurinaiensis israelensis (Bti) by the genes encoding P43 toxins (also referred to as "41.9 kDa protein" with reference to its molecular weight calculated from the amino acid sequence: 41.9 kDa) and the P56 toxin (calculated molecular weight: 51.4 kDa) was carried out according to the protocol described in an article of
HEIERSON et al (2). However, modifications to the transformation protocol described by HEIERSON et al were carried out in order to adapt it to the introduction of the genes coding for the P43 and P56 proteins of B.shaericus in B.thurinqiensis israelensis. These arrangements are presented in the summary of the steps of the adapted process which follows - is grown on a medium as described by HEIERSON Bti bacteria to obtain an optical density of 1.5 (OD 1.5). centrifugation culture for 10 minutes at room temperature and with a rotation speed of 3700 rpm; then washed in 50mM Tris buffer at pH 7.5 (1 / 2V). The product is then resuspended in Tris HCl buffer pH 8.9, containing 30% sucrose so as to obtain an optical density of 0.8 (OD 0.8). The suspension is then distributed in 5 ml in Erlenmeyer flasks and gently stirred for 30 minutes (75 rpm) at 37.degree. The bacteria are then subjected to centrifugation for 10 nm at 3700 rpm and then resuspended in 0.5 ml of LBP medium.

Then, 100 μg of DNA dissolved in 100 μl of medium containing TE buffer and LBP medium (in 1: 1 ratio) are added. 1.5 ml of polyethylene glycol (40% PEG in Tris buffer) is then added, then gently mixed and incubated for 10 nm with gentle stirring (75 rpm) at 37 ° C. A new centrifugation for 10 minutes at 3700 rpm and at room temperature. Ambient is performed, then the cells are resuspended in 5 ml of Luria medium.

After a further incubation for 1 h 30 to 37 ° C. with gentle shaking (75 rpm), the cells are plated (0.5 ml) Luria on-box containing 2.5 agar, 2.5% gelatin and an antibiotic to select the bacteria effectively transformed with the recombinant plasmid carrying B. sphaericus genes; an antibiotic, such as tetracycline (30g / ml) or chloramphenicol (5pg / ml) or the
Kanamycin (25 μg / ml) according to the vector plasmid used.

 For more details concerning the principle of this transformation, it will be easy to refer to the aforementioned article by HEIERSON, the content of which is incorporated in the present specification.

 The transformation protocol previously described was implemented to transform two strains of Bti, strains 4Q2-81 and 4Q2-72 which are respectively a "crystal" strain and a "crystal +" strain.

A "crystal +" strain is able to produce crystals containing Bti toxins, unlike a "crystal" strain
The strain 4Q2-81 is derived from strain n 4Q2 (Bacillus Genetic stock Center the Ohio State
University Columbus) lacking a plasmid (also referred to as "plasmid -") that does not synthesize crystals and is nontoxic to mosquito larvae.

 Strain 402-72 is derived from the same strain 402: it is capable of producing crystals, it has a plasmid of 72 MDa bearing all the genes of the Bti crystal proteins, it is a deprive of the small plasmids present at the origin in strain 4Q2.

 To transform these two strains, plasmids pGSP10 and pGSP11 containing the B.sPhaericus toxin genes recloned in the pBU4 vector were used.

The vector pBU4 shown in FIG. 1 was obtained by fusing the vectors pUC11 and pBC16.1. This plasmid pBU4 was carried out after the following steps - hydrolysis of the plasmid pUC79 using the Eco 0109 enzyme, then repair of the site by the Klenow fragment, - hydrolysis of the plasmid pBC16.1 by the Eco RI enzyme, then repair of the site by the Klenow fragment. Both plasmids were then ligated to form plasmid pBU4 or plasmid pBU5.

 The B.shaericus DNA sequence (shown in Figure 3) was cloned at the Hind III site of the multiple cloning site (polylinker) of the constructed vector.

Two plasmids were prepared according to the insertion direction of the B.sphaericus DNA sequence in pBU4 pGSP10 resulting from a construct in which the B. sDhaericus genes are transcribed in the sense of the lacZ (placZ) promoter and the pGSP11 plasmid results from an inverted construct with respect to the lacZ promoter. Plasmids pGSP10 and pGSP11 were isolated from E. coli clones. Plasmids were then introduced into Bti4Q2-81 and Bti4Q2-72-by transformation. The recombinant plasmids were stable in both cases.

 The recombinant clones were analyzed by the immunodetection method designated by WESTERN BLOT to characterize the protein extracts produced.

 Transformants 402-81 (pGSP10) and 402-81 (pGSP11) synthesized both the 41.9 kDa and 51.4 kDa proteins in an amount large enough for the detection of these proteins to be done by assay. bacterial extracts by SDS polyacrylamide gel electrophoresis and staining with Coomassie blue (see Figure 4, lines 4 and 5). Similarly, recombinant clones formed from strain Bti4Q2-72, synthesize B. sphaericus proteins in addition to their own toxins (Figure 4, lines 1 and 2).

 As shown in Fig. 4, protein expression was greater in transformants containing plasmid pGSP10R in which transcription of B. sPhaericus genes is performed in the same direction as the transcriptional direction of the lacZ promoter of the vector. This finding was essentially verified for the 51.9 kDa polypeptide. In all the transformants, this polypeptide appeared in a smaller amount than the 41.9 kDa polypeptide.

The presence of B.sPhaericus crystal proteins in Bti transformants was confirmed by immunodetection using an antiserum directed against an alkaline extract of B.sphaericus spores (see Figure 5). High molecular weight proteins were also detected in addition to the 41.9 and 51.4 kDa proteins. They can represent undissociated polymers composed of the proteins of the crystal of
B. sPhaericus.

 It has been noted that the presence of the 51.4 kDa protein is required to induce toxicity of the 41.9 kDa protein, when their respective genes are expressed in E. coli. These results suggest that the B sphaericus 51.4 kDa protein could possess a proteolytic activity capable of activating the 41.9 kDa protein when these two proteins are expressed in E. coli.

 At the same time, the presence of Bti crystal proteins in transformants 402-72 was monitored by immunodetection with antiserum directed against the purified Bti crystals (see Figure 6, lines 1, 2, 3). No crystal protein was detected in transformants 402-81 (see Figure 6, lines 4, 5, 6), this strain being "crystal-".

 Weak cross-reactions were observed between strains 402-81 (pBU4) and 402-81 (pGSp11). However, their absence in strain 4Q2-81 (pGSP11) suggested that these reactions are not specific. In addition, hybridization experiments carried out between the total DNA of strain 4Q2-81 and the 125 kDa protein gene isolated from strain 4Q2-72, and used as a probe, indicated that this strain of Bti does not contain the genes corresponding to 125 and 135 kDa proteins.

 Bti transformed with ss crystal protein genes. sPhaericus has crystal structures. Electron microscopic observations of transformants 402-81 (pGSP10) showed that this clone produced inclusions with a regular tall-crystalline configuration (Figure 7).

 In transformant 402-72 (pGSP10), similar inclusions were detected in addition to irregular crystals typical of Bti (see Figure 8). It is possible that transformants 4Q2-72 (pGSP10), which contain large inclusions of Bti crystal also contain B crystal proteins.

sphaericus. In all cases, these inclusions were localized outside the exosporium, as usually in the Bti crystals whereas the inclusions of B.sPhaericus are localized between two membranes of the exosporium. However, it is reasonable to assume that these structures must be attributed to
B. sPhaericus which are produced in a large quantity in transformed strains.

Example 2: Biological activity of transformed strain
The biological activity of the transformants was determined on three mosquito species, Aedes segypti, Anophele stephensi and Culex pipiens.

The results of Table I show that the transformants 402-81 (pGSP10) and 402-81 (pGSP11) which express only B. snhaericus proteins, were like wild B. sPhaericus, the most strongly active against Culex. pipiens and Anopheles stePhensi. Slight toxicity to Aedes aeavPti was also observed. In contrast, the transformants 4Q2-72 (pGSP10) and 4Q2-72 (pGSP11) that produced crystal proteins of both Bti and
B. sPhaericus were very active against the three species of mosquitoes. This result is in line with the specificity associated with Bti.De crystals. Moreover, transformants expressing both types of toxins were more active than the recipient strain.
TABLE I

<tb><SEP><SEP> strains Aedes <SEP> aegypll <SEP> Anopheles <SEP> stephensi <SEP> Culex <SEP> pipiens <SEP>
<tb><SEP> Dilution <SEP> 24 <SEP> H <SEP> 48 <SEP> H <SEP> 72 <SEP> H <SEP> 24 <SEP> H <SEP> 48 <SEP> H <SEP> 72 <SEP> H <SEP> 24 <SEP> H <SEP> 48 <SEP> H <SEP> 72 <SEP> H
<tb><SEP> 1x10-2 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 5 <SEP> 20 <SEP> 20 <SEP> NT <SEP> NT <SEP> NT
<tb><SEP> 0.5x10-2 <SEP> 5 <SEP> 5 <SEP> 5 <SEP> 20 <SEP> 20 <SEP> 20 <SEP> NT <SEP> NT <SEP> NT
<tb><SEP> 4Q2-81 <SEP> 1x10-3 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 0 <SEP> 0 <SEP> 0
<tb><SEP> / pBU4 <SEP> 0.5x10-3 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 5 <SEP> 10
<tb><SEP> 1x10-4 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 25 <SEP> 30 <SEP> 30 <SEP> 0 <SEP> 0 <SEP> 5
<tb><SEP> 0.5x10-4 <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> 0 <SEP> 5 <SEP> 5
<tb><SEP> 1x10-5 <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> 0 <SEP> 0 <SEP> 0
<tb><SEP> 1x10-2 <SEP> 30 <SEP> 75 <SEP> 85 <SEP> 95 <SEP> 100 <SEP> 100 <SEP> NT <SEP> NT <SEP> NT
<tb><SEP> 0.5x10-2 <SEP> 50 <SEP> 90 <SEP> 90 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> NT <SEP> NT <SEP> NT
<tb><SEP> 4Q2-81 <SEP> 1x10-3 <SEP> 35 <SEP> 85 <SEP> 85 <SEP> 90 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> pGSP10 <SEP> 0.5x10-3 <SEP> 25 <SEP> 60 <SEP> 60 <SEP> 85 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> 1x10-4 <SEP> 30 <SEP> 35 <SEP> 35 <SEP> 80 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> 0.5x10-4 <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> 1x10-5 <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> 85 <SEP> 95 <SEP> 95
<tb><SEP> 1x10-2 <SEP> 25 <SEP> 65 <SEP> 80 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> NT <SEP> NT <SEP> NT
<tb><SEP> 0.5x10-2 <SEP> 10 <SEP> 75 <SEP>'<SEP><SEP> 85 <SEP> 90 <SEP> 100 <SEP> 100 <SEP> NT <SEP> NT <SEP> NT <SEP>
<tb><SEP> 402-81 <SEP> 1x10-3 <SEP> 15 <SEP> 35 <SEP> 35 <SEP> 85 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> pGSP11 <SEP> 0.5x10-3 <SEP> 0 <SEP> 5 <SEP> 10 <SEP> 70 <SEP> 90 <SEP> 90 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> 1x10-4 <SEP> 5 <SEP> 5 <SEP> 0 <SEP> 25 <SEP> 80 <SEP> 85 <SEP> 80 <SEP> 95 <SEP> 95
<tb><SEP> 0.5x10-4 <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> 85 <SEP> 90 <SEP> 90
<tb><SEP> 1x10.5 <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> NT <SEP> 0 <SEP> 10 <SEP> 10
<tb><SEP> 1x10-4 <SEP> 10 <SEP> 10 <SEP> 15 <SEP> 75 <SEP> 95 <SEP> 95 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> 0.5x10-4 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 40 <SEP> 85 <SEP> 95 <SEP> 100 <SEP> 100 <SEP> 100
<tb> B.sphaericus <SEP> 1x10-5 <SEP> O <SEP> 0 <SEP> 0 <SEP> 20 <SEP> 35 <SEP> 35 <SEP> @ <SEP> 90 <SEP> 90 <SEP > 100
<tb><SEP> 0.5x10-5 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 25 <SEP> 50 <SEP> 50 <SEP> 45 <SEP> 85 <SEP> 95
<tb><SEP> 0.5x10-5 <SEP> 15 <SEP> 85 <SEP> 95 <SEP> 15 <SEP> 20 <SEP> 20 <SEP> 0 <SEP> 5 <SEP> 5
<tb><SEP> 402-72 <SEP> fx10-6 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 5 <SEP> 5 <SEP> 10 <SEP> 5 <SEP> 10 <SEP> 10
<tb><SEP> 0.5x10-6 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 5 <SEP> 5 <SEP> 10
<tb><SEP> 1x10-7 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 5 <SEP> 10 <SEP> 15 <SEP> 0 <SEP> 5 <SEP> 5
<tb><SEP> O.5x10-5 <SEP> 100 <SEP> 100 <SEP> 100 <SEP> 35 <SEP> 55 <SEP> 60 <SEP> 100 <SEP> 100 <SEP> 100
<tb><SEP> 4Q2-72 <SEP> 1x10-6. <SEP> 10 <SEP> 25 <SEP> 25 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 25 <SEP> 50 <SEP> 55
<tb><SEP> pGSP10 <SEP> 0.5x10-6 <SEP> 0 <SEP> 5 <SEP> 10 <SEP> 15 <SEP> 25 <SEP> 25 <SEP> 0 <SEP> 5 <SEP> 20
<tb><SEP> 1x10-7 <SEP> O <SEP> a <SEP> o <SEP> 15 <SEP> 15 <SEP> 20 <SEP> o <SEP> o <SEP> 5
<tb><SEP> 0.5x10-5 <SEP> 95 <SEP> 100 <SEP> 100 <SEP> 35 <SEP> 35 <SEP> 35 <SEP> 90 <SEP> 95 <SEP> 100
<tb><SEP> 402-72 <SEP> 1x10-6 <SEP> 0 <SEP> 15 <SEP> 20 <SEP> 10 <SEP> 10 <SEP> 10 <SEP> 20 <SEP> 30 <SEP> 30
<tb><SEP> pGSP11 <SEP> O.5x10-6 <SEP> 0 <SEP> 10 <SEP> 10 <SEP> 15 <SEP> 15 <SEP> 15 <SEP> 0 <SEP> 0 <SEP> 15
<tb><SEP> 1x10-7 <SEP> O <SEP> 0 <SEP> - <SEP> 0 <SEP> 0 <SEP> 5 <SEP> 5 <SEP> 0 <SEP> 0 <SEP> 0
<Tb>
BIOLOGICAL ACTIVITY OF TRANSFORMANTS
BIBLIOGRAPHY (1) BAUMANN et al., J. of Bacteriol., Vol 170 2045-2050.

(2) YAMSURA, DG et al (1984), Proc. Nat. Acad. Sci.
USA, 81: 439-443.

(3) Klier, A et al (1987), Mol. Microbiol. 1 (2) 233-241.

(4) PADUA et al (1984), J. Invertebr. Pathol., 44 12-17.

(5) SULLIVAN et al. New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene 29 (1984) 21-26 (6) MITEVA et al. Construction of a bifunctional genetically labeled plasmid for Bacillus thuringiensis subsp. israelensis. Arch Microbiol (1988) 150: 496-498.

(7) YANMISCH et al (1985), Gene, 33: 103-119.

(8) POLAK, J. et al (1982), Plasmid, 7: 152-162.

(9) LUCHANSKY et al (1988), Mol. Microbiol., 2637-646.

(10) GONZALEZ et al (1982), Proc. Nat. Acad. Sci., USA, 79: 6951-6955.

(11) LERECLUS et al (1983), Mol. Gen. Genet., 191 307-313.

(12) HEIERSON et al. Transformation of Vegetative Cells of Bacillus thuringiensis by Plasmid DNA. Journal of
Bacteriology. Mar. 1987. p.1147-1152

Claims (16)

A recombinant bacterium having a larviciding activity against Diptera larvae, particularly against mosquito and Simulia larvae, as obtained by genetic modification of a bacterium wild-type belonging to the Bacillaceae family characterized in that - it is different from B.sPhaericus, - it comprises, possibly under the control of an exogenous promoter, a DNA sequence foreign to the genome of the wild-type bacterium, coding for minus the active part of a protein that is toxic to Diptera larvae, as produced by B.sDhaericus, they are capable of expressing both the above foreign DNA sequence and the one or more genes specific to the wild-type bacterium responsible for its larvicidal activity vis-à-vis Diptera larvae, either during the process of sporulation of the recombinant bacteria formed, or during the vegetative phase of growth. of this recombinant bacterium when the genes specific to the wild-type bacterium and the foreign DNA sequence are placed under the control of a vegetative phase functional promoter in the host bacterium, so as to obtain an expression of the toxicity, comparable to - that observed during the course of sporulation. 2 / recombinant bacterium according to claim 1, characterized in that it is sporulating. 3 / Recombinant bacterium according to claim 1, characterized in that the genes or genes specific to the wild-type bacterium responsible for the larvicidal activity and the foreign DNA sequence are placed under the control of a vegetative promoter, allowing their expression during the vegetative phase of growth. 4 / Bacterium according to one of claims 1 to 3, characterized in that the DNA sequence foreign to its genome encodes at least the active part of a toxic protein such as P43 protein produced by B sphaericus. 5 / bacterium according to one of claims 1 to 3, characterized in that the DNA sequence foreign to its genome encodes at least the active part of a toxic protein such as the P56 protein produced by B sphaericus. 6 / A bacterium according to one of claims 1 to 3, characterized in that the DNA sequence foreign to its genome encodes at least the active part of toxic proteins, such as P43 and P56 proteins taken together, of B. sPhaericus 7 / recombinant bacterium according to any one of claims 1, 2, 4, to 6, characterized in that the foreign DNA sequence inserted into the wild-type bacterium is placed under the control of a strong promoter, including a promoter sporulation from B.sDhaericus. Recombinant bacterium according to claim 1, wherein the wild-type bacterium is a B. Diptera of the family Mosquitoes or Simulia, in particular of the genus Culex, Aedes or Anopheles. 9 / Recombinant bacterium according to claim 8, characterized in that the B.thurinaiensis strain belongs to the variety israelensis or morrisoni. 10 / Recombinant bacterium according to any one of claims 7 to 9, characterized in that it is clone 402-72 / pGSP10 or clone 402-72 / pGSP11. 11 / recombinant expression and cloning vector containing a foreign DNA sequence as defined in any one of claims 1, 3 to 7. 12 / recombinant vector according to claim 11, characterized in that it is the plasmid pBU4 modified by the DNA sequence coding for at least the active part of the P43 protein or the P56 protein or both proteins together. B. sphaericus. 13 / Recombinant vector according to claim 12, characterized in that it is the plasmid pGSP10. 14 / Recombinant vector according to claim 12, characterized in that it is the plasmid pGSP11. 15 / recombinant vector according to claim 11, characterized in that it is a resident plasmid of B.thurinaiensis, modified by the DNA sequence of B.sphaericus encoding at least the active portion of the P43 protein or the P56 protein or both proteins together. 16 / larvicidal composition comprising recombinant bacteria according to any one of claims 1 to 10, in an amount sufficient to exhibit a larvicidal activity vis-à-vis Diptera larvae including the family of Simulia and / or Mosquitoes, preferably on all larvae of the genus Aedes, Culex and Anopheles. 17 / larvicidal composition according to claim 16, characterized in that it is in the form of powder, oily suspension or briquettes. 18 / Process for the production of recombinant bacteria according to any one of claims 1 to 10, characterized by the following steps - contacting the recombinant vector according to any one of claims 11 to 15 with sporulating wild-type bacteria chosen from Bacillaceae and compatible with the expression of the foreign DNA sequence contained in said recombinant vector, under conditions allowing the transformation of said bacteria by said recombinant vector, - selection of the recombinant bacteria obtained, by any appropriate physical or chemical means.