Novel regulators VipR in bacillus strains

Abstract

The present invention relates to a novel modulator VipR capable of modulating gene expression, and more particularly, the expression of vip3A. The invention also relates to an expression system that allows for the expression of a protein of interest by VipR modulation. The expression system can be used for transformation of bacillus strains.

Description

[0001] The present invention relates to a novel regulator VipR capable of regulating gene expression, and more specifically, capable of regulating the expression of vip3A. The present invention also relates to an expression system that allows the expression of a target protein to be regulated by VipR. Such an expression system can be used for the transformation of a Bacillus strain.

[0002] The species Bacillus thuringiensis includes a large number of Gram-positive spore-forming bacterial strains that belong to the Bacillus cereus group and are characterized by the production of parasporal crystal inclusions (1). Many strains of Bacillus thuringiensis are insect pathogenic, and their insecticidal properties are mainly due to the presence of crystalline proteins composed of Cry and Cyt toxins encoded by plasmid genes (2-4). The presence of cry genes is a common feature of all strains of Bacillus thuringiensis species, and the expression of most cry genes depends on the sporulation-specific factors σE and σK, which act in the mother cell compartment during sporulation. However, a few cry genes do not follow the same regulatory pathway (5). During sporulation, they are also expressed in the mother cell or non-sporulation subpopulations, but are not related to the sporulation sigma factor. Of note, these genes are the cry3 gene encoding a toxin active against coleopteran insects (6) and the cry genes of strain LM1212, which are expressed under the control of the specific transcriptional activator CpcR encoded by a plasmid gene (7). Different combinations of toxins can be found in crystal inclusions depending on the strain of Bacillus thuringiensis. As an example, the commercial strain kurstaki HD1 contains six cry genes: cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa and cry2Ab (http: / / www.lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / ). All of these cry genes encode proteins active against lepidopteran insect larvae and provide a broad insecticidal spectrum in this order (8). However, the pathogenicity of Bacillus thuringiensis to some insects and other arthropods also depends on various chromosomal factors, including those belonging to the PlcR toxicity regulon (9, 10). In addition, several B. thuringiensis strains carry plasmid genes encoding insecticidal proteins other than Cry toxins. Notably, the presence of the vegetative insecticidal protein Vip3A toxin contributes significantly to the overall insecticidal activity of B. thuringiensis strains (11). This type of insecticidal toxin was first discovered in the culture supernatant of B. thuringiensis strain AB88, and its production was found from the vegetative growth phase to the end of the stationary phase and sporulation (12).

[0003] It has been shown that the vip3A gene is present in about 50% of the Bacillus thuringiensis strains tested and is located on a large plasmid that also carries the cry1I and cry2A genes (13, 14). Complete sequence analysis of the Bacillus thuringiensis strain Kustak HD1 showed that the vip3A gene is located on a large plasmid pBMB299, which also carries the cry1Aa, cry1Ia, cry2Aa and cry2Ab genes (15). The Vip3A toxin is specifically toxic to Lepidoptera insects belonging to the Noctuidae family (including important agricultural pests such as Spodoptera exigua and Spodoptera frugiperda), which are less sensitive to Cry1A and Cry2A toxins (16, 17). In addition, the specific receptors recognized by the Vip3A toxin in the insect intestine are different from those recognized by the Cry toxins (18-20). These properties mean that the vip3A gene is often used in combination with cry genes in plant transgenic pyramid strategies to improve plant resistance to pests, but also to circumvent the resistance of some insects to Cry toxins (21).

[0004] Surprisingly, the inventors found that the upstream region of vip3A was not sufficient to induce the expression of genes fused to this region in Bacillus thuringiensis strain Kustak HD73 Cry-. Then, the inventors have shown that in the HD73 strain containing pBMB299, the expression of Vip3A is controlled by a regulator whose gene is located on the same plasmid. This regulator gene, called vipR, is responsible for the transcription of the vip3A gene during the stationary phase. They also demonstrated that the transcription of vipR is positively self-regulated, and the determination of vipR and vip3A promoters determined the putative vipR target upstream of the vip3A and vipR promoter σA-specific-10 region. Surprisingly, this conserved sequence is also found upstream of cry1I and cry2 genes. Finally, they show that in the Δspo0A mutant that cannot initiate sporulation, the expression of vip3A and vipR is greatly increased. In summary, novel regulators involved in insect pathogenicity of Bacillus thuringiensis through a sporulation-independent pathway have been characterized.

[0005] The inventor has subsequently developed an expression system comprising a regulator VipR and a promoter PvipR thereof, a promoter regulated by VipR and a gene encoding a target protein. In this expression system, the promoter regulated by VipR allows the gene encoding the target protein to be transcribed. This expression system can be used to transform a bacillus strain, allowing it to produce a target protein.

[0006] The present invention relates to the use of the vipR regulator gene in a Bacillus strain having at least 90%, preferably 95% and more preferably at least 98% or 100% identity to the sequence SEQ ID N° 1 for directing the expression of at least one promoter comprising the following sequence:

[0007] 5'TTC-NNNN-AT-NGNN-GAA-NN-TAT-NTNTN-CTTT 3' (SEQ ID N°4), wherein N is A or T or C or G, and wherein the sequence of SEQ ID N°4 is positioned between 15 nucleotides and 21 nucleotides, more preferably 18 nucleotides upstream of the σA-specific-10 box before the start codon of the gene encoding the target protein.

[0008] According to a specific embodiment, the vipR regulator gene is used together with the promoter PvipR (SEQ ID N°5) that directs the expression of the VipR regulator. In such an embodiment, the vipR regulator gene and its promoter PvipR (SEQ ID N°5) can be combined in an expression system.

[0009] The σA-specificity-10 box has the following sequence: TNNNNT (N is C or A or G or T), preferably TATAAT or TATACT or TATGCT or TATCGT or TATCTT or TATATT or TATTAT or TATAGT or TATGAT, and more preferably TATAAT or TATACT.

[0010] Preferably, SEQ ID N°4 is the following sequence: 5'TTC-X1-X2-X3-X4-AT-X5-G-X6-X7-GAA-X9-X4-TAT-X8-T-X9-T-X8-CTTT 3' or more preferably 5'TTC-X1-X2-X3-X4-AT-X5-G-X6-X7-GAA-X6-X4-TAT-X8-T-X9-T-X8-CTTT 3', wherein X1 is A or T or C; X2 is C or T; X3 is C or G or T; X4 is A or T; X5 is A or C or G; X6 is A or G; X7 is T or G; X8 is G or C; X9 is A or C; in a preferred embodiment, the sequence is SEQ ID N°4: 5'X1-TTC-X2-X1-X3-X4-AT-X5-G-X6-X7-GAA-X9-X4-TAT-X8-T-X9-T-X8-CTTT-X4 3' or more preferably 5'

[0011] The transcriptional VipR regulator has at least 90%, preferably 95% and more preferably at least 98% or 100% identity to the sequence SEQ ID N°2.

[0012] The promoter regulated by the transcriptional VipR regulator can be selected from the group consisting of: PvipR (SEQ ID N°5), Pvip3 (SEQ ID N°15 or SEQ ID N°3), Pamidase-1 (SEQ ID N°6), Pamidase-2 (SEQ ID N°7), Pcry1I (SEQ ID N°8 or SEQ ID N°76), Pcry2Ab (SEQ ID N°9), Pamidase pBMB65 (SEQ ID N°72 or SEQ ID N°73), P amidase pBMB95 (SEQ ID N°65 or SEQ ID N°74) and P amidase pHT73 (SEQ ID N°66 or SEQ ID N°75), preferably, the promoter is selected from the group consisting of PvipR, Pvip3 and Pcry1I, and more preferably the promoter is Pvip3.

[0013] The term "target protein" means an endogenous protein or a protein not naturally expressed by the bacterial strain according to the invention, also referred to as a heterologous protein. Preferably, the target protein is a cytotoxic protein or an industrial target protein naturally expressed by a strain of the genus Bacillus, such as an enzyme (e.g., a protease, a lipase, an amylase), a hormone, an antigen (e.g., a protein that can be used as an immunogen, a polypeptide or for therapeutic use); thus, the target protein can be used in crop protection, vector control, commercial production of enzymes and the pharmaceutical industry, in particular for the production of vaccines.

[0014] In a specific embodiment, the transcriptional VipR regulator activates the expression of a gene encoding a target protein, which is selected from the group consisting of: Vip3A (SEQ ID N°10), Cry1I (SEQ ID N°11) and Cry2Ab (SEQID N°12), preferably the target protein is selected from the group consisting of Vip3A and Cry1I, and more preferably the target protein is Vip3A.

[0015] The transcriptional VipR regulon activates expression of the gene during stationary phase, at high levels for several hours when the bacteria are grown in a rich medium such as LB, and activation preferably occurs at the onset of stationary phase.

[0016] Preferably, the Bacillus strain is selected among Bacillus thuringiensis, Bacillus cereus, Bacillus weihenstephanensis, Bacillus subtilis, Bacillus megaterium, Bacillus brevis, and more preferably Bacillus thuringiensis.

[0017] Therefore, the present invention also relates to an expression system for the protein of interest. The expression system is understood to be a system comprising several components (sequences) which allows the regulator to activate the expression of the protein of interest.

[0018] The present invention also relates to an expression system of a target protein in a Bacillus strain, the expression system comprising:

[0019] - at least one polynucleotide sequence encoding the protein of interest;

[0020] - at least one first promoter comprising the following sequence:

[0021] 5'TTC-NNNN-AT-NGNN-GAA-NN-TAT-NTNTN-CTTT 3'(SEQ ID N°4),

[0022] Wherein, N is A or T or C or G, and

[0023] wherein the SEQ ID N°4 sequence is located between 15 nucleotides and 21 nucleotides, and more preferably 18 nucleotides upstream of the σA-specific-10 box before the start codon of the gene encoding the target protein;

[0024] allowing expression of the polynucleotide sequence encoding the protein of interest; and

[0025] - a vipR regulator gene having at least 90%, preferably at least 95% and more preferably at least 98% or 100% identity to SEQ ID N°1 directing the expression of said at least one first promoter, and a second promoter PvipR of SEQ ID N°5 directing the expression of said VipR regulator.

[0026] Preferred embodiments of the σA-specificity-10 cassette and the first promoter of SEQ ID N° 4 are as described above.

[0027] Preferably, the Bacillus strain is selected among Bacillus thuringiensis, Bacillus cereus, Bacillus weihenstephensis, Bacillus subtilis, Bacillus megaterium, Bacillus brevis, and more preferably Bacillus thuringiensis.

[0028] In a preferred embodiment, the target protein can be selected from the group consisting of Vip3A (SEQ ID N°10), Cry1I (SEQ ID N°11) and Cry2Ab (SEQ ID N°12), preferably the target protein is selected from the group consisting of Vip3A and Cry1I, and more preferably the target protein is Vip3A.

[0029] The first promoter can be selected from the group consisting of: Pvip3 (SEQ ID N°15 or SEQ ID N°3), Pamidase-1 (SEQ ID N°6), Pamidase-2 (SEQ ID N°7), Pcry1I (SEQ ID N°8 or SEQ ID N°76), Pcry2Ab (SEQ ID N°9), Pamidase p BMB65 (SEQ ID N°72 or SEQ ID N°73), P amidase pBMB95 (SEQ ID N°65 or SEQ ID N°74) and P amidase p HT73 (SEQ ID N°66 or SEQ ID N°75), preferably the promoter is selected from the group consisting of Pvip3 and Pcry1I and more preferably the promoter is Pvip3.

[0030] Preferably, the expression system of the target protein further comprises:

[0031] - a stabilizing sequence of mRNA, which is located downstream of the promoter and upstream of the gene sequence encoding the protein of interest; preferably, it is the STAB-SD of the sequence SEQ ID N°13; preferably, the STAB-SD sequence is located downstream of transcription +1 and upstream of the ribosome binding site at a position comprising between about 100 nucleotides and 500 nucleotides, preferably between 100 nucleotides and 300 nucleotides, more preferably between 100 nucleotides and 150 nucleotides;

[0032] The terminator sequence of the -cry1Ac gene (named Tcry1Ac) has at least 90% identity with SEQ ID N°14; this sequence is located downstream of the gene sequence encoding the target protein.

[0033] However, the choice of these sequences should not be considered limiting, as one skilled in the art will be able to replace these sequences with functionally equivalent sequences.

[0034] In a specific embodiment, the expression system of the target protein comprises:

[0035] (i) at least one polynucleotide sequence encoding a target protein; the target protein may be selected from the group consisting of Vip3A, Cry1I and Cry2Ab, preferably Vip3A and Cry1I, and more preferably Vip3A;

[0036] (ii) at least a first promoter comprising the following sequence:

[0037] 5'TTC-NNNN-AT-NGNN-GAA-NN-TAT-NTNTN-CTTT 3' (SEQ ID N°4), wherein N is A or T or C or G; and

[0038] wherein the SEQ ID N°4 sequence is located between 15 nucleotides and 21 nucleotides, and more preferably 18 nucleotides upstream of the σA-specific-10 box before the start codon of the gene encoding the target protein;

[0039] The first promoter can be selected from the group consisting of: Pvip3, Pamidase-1, Pamidase-2, Pcry1I, Pcry2Ab, Pamidase pBMB65 , P-amidase pBMB95 and P-amidase pHT73 , preferably Pvip3 and Pcry1I and more preferably Pvip3;

[0040] (iii) vipR regulator gene;

[0041] (iv) the second promoter PvipR;

[0042] (v) an optional mRNA stabilizing sequence (preferably STAB-SD) and / or the terminator sequence Tcry1Ac of the cry1Ac gene.

[0043] or

[0044] (i) at least one polynucleotide sequence encoding a target protein; the target protein may be selected from the group consisting of Vip3A, Cry1I and Cry2Ab, preferably Vip3A and Cry1I, and more preferably Vip3A;

[0045] - at least one first promoter comprising the following sequence:

[0046] 5'TTC-NNNN-AT-NGNN-GAA-NN-TAT-NTNTN-CTTT 3' (SEQ ID N°4), wherein N is A or T or C or G; and

[0047] wherein the SEQ ID N°4 sequence is located between 15 nucleotides and 21 nucleotides, and more preferably 18 nucleotides upstream of the σA-specific-10 box before the start codon of the gene encoding the target protein;

[0048] The first promoter can be selected from the group consisting of: Pvip3, Pamidase-1, Pamidase-2, Pcry1I, Pcry2Ab, Pamidase pBMB65 , P-amidase pBMB95 and P-amidase pHT73 , preferably Pvip3 and Pcry1I and more preferably Pvip3;

[0049] (iii) vipR regulator gene;

[0050] (iv) the second promoter PvipR;

[0051] (v) STAB-SD sequence.

[0052] or

[0053] (i) at least one polynucleotide sequence encoding a target protein; the target protein may be selected from the group consisting of Vip3A, Cry1I and Cry2Ab, preferably Vip3A and Cry1I, and more preferably Vip3A;

[0054] - at least one first promoter comprising the following sequence:

[0055] 5'TTC-NNNN-AT-NGNN-GAA-NN-TAT-NTNTN-CTTT 3' (SEQ ID N°4), wherein N is A or T or C or G; and

[0056] wherein the SEQ ID N°4 sequence is located between 15 nucleotides and 21 nucleotides, and more preferably 18 nucleotides upstream of the σA-specific-10 box before the start codon of the gene encoding the target protein;

[0057] The first promoter can be selected from the group consisting of: Pvip3, Pamidase-1, Pamidase-2, Pcry1I, Pcry2Ab, Pamidase pBMB65 , P-amidase pBMB95 and P-amidase pHT73 , preferably Pvip3 and Pcry1I and more preferably Pvip3;

[0058] (iii) vipR regulator gene;

[0059] (iv) the second promoter PvipR;

[0060] (v) The terminator sequence Tcry1Ac of the cry1Ac gene.

[0061] or

[0062] (i) at least one polynucleotide sequence encoding a target protein; the target protein may be selected from the group consisting of Vip3A, Cry1I and Cry2Ab, preferably Vip3A and Cry1I, and more preferably Vip3A;

[0063] - at least one first promoter comprising the following sequence:

[0064] 5'TTC-NNNN-AT-NGNN-GAA-NN-TAT-NTNTN-CTTT 3' (SEQ ID N°4), wherein N is A or T or C or G; and

[0065] wherein the SEQ ID N°4 sequence is located between 15 nucleotides and 21 nucleotides, and more preferably 18 nucleotides upstream of the σA-specific-10 box before the start codon of the gene encoding the target protein;

[0066] The first promoter can be selected from the group consisting of: Pvip3, Pamidase-1, Pamidase-2, Pcry1I, Pcry2Ab, Pamidase pBMB65 , P-amidase pBMB95 and P-amidase pHT73 , preferably Pvip3 and Pcry1I and more preferably Pvip3;

[0067] (iii) vipR regulator gene;

[0068] (iv) the second promoter PvipR;

[0069] (v) STAB-SD sequence and the terminator sequence Tcry1Ac of the cry1Ac gene.

[0070] Expression system and components of the expression system for the protein of interest can be inserted into at least one vector, such as a plasmid.

[0071] Preferably, the plasmid exhibits high isolation stability (for example, the vector must be stained in the bacterial strain for at least 25 generations) and / or structural stability (the vector does not reassociate with the chromosomal DNA of the bacterium to which it is associated); it can be selected, for example, among the high copy number vectors pHT304, pHT315 and pHT370, or, for example, among the low copy number vectors pHT73 or pBMB299.

[0072] The several components of the expression system do not need to be carried by the same vector. Thus, the VipR regulator and its promoter can be located on a first vector, and the protein of interest regulated by VipR and the promoter can be located on a second vector.

[0073] In another embodiment, the expression system of the target protein can be used to produce at least two target proteins. Here, the components of the expression system can be located on the same vector or on different vectors again. Preferably, the expression system of the target protein includes sequences of several genes encoding cytotoxic proteins, and more preferably, the expression system includes a gene encoding Vip3A and a gene encoding at least one Cry toxin.

[0074] The vector according to the present invention can be introduced into the host bacteria according to the technology known to those skilled in the art; Specifically, the transformation of the host bacteria can be carried out by electroporation (Lereclus et al., 1989) or by heterologous conjugation (Tieu-Cuot et al., 1987). The expression system can also be introduced into the bacterial chromosome or on the retention plasmid by homologous recombination (Lereclus et al., 1992).

[0075] The present invention also relates to recombinant strains of Bacillus, preferably, the strains of Bacillus are non-spore-forming.

[0076] In order to inhibit the sporulation activity of strains of the genus Bacillus, the genes necessary for sporulation can be inactivated, such as the expression of the transcriptional regulator Spo0A responsible for the initiation of sporulation, or the genes involved in the expression of the σ sporulation factors SigE, SigF, SigH and SigK; preferably, the inactivated genes are spo0A, sigE, sigH or sigK. The inactivation of these genes can be obtained by interrupting or modifying the coding sequence or by deleting all or part of the gene. The deletion is obtained by double crossing over between adjacent regions upstream and downstream of the gene, using a plasmid with a temperature-sensitive replicon (e.g., plasmid pRN5101 (Lereclus et al., 1992) or pMAD (Arnaud et al., 2004)) and using the schemes described in these articles. The deletion of the spo0A gene (named as Δspo0A) has a very early effect when the bacteria enter the stationary phase, particularly preventing the bacteria from participating in the sporulation process (Lereclus et al., 1995). Deletion of the sigE gene (designated ΔsigE) had a late effect, which prevented the progression of sporulation (Bravo et al., 1996). In both cases, the Bacillus strains failed to sporulate, died and contained almost exclusively the protein of interest.

[0077] Preferably, the strain used is Bt Kustak HD73Δspo0A or Bt 407ΔsigE.

[0078] The target protein produced in the non-spore-forming Bacillus strain is encapsulated in the dead bacteria, facilitating the recovery of the protein.

[0079] According to another embodiment of the present invention, the Bacillus strain of the present invention does not express amidase 1 or amidase 2, for example by deleting the corresponding genes, or expressing an inactivated amidase 1 or amidase 2 protein.

[0080] The present invention also relates to a method for preparing a recombinant Bacillus strain, the method comprising the following steps:

[0081] a-Preparing a vector including an expression system;

[0082] b- Transforming the strain with the vector obtained in step (a).

[0083] The cultivation of the bacterial strain is carried out on a culture medium (medium) containing at least one nitrogen source and glucose at appropriate concentrations, preferably at a temperature between 25°C and 35°C, preferably at 30°C; for example, the culture medium is LB medium.

[0084] The present invention also relates to a method for producing a target protein, the method comprising the following steps:

[0085] a- preparing the bacterial strain according to the present invention;

[0086] b- cultivating said bacterial strain in stationary phase, preferably at a temperature ranging from 25 to 37°C, preferably from 30°C to 35°C; and

[0087] c-Optionally, purify the protein of interest.

[0088] Purification of the protein of interest can be performed by centrifugation; in addition, it can be performed using methods of size exclusion chromatography, ion exchange chromatography or even affinity chromatography.

[0089] According to one embodiment, the target protein produced is an insecticidal protein of Bacillus thuringiensis.

[0090] It is known that such proteins can be used as biological insecticides in formulations traditionally comprising insecticidal proteins in crystalline form and bacterial spores to control crop pests and disease vectors (such as mosquitoes). In particular, these may be proteins of the Cry family (crystalline proteins) or proteins of the Cyt family (cytolytic insecticidal toxins) or Vip3 proteins (toxins active against Lepidoptera), and more preferably proteins of the Cry family.

[0091] Therefore, the present invention relates to the use of the recombinant Bacillus strain expressing the insecticidal protein according to the present invention as a biological insecticide.

[0092] After their expression, the proteins are protected from degradation by the bacterial membrane constituting the envelope of the bacterial spherule. Furthermore, the non-spore-forming nature of the bacteria gives the invention the advantage of not spreading spores in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 -Vip3A in HD73 - The strain does not express.

[0094] A-Genetic organization of the region of the Bt HD1 pBMB299 plasmid containing the vip3A gene (NZ_CP004876.1). Asterisks indicate truncated genes containing nonsense mutations. Schematic representation of the ability of the screened DNA fragments to produce transcriptional activity.

[0095] B-HD73 - The (pHT-Pvip3) strain was isolated on LB X-galactose (50 μg / mL) plates and grown at 37°C for 24 h.

[0096] C-HD73 carrying pHT-Pvip3, pHT-Pvip3 med1, pHTPvip3med2 or pHT-Pvip3long - Strains were isolated on LB X-galactose (50 μg / mL) plates and grown at 37°C for 24 hours.

[0097] Figure 2 - Transplant pBMB299 to HD73 - Conjugative transfer of the strains allows the cells to produce Cry crystals.

[0098] Bt HD73 - Phase contrast microscopy images of SmR(pBMB299) sporulating cells. Cells were grown on HCT plates at 30°C for 4 days. Spores are refracted structures indicated by arrows. Crystals are indicated by circles.

[0099] Figure 3 -Expression of vip3A in Bacillus thuringiensis Kustak HD73 Cry strain.

[0100] Bacillus thuringiensis HD73 - Wild type (WT)- and HD73 -Western blot analysis of Vip3Aa produced in the SmR(pBMB299)-pBMB- strain. The strain was cultured in LB medium at 37°C and samples were collected 1 h (T1) and 4 h (T4) after entering the stationary phase. Supernatant and cell pellet proteins were prepared as described in M&M. 20 μg of protein were loaded into each well. 0.1 μg of purified Vip3Aa was used as a control group. C-control group, M-protein molecular weight marker.

[0101] Figure 4 - Characterization of the DNA region required for vip3A gene expression.

[0102] A-Schematic representation of genes located upstream and downstream of the vip3A ORF and the ability of the screened DNA fragments to generate transcriptional activity. Asterisks indicate genes containing nonsense mutations.

[0103] B- Bacillus thuringiensis HD73 carrying pHT-Pvip3, pHT-Pvip3med1, pHT-Pvip3med2 or pHT-Pvip3long plasmid - β-Galactosidase activity of strain Bacillus thuringiensis HD73 - The activity of the strains was determined when they were grown in LB at 37° C. Data are mean ± SEM, n=2.

[0104] Figure 5 - Characterization of the vip3A promoter elements.

[0105] Schematic diagram of the vip3A locus (SEQ ID N°50). The transcription start site is indicated by an arrow at position -403 relative to the vip3A start codon. Asterisks indicate genes containing nonsense mutations. The DNA sequence containing the vip3A promoter element is highlighted below. The TSS identified by RACE PCR is in bold. The DNA sequence corresponding to the assumed -10 box is in italics. The palindromic sequence predicted by the mFold software to form a hairpin structure is underlined. The RNA used for RACE-PCR was prepared from Bacillus thuringiensis HD1 cells grown in LB medium and collected at T2.

[0106] Figure 6 - Mutation of ViPR HTH.

[0107] Alignment of A-VipR (SEQ ID N°51) and MgaSpn (SEQ ID N°43) protein sequences. The sequence corresponding to the HTH domain of S. pyogenes Mga is boxed. Arrows point to the two amino acids selected for mutation.

[0108] B- 3D model of VipR structure modeled by Phyre2 web server. W113 and S114 AA are indicated in pink. M1 and N471 amino acids at the N- and C-termini are indicated.

[0109] Figure 7 -orf-HTH gene mutation disrupts Pvip3long transcriptional activity.

[0110] A - The codons specifying amino acids W113 and S114 were modified to each codon for alanine. The mutated bases are in bold.

[0111] B-HD73 with pHT-Pvip3long or pHT-Pvip3long-mut - β-Galactosidase activity of strains. Bacteria were grown in LB at 37°C. Time 0 corresponds to the bacteria entering stationary phase. Data are mean ± SEM, n = 2

[0112] Figure 8 - The vip3 promoter is activated by VipR.

[0113] A - Schematic diagram of the concept used to study the regulation of the vip3 gene. Asterisks indicate genes containing nonsense mutations.

[0114] B-Bacillus thuringiensis HD73 - β-Galactosidase activity of (pHT-Pvip3, pPxyl-vipR) cells grown in the absence (20 mM) and presence (20 mM) of xylose. Bacteria were cultured in LB at 37°C.

[0115] DNA sequence of C-vip3A promoter (SEQ ID N°52). The bases forming the palindromic sequence are underlined. The mutated bases are in bold (SEQ ID N°48).

[0116] D-Bacillus thuringiensis HD73 grown in the presence of xylose (20 mM) - (pHT-Pvip3, pPxyl-vipR) and HD73 - β-galactosidase activity of (pHT-Pvip3-mut, pPxyl-vipR) cells. Bacteria were cultured in LB at 30°C, time 0 corresponds to the bacteria entering the stationary phase. Xylose was added at T-1. Data are mean ± SEM, n = 2

[0117] Fig. 9 -VipR is a self-regulated transcriptional activator.

[0118] A - Schematic diagram of the concept used to study vipR gene regulation.

[0119] B- Bacillus thuringiensis HD73 grown in LB at 37°C - β-galactosidase activity of (pHT-PvipR) cells.

[0120] C-HD73 - (pHT-PvipR, pPxyl-vipR) and HD73 - β-Galactosidase activity of (pHT-PvipR, pPxyl) cells grown in the presence of xylose (20 mM) at 30°C. Time 0 corresponds to the entry of bacteria into stationary phase. Xylose was added at T-1. Data are mean ± SEM SEM, n=2.

[0121] Schematic diagram of the genetic makeup of D-vipR (SEQ ID N°53). The transcription start point is indicated by an arrow. P1 and P2 are located at positions -575 and -988 of the vip3A start codon, respectively. The DNA sequence containing the vipR promoter element is highlighted. The TSS identified using RACE-PCR is in bold. The DNA sequence corresponding to the putative -10 box is in italics. The palindrome in P1 is underlined. The RNA used for RACE-PCR was obtained from HD73 grown in LB medium. - pHT-Pvip3long cells were prepared and collected at T2.

[0122] Alignment of the E-vipR (SEQ ID N°55) and vip3A (SEQ ID N°54) promoter DNA sequences highlighted conserved palindromic sequences.

[0123] Fig.10 Northern blot analysis of -vipR transcription showed that HD73 - There are two transcripts in the (pHT-Pvip3long) strain.

[0124] 10 μg RNA was separated on 1% agarose gel and transferred overnight on a nylon membrane (GE Healthcare, #RPN303B) in SSC 10X buffer by capillary. RNA was then UV-crosslinked to the membrane using a Stratalinker device. According to the manufacturer's instructions, a membrane with a DNA digoxin (dig) labeled probe was generated and incubated using a high-efficiency digoxin DNA labeled primer (Dig-HighPrimeDNA) labeling and detection initiator kit II (Roche, #11585614910). The primers used to generate the DNA probe were NB-vipR-fw (GCATAAGTTCAATTATATGCGAATTG, SEQ ID N°41) and NB-vipR-fw (TTTCAGAAGATATTGTTTGGAATAAATGT, SEQ ID N°42). The membrane was washed twice in 2x SSC 0.1% SDS and once in 1x SSC 0.1% SDS at 65°C for 10 min. Immunoprecipitation was performed using the Chemidoc system (Bio-Rad) according to the kit instructions. The numbers on the left show the size (in bases) of the single-stranded RNA transcripts used in the RiboRuler high-end RNA ladder (ThermoScience)

[0125] Fig.11 - Alignment of conserved sequences found in the pBM299 plasmid.

[0126] The names of genes putatively controlled by VipR (SEQ ID N° 49, 56-64) are shown on the left. The distance between the putative -10-box and the last nucleotide of the conserved motif is indicated. The consensus is shown at the top in the form of sequence logos, where the height of the letters (in bits) is proportional to their frequency.

[0127] Fig.12 - Increased expression of vip3A in Bt HD73 Cry-Spo0A-.

[0128] A - Schematic representation of the concept used to study the regulation of vip3 and vipR genes in sporulation mutant strains.

[0129] B-Bacillus thuringiensis HD73 - (pHT-PvipR-vipR) and HD73 - β-Galactosidase activity of Spo0A-(pHT-PvipR-vipR) cells.

[0130] C-HD73 -(pHT-PvipR-vipR-Pvip3) cells and HD73 - β-Galactosidase activity of Spo0A-(pHT-PvipR-vipR-Pvip3) cells.

[0131] D-HD73 - (pHT-PvipR) and HD73 - β-Galactosidase activity of Spo0A-(pHT-PvipR) cells. Strains were grown in LB at 37°C. Time 0 corresponds to the bacteria entering stationary phase. Data are mean ± SEM, n = at least 2.

[0132] Fig.13 - Expression of the lacZ gene fused to three different promoters in the presence or absence of the vipR gene.

[0133] Colonies of Bacillus thuringiensis Kustak HD73 strain harboring a transcriptional fusion between the promoter of the cry1I or amidase gene and the lacZ gene in the presence (left) or absence (right) of the vipR gene. Bacteria were grown on LB medium (top and middle panels) or HCT-glucose-xylose containing X-galactose (bottom panel). Example

[0134] Materials and methods

[0135] Strains and plasmid construction.

[0136] In this study, Bacillus thuringiensis HD73 Cry-, belonging to serotype 3 (22), was used as the heterologous host and was named HD73 - . Escherichia coli DH5α was used as the host bacteria for plasmid construction. Vip3Aa protein was produced using Escherichia coli strain BL21λDE3 (Invitgen). Demethylated DNA (51) was prepared using Escherichia coli strain ET12567 before electroporation (52, 53) in Bacillus thuringiensis. The plasmids and bacterial strains used in the study are listed in Tables 1 and 2, respectively. Bacteria were routinely grown in LB medium at 37°C, and Bacillus thuringiensis cells were cultured at 30°C when indicated. Time 0 was defined as the beginning of the transition phase between the exponential phase and the stationary phase of Bacillus thuringiensis growth. The following concentrations of antibiotics were used for the selection of Bacillus thuringiensis: erythromycin 10 μg / mL, streptomycin 200 μg / mL, and kanamycin 200 μg / mL. Escherichia coli was screened with kanamycin 20 μg / mL and ampicillin 100 μg / mL. When necessary, 20 mM xylose was added to the culture.

[0137] Table 1: Plasmids used

[0138]

[0139]

[0140]

[0141] Table 2: Strains used

[0142]

[0143]

[0144] DNA manipulation.

[0145] Plasmids were extracted from E. coli cells by alkaline lysis using the Promega DNA extraction kit. DNA fragments were purified using the Promega gel and PCR clean-up system. Chromosomal DNA was extracted from exponentially growing Bacillus thuringiensis HD1 cells using the Qiagen Puregene Yeast / Bacteria kit. Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs and used according to the manufacturer's protocol. Primers used in the study (Table 3) were synthesized by Eurofins Genomics. PCR was performed using a 2720 thermal cycler (Applied Biosystems) or a master cycler Nexus X2 (Eppendorf). All constructs were verified by PCR and sequencing. Sequencing was performed by Eurofins Genomics.

[0146] Table 3: Primers used

[0147]

[0148]

[0149] Bioinformatics analysis.

[0150] The DNA sequences of vip3A plasmids of nine strains of Bacillus thuringiensis were compared by blast. Using Bacillus thuringiensis HD1pBMB299 plasmid (NZ_CP004876.1) as a reference, 17 genes (of which vip3A and three cry genes have the same coding orientation) were defined as insecticidal toxin islands. Each gene of the insecticidal toxin island of pBMB299 was compared with the corresponding gene on the vip3A plasmid of the following strains using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The structure of the protein (WP_000357137.1) encoded by orf-HTH was predicted using the online available Phyre2 software (27) and the HHpred server (54) for detecting structural homology. The intergenic region upstream of the vip3A coding sequence was analyzed for the presence of secondary structures in the nucleic acid sequence using the Mfold web server (28). The same sequence was also analyzed for the presence of potential Rho-independent transcriptional terminators using the ARNold web server (http: / / rssf.i2bc.paris-saclay.fr / toolbox / arnold / ).

[0151] Production and purification of Vip3A protein.

[0152] BL21 (vip3) strains were grown in LB medium supplemented with 20 μg / ml kanamycin at 37°C, with an OD600 nm of 0.6. Vip3A expression was induced by adding 1mM IPTG, and continued to grow for 4h at 37°C. Bacterial cells were collected by centrifugation and resuspended in 5% of the initial culture volume of lysis buffer (50mM Tris, 300mM NaCl, 7.5% glycerol, pH8.0). Bacteria were treated with 1mg / ml lysozyme on ice for 60min, and then solubilized by ultrasonic cells. The suspension was centrifuged at 5095×g for 10min to remove bacterial debris. The supernatant containing Vip3A protein was loaded onto 1ml of Ni-NTA agarose resin pre-equilibrated with lysis buffer. The resin of Vip3A-bound protein was continuously washed with 4mL lysis buffer containing 25- and 50-mM imidazole. Vip3A protein was eluted with 1.5 mL of lysis buffer containing 250- and 500-mM imidazole. To remove imidazole, the protein buffer was exchanged with PBS using a PD-10 column (Sigma). The purified protein was stored at -80°C before use.

[0153] Conjugative transfer of vip3A encoding plasmid pBMB299.

[0154] Bacillus thuringiensis HD1 (pHT1618K) was used as the pBMB299 plasmid donor strain, and streptomycin-resistant HD73 - SmR was used as the recipient strain. The donor and recipient strains were grown at 37°C until OD600nm 0.7. Then 5×10 6 cells in 2 ml of BHI broth. The filter support transferred the bacterial suspension onto a 0.45 μm membrane. The membrane was then placed on a BHI plate and incubated overnight at 37°C. The bacteria were collected by scraping and resuspended in physiological water. The suspension was then diluted and placed on an LB plate containing 200 μg / mL of kanamycin and 200 μg / mL of streptomycin to select for proconjugative bacteria. The presence of the pBMB299 plasmid in colonies receiving pHT1618K was confirmed by PCR using the primer pair vip3-fw / vip3-rev targeting the vip3A gene. Strain HD73 -SmR (pHT1618K, pBMB299) was finally solidified with pHT1618K as described (25). Briefly, the strain was grown on HCT medium at 30°C for 3 days until sporulation and spores were plated on LB medium containing 200 μg / mL streptomycin. Isolated colonies were screened for loss of kanamycin resistance on the plates. HD73 was confirmed by PCR using the primer pair vip3-fw / vip3-rev. - Presence of pBMB299 in the SmR KanS clone.

[0155] Western immunoblotting assay.

[0156] For Western blot experiments, Bacillus thuringiensis HD1 and HD73 - The SmR (pBMB299) strain was cultured in LB medium at 37°C with stirring. For each time point, 50 mL of culture was collected. Bacteria were separated from the growth medium by centrifugation at 5095 × g for 10 min. The bacterial cell pellet was resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, 7.5% glycerol, pH 8.0). It was then treated with 1 mg / mL of lysozyme on ice for 1 h and sonicated. Proteins in the culture medium were precipitated according to the following steps: 100 mM dithiothreitol was added to the suspension to prevent protein oxidation, then 19.62 g (NH4)2SO4 was slowly added to 45 ml of the sample to reach 70% saturation (0°C), and incubated overnight with slow stirring at 4°C. The precipitated protein was collected by centrifugation at 5095 × g for 10 min and resuspended in 200 μL lysis buffer. The protein concentration of the pellet and precipitate supernatant samples was determined using the Bradford method (55). 20 μg protein and 0.05 μg purified Vip3A from each sample were separated by SDS-PAGE on a 7.5% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue or subjected to Western blotting. For Western blotting, the proteins were electrotransferred to a PVDF membrane ( PVDF membrane, Bio-Rad). The membrane was blocked in 5% skim milk dissolved in TBS-T buffer (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 8.0) for 1 h and then treated with anti-Vip3Aa11 polyclonal antibody diluted 1:100,000 in 5% skim milk TBS-T buffer for 1 h at room temperature (56). The membrane was washed 3 times with 15 ml TBS-T buffer and then treated with goat anti-rabbit antibody (Invitgen G21234) diluted 1:20,000 for 1 h. The membrane was washed 3 times with TBS-T buffer and then developed using SuperSignalTM West PicoChemilLight substrate (Thermo Fisher Scientific) according to the manufacturer's instructions and imaged using the Chemidoc system (Bio-Rad).

[0157] β-Galactosidase assay.

[0158] β-galactosidase activity was monitored using qualitative and quantitative methods. For the qualitative test, cells were stained on LB plates containing 50 μg / mL X-galactose and incubated for a period of time at 37°C. The blue coloration reflects the activity of the β-galactosidase. For the quantitative method, the strains were pre-cultured in 10 ml of LB medium from the newly isolated strains on the plate until OD600nm 0.6-1.0. Then, the pre-culture was used to inoculate 50 ml of LB medium at OD600nm=0.005, and the bacteria were allowed to grow until harvested by centrifugation at the specified time point. Activity was measured as described above and expressed in units of per milligram of protein (57).

[0159] mRNA extraction and determination of vip3a and vipR transcription start sites.

[0160] RNA was extracted from Bacillus thuringiensis HD1 strain grown in LB medium at 37°C and harvested at T2. RNA samples were prepared as described (7) and stored at -80°C until use. RACE-PCR was performed using the 5'RACE system (Invitrogen, catalog number: 18374058) according to the instruction manual. cDNA was generated using specific primer GSP1. Subsequent PCR steps were performed using nested gene-specific GSP2 and GSP3 primers. PCR products were sequenced using GSP3 to determine the transcription start site of vip3A. To determine the vipR transcription start site, Bacillus thuringiensis HD73 grown in LB medium at 37°C was cultured in 4% CO2 and 1% CO2 was added. -RNA was extracted from the strain and harvested at T2. cDNA was generated using the specific primer vipR-GSP1. Subsequent PCR steps were performed using nested gene-specific vipR-GSP2 and vipR-GSP3 primers. The PCR product was sequenced using primer vipR-GSP3 to determine the P1 transcription start site of vipR, and the PCR product was sequenced using primer midvipR-GSP3 to determine P2.

[0161] result

[0162] Expression of the vip3A gene requires the presence of plasmid pBMB299.

[0163] To study the regulation of vip3A gene expression, the inventors used the Bacillus thuringiensis strain HD73 as a heterologous and natural host, which does not naturally carry the vip3A gene (22). - The Kustak strain was cured from the pHT73 plasmid carrying the cry1Ac gene (23). The transcriptional activity of the DNA fragment upstream of the vip3A gene of the pBMB299 plasmid (NZ_CP004876.1) of the Bacillus thuringiensis Kustak HD1 strain was analyzed. The 709 bp DNA fragment was fused to the lacZ gene in pHT304.18Z (24), and the resulting plasmid pHT-Pvip3 ( Figure 1 A) Convert to HD73 - HD73 was isolated on LB plates containing 50 μg / ml X-galactose. - (pHT-Pvip3) bacteria and observed the plates after 24 h of growth at 37°C. No blue colonies were observed, indicating that the bacterial cells did not produce β-galactosidase activity ( Figure 1 B) The results show that in HD73 - The strain lacked a specific regulator required for vip3A expression, which was present in the parental HD1 strain. The inventors hypothesized that this regulator was encoded by a gene in pBMB299. To prove this hypothesis, they transferred this plasmid into HD73 - SmR strain. Computer analysis of the genes encoded on pBMB299 revealed the presence of a putative conjugative transfer protein (TraG) and indicated that the plasmid was conjugative. They introduced pHT1618K into the HD1 strain by electroporation to select for conjugative events and thus increase the number of HD73 strains that obtained pHT1618K through the mobilization process. -The probability of finding a clone carrying pBMB299 among the SmR KMR exconjugants. In fact, it has been shown previously that the Gram-positive pBC16 replicon constituting pHT1618 is capable of being mobilized by conjugation between Bacillus thuringiensis strains (25). The received SmR KMR exconjugant clones of pHT1618K were screened for pBMB299 using the primer pair vip3-Fw / vip3-rev targeting the vip3A gene. 14% of the exconjugants contained pBMB299, and one clone, HD73, was selected. - SmR KMR (pBMB299, pHT1618K). Consistent with the transfer of pBMB299, the preconjugative clones produced bipyramidal crystals when grown on HCT plates at 30°C for 4 days ( Figure 2 ). The strain was then solidified by growing the bacteria on HCT plates until spores formed as described previously (26). Spores were then plated on LB plates and SmR KMS colonies that had lost pHT1618K were selected and the presence of pBMB299 was confirmed by PCR as above. The inventors then analyzed the production of Vip3A protein by Western blotting using anti-Vip3A11 antibodies. HD73 - SmR(pBMB299) and HD73 - The strain was cultured in LB at 37°C, and bacterial cells and culture supernatants were collected at T1 and T4. Anti-Vip3A antibody was used in HD73 - The samples produced by the SmR strain did not show Vip3A ( Figure 3 ). However, in HD73 - Vip3A protein was detected in both the supernatant and cell protein extract of the SmR(pBMB299) strain. These results indicate that the HD73 strain is able to produce and secrete toxins when carrying pBMB299, and that the plasmid itself is able to synthesize Vip3A specifically in strain HD73. Therefore, they believe that the regulator is encoded by the gene present on pBMB299.

[0164] Identification of genes involved in vip3A expression.

[0165] To determine the involvement of pBMB299 genes in vip3A expression, the inventors performed a bioinformatics analysis of plasmids carrying vip3A from different Bacillus thuringiensis strains (HD1, BGSC4C1, CT-43, HD12, HD29, IS5056, L7601, YBT-1520, YC-10). All of these were large plasmids, ranging in size from 267 to 299 kb. In the HD1 strain, vip3A was located in a genetic context containing multiple insertion sequences or transposase pseudogenes. Notably, vip3A was surrounded by two truncated DNA sequences, showing similarity to the tnpB gene of the IS1341 family. The IS232 mobile element and the gene encoding the protein were located in the vip3A ( Figure 4 A), the protein contains an N-terminal helix-turn-helix (HTH) domain, putatively involved in DNA binding and annotated as a transcriptional anti-terminator. The orf-HTH gene is present in the same position in all nine Bacillus thuringiensis strains studied, and the gene sequence shows 90%-100% identity between strains (not shown). This gene was previously identified by genomic analysis of Bacillus thuringiensis strain YBT1520 (15). Structural predictions of the protein encoded by the orf-HTH gene using Phyre2 software (27) and HHpred analysis showed that, although the sequence identity was low (17% and 19%, respectively), it was similar to the anthrax virulence regulator Atxa and the Streptococcus pneumoniae virulence regulator Mga. The presence of this HTH-containing protein gene near vip3A in all strains led us to study its role in transcriptional activation of the vip3A promoter (Pvip3). They constructed different transcriptional fusions with lacZ using DNA fragments of varying lengths that included Pvip3 and extended upstream to the end of the IS232 ATPase coding sequence, such as Figure 4 A. Plasmids pHT-Pvip3med1, pHT-Pvip3med2, and pHT-Pvip3long carrying these transcriptional fusions were transformed into strain HD73 - The obtained strain was inoculated on an LB plate containing X-galactose ( Figure 1 C) Only available on HD73 - β-galactosidase activity (blue) was detected in colonies of the (pHT-Pvip3long) strain, indicating that the DNA fragment containing the orf-HTH coding sequence is required for the production of β-galactosidase. These results were confirmed by measuring the expression kinetics of four transcriptional fusions, Pvip3-lacZ, Pvip3med1-lacZ, Pvip3med2-lacZ, and Pvip3long-lacZ ( Figure 4 B). Strain HD73 -(pHT-Pvip3), HD73 - (pHT-Pvip3med1) and HD73 - (pHT-Pvip3med2) produces very low amounts of β-galactosidase throughout bacterial growth ( Figure 4 B). In contrast, the strain carrying pHT-Pvip3long had higher β-galactosidase activity during vegetative growth and increased significantly 1 h after the start of stationary phase. These results suggest that the orf-HTH gene is involved in the expression of the vip3A gene.

[0166] Analysis of the vip3A promoter region.

[0167] The inventors determined the transcription start site (TSS) of the vip3A gene using total RNA samples harvested from HD1 cells at T2. The 5' end of the vip3A transcript (designated as the putative TSS) was identified 403 bp upstream of the vip3A start codon ( Figure 5 ). Before the start, there was a potential -10 box (TATAAT), but no canonical SigA-35 box was identified. Instead, analysis of the Pvip3 DNA sequence using mFold software (28) revealed a palindromic DNA sequence with the potential to form a stem-loop structure, ending 29 bp upstream of the TSS ( Figure 5 These data are consistent with those obtained by transcriptional fusion of Pvip3long-lacZ ( Figure 4 B) The obtained β-galactosidase activity strengthens the hypothesis that the protein encoded by the orf-HTH gene activates the Pvip3 promoter by binding to the stem-loop structure.

[0168] Characterization of regulators of vip3A expression.

[0169] Structural prediction of the protein encoded by the orf-HTH gene showed that it has similarities with the virulence regulator Mga of Streptococcus pneumoniae. Alanine substitution of two amino acids in the HTH domain of Mga abolished its DNA binding activity and regulation of its target genes (29). In order to prove that the protein (SEQ ID N°45) encoded by the orf-HTH gene (SEQ ID N°44) is responsible for the activation of the vip3A promoter, it was modified according to its alignment with the HTH domain of the protein Mga ( Figure 6 ). Two residues W113 and S114 of the potential HTH domain were replaced by two alanines ( Figure 7A) (SEQ ID N°46 and 47). A transcriptional fusion between the Pvip3long fragment containing the mutation and the lacZ gene was constructed in the pHT304.18Z plasmid, and the resulting pHT-Pvip3longmut plasmid was introduced into HD73 - strains to produce HD73 - (pHT-Pvip3long-mut) strain. Compare HD73 - (pHT-Pvip3long-mut) and HD73 - The β-galactosidase activity of the (pHT-Pvip3long) strain showed that the mutation of the two amino acids completely abolished the transcriptional activity of the Pvip3long fragment ( Figure 7 B). The results indicate that the protein encoded by the orf-HTH gene is involved in the activation of the Pvip3A promoter in the early stationary phase. This transcriptional activator was named VipR, i.e., VIP regulator. To confirm the activation of vip3A expression by VipR and to prove that the lacZ transcription produced by the Pvip3long-lacZ transcriptional fusion is from the promoter Pvip3, they inoculated HD73 containing the pPxyl-vipR plasmid. - pHT-Pvip3 was introduced into the strain. In this strain, vipR is expressed from the xylose-inducible promoter Pxyl( Figure 8 A) HD73 was obtained in the presence or absence of xylose in the culture. - The (pHT-Pvip3, pPxyl-vipR) strain was cultured in LB and the β-galactosidase activity of the cells was measured throughout growth from T-1 to T7. They observed that the addition of xylose to the culture significantly increased the β-galactosidase production in the stationary phase ( Figure 8 B). This indicates that the production of VipR activates lacZ transcription of Pvip3. To determine the location of the vip3A TSS ( Figure 5 ) is involved in the control of vip3A expression, and mutations were introduced to prevent the DNA sequence from forming a stem-loop structure ( Figure 5 C) A transcriptional fusion between the mutant Pvip3 promoter and lacZ was created in pHT304.18Z, and the resulting plasmid pHT-Pvip3-mut was transformed into strain HD73 - (pPxyl-vipR). For strain HD73 - (pPxyl-vipR, pHT-Pvip3) and HD73 - The β-galactosidase activity assay of the mutant promoter (pPxyl-vipR, pHT-Pvip3-mut) showed that the transcriptional activity of the mutant promoter was significantly lower than that of the wild-type promoter ( Figure 8 D). This indicates that the palindromic sequence is required for VipR to fully activate vip3A transcription.

[0170] The vipR gene is self-regulated.

[0171] The expression kinetics of vipR were studied using a 1581 bp DNA fragment (PvipR) that included the end of IS232 and the first 355 bp of the vipR coding sequence ( Fig. 9 A). The DNA fragment was transcribed and fused with lacZ in the pHT304.18Z plasmid, and the resulting pHT-PvipR plasmid was transferred to HD73 - In the bacterial growth cultured in LB medium, strain HD73 was measured - β-galactosidase activity of (pHT-PvipR) Fig. 9 B) The results show that the expression of PvipR is low during the exponential growth phase and increases 4-fold from T0 to T3. To determine whether the expression of vipR is self-regulated, the inventors introduced the plasmid pHT-PvipR into HD73 - (pPxyl) and HD73 - (pPxyl-vipR) strain and compared the β-galactosidase activities of the two strains ( Fig. 9 C) The results showed that when VipR was produced in bacterial cells under the control of Pxyl, the transcription of vipR increased significantly. Therefore, VipR exhibited the characteristics of a self-regulating transcriptional activator. Then, they used HD73 harvested at T2 - Total RNA samples of (pHT-Pvip3long) bacteria were used to determine the TSS of the vipR gene. The proximal vipR TSS named P1 was identified 575 bp upstream of the vipR start codon ( Fig. 9 D). There is a potential -10 box (TATCTT) before the start. As for vip3A, no canonical SigA-35 box was identified, but there was a 16 bp palindromic sequence 32 bp upstream of this TSS. Aligning this sequence with the palindromic sequence of the vip3A promoter allowed us to identify a conserved DNA sequence that could be a VipR binding site ( Fig. 9E). This indicates that P1 is a self-regulated vipR promoter. 5'-RACE PCR analysis of the vipR promoter showed that there is another TSS 998 bp upstream of the vipR start codon. This distal TSS, designated P2, is preceded by the canonical -10 box (TAAAGT). The putative SigA-35 box (TGTAAAA) is correctly located 17 bp upstream of the -10 box, indicating that vipR transcription from the P2 promoter is SigA-dependent. The recognition of two vipR promoters confirms the results of the Northern blot hybridization analysis of vipR transcription, which is shown in HD73. - Two transcripts were detected in the RNA sample of the strain (pHT-Pvip3long) Fig.10 ). In HD1 and HD73 - No discrete bands corresponding to the two transcripts were detected in the (pBMB299) RNA sample. However, a slight smear appeared at the same location. - The higher abundance of vipR transcripts in the (pHT-Pvip3long) strain may be due to the higher copy number of pHT-Pvip3long than pBMB299.

[0172] Identification of putative VipR regulon genes on plasmids of HD1 and HD73 strains.

[0173] The inventors searched for DNA sequences similar to the putative VipR binding site in plasmids pBMB299, pBMB65, and pBMB95 of HD1 strain and plasmid pHT73 of HD73 strain, and identified a total of 10 conserved sequences located in the 5' untranslated region of the coding sequence ( Fig.11 ). The relevance of these sequences as potential VipR binding sites was greatly strengthened by the presence of a putative SigA-10 box 17 bp downstream of the last nucleotide of the consensus sequence. This result strongly suggests that the transcription of these 10 genes is at least partially controlled by VipR. In addition to vip3A and vipR, the expression of the cry1I genes also seems to be controlled by VipR. This result is consistent with the observation that Cry1I toxins (formerly known as CryV) are produced and exported in the early stationary phase, like Vip3A (30). Even more surprisingly, the two cry2A genes (formerly known as cryB or cryII) are also located downstream of the putative VipR box. These genes are known to be transcribed by the sporulation-specific σ factor (31, 32), and their VipR-dependent expression means that Cry2A toxins are also produced before the sporulation process and, therefore, before the formation of parasporal crystal inclusions. Finally, the genes encoding N-acetylmuramoyl-L-alanine amidases (named amidase 1, amidase 2, and amidase II) were expressed in vitro and in vitro. pBMB65 , amidase pBMB95and amidase pHT73 ) are also located downstream of the putative VipR box ( Fig.11 ). The involvement of VipR in the activation of the three promoters was investigated using lacZ transcriptional fusions. In pHT304.18Z, a 381 bp DNA fragment upstream of the cry1I gene of pBMB299, a 199 bp DNA fragment upstream of the amidase gene present in the pBMB95 plasmid, and a 343 bp DNA fragment upstream of the amidase gene on pHT73 were transcriptionally fused with lacZ, and the resulting plasmids were transferred to HD73 - (pPxyl) and HD73 - The strains were plated on LB plates containing 50 μg / ml X-galactose or HCT-glucose plates containing 100 μg / ml X-galactose and 20 mM xylose, and the plates were observed after 24 h of growth at 37°C. β-galactosidase activity (blue) was only detected in HD73 carrying the pPxyl-vipR plasmid. - strain, and thus only detected when vipR was expressed in the cells ( Fig.13 ). This indicates that VipR is required for the expression of the cry1I gene and the two amidase genes.

[0174] In the Δspo0A genetic background, the expression of vip3A and vipR was significantly increased.

[0175] At the beginning of the stationary phase, the expression of vipR and vip3A is activated, allowing the inventors to study the role of key regulators in the transition phase in Bacillus Spo0A (33). The expression of vipR in spo0A mutants was studied using a 2644-bp DNA fragment (including the 5' untranslated region upstream of vipR and the vipR coding sequence). This DNA fragment was cloned upstream of the lacZ gene in pHT304.18Z, and the resulting plasmid pHT-PvipR-vipR ( Fig.12 A) Introduced to HD73 - and HD73 - The Δspo0A strain was used to compare the transcription of vipR in these two genetic backgrounds. The levels of β-galactosidase produced by the bacteria showed that in the presence of vipR, the transcription of vipR was increased 40-fold in the Δspo0A mutant ( Fig.12 B) To determine whether increased vipR transcription leads to increased vip3A expression, the inventors measured HD73 -transcriptional activity of Pvip3A in the Δspo0A mutant. In the Pvip3long DNA sequence, the vipR and vip3A orfs were in the same orientation, and no terminator sequence was found downstream of vipR. Therefore, they could not rule out the possibility that vipR transcription produces a polycistronic mRNA including vip3A. Therefore, in order to disconnect vipR transcription from Pvip3A transcriptional activity, they constructed a plasmid carrying a vipR expression cassette in the opposite orientation to the Pvip3A-lacZ transcriptional fusion. In the pHT304.18Z plasmid, a 2642-bp DNA fragment was reversely cloned upstream of the Pvip3 DNA fragment, which included the 5' untranslated region upstream of vipR and the vipR coding sequence ( Fig.12 A) The resulting plasmid pHT-PvipR-vipR-Pvip3 was introduced into HD73 - and HD73 - Δspo0A strain, and compared the β-galactosidase activity of the two strains ( Fig.12 C) They observed that, compared with HD73 - In comparison, HD73 - The expression of vip3 was significantly increased in the Δspo0A strain. Bioinformatic analysis of the DNA sequence upstream of the vipR coding sequence did not allow us to identify the DNA sequence corresponding to the Bacillus subtilis Spo0A cassette (34). However, to determine whether Spo0A controls the expression of vipR at the transcriptional level, the pHT-PvipR plasmid was introduced into HD73 - The Δspo0A strain was used to compare the β-galactosidase activity of this strain with that of HD73 - The results showed that in the absence of VipR, vipR transcription in the two strains was similar ( Fig.12 D), indicating that Spo0A does not affect the transcriptional activity of the distal promoter P2.

[0176] Sequence Listing:

[0177] SEQ ID N°1: vipR gene

[0178]

[0179] SEQ ID No. 2: VipR protein

[0180] MDKFITDLIQDKSIVRQLQILETLIDSNEIKSSKDLSQSLKCTSRTIINDISQLKLALPENWDLISVQSKGYLLKRDFSNDFSELIIPYLMNSELYTILIGIFNQKYYSLEKWSQLLYLDKITLKKMLKNFRKILVNFGLDFNFRTIKLIGQEINLRYFYIMFFYNIQKYKEVINLDSRLQEKIKSITRIHNVEIDYNMLAVIISVSIKRIANKHYMSEVLEFLPILDTNNLNCISSIICELEIFFNIKFTEYELSYFKNAFSIILEWNIEEKNRITDYYYKINKKTYDKNKHLFQTISSEINVCLEIKEKIKHDLYFILHKIYKFQKYGLSIGAFGNEFDTVHHEFSEGHNRIYPLISSWNKKINKSRLTNDEINYIIYHILFIVHSNHNKKGLLLLSGSSALKKFIYYKLNHELGDFVTLQQKPDCMHKFDVILTNYQIPNTPIPIIRISNKTIQKDSSYVRKVLSPFN

[0181] SEQ ID No. 3: Promoter Pvip3 (intergenic sequence)

[0182] GACTGTCCTTCTTATCTTACACGCTATAATGCGGATATTAACTCAATAAGCCATAAGGCTTCTAATCGCAAGGACTGCTGGGGTAGTTCAATAGCTCTGGTATAAATAGCAGTAAAATAACCTTTGGTACAATTCGATAGTTACTAGACGTAACGATTAGCCGAGAAATCCCCACTTTAGAGAGCACGTTAGGAGGTTAAGTGAAGGGTATTTCACAAATGATCGATTATATTTATAGGAAAAGGATAGATCACTTCATCTATAGATGAAATTATCTATCCTTTTATTTGTTAGATAGGAGA TATAATTTACTCATAGCCCCAGAGAAAGTGTTAACAGGTGATAGCATCCAATTTTATAGTAAGGGAGGTTTTACTAAGTTCTAATAATTTTCTGGGTAATTAGTCCTTACAACGACTTACATTTAGGAAATTGCCTGTTAATAAAATTTATATAGTAAAGGAGTGCTTTAATGTAAAAAAATAGGGATAAATGAATTTTTGTGAATAGGAAGACCATTTGATAGGTAAATATTATAATTCAAAAAGTAGAATAAGCAAAATTGAGTAACACATTAATGATATAAACTAATTTTATACAATTGAAATTGATAAAAAGTTATGAGTGTTTAATAATCAGTAATTACCAATAAAGAATTAAGAATACAAGTTTACAAGAAATAAGTGTTACAAAAAATAGCTGAAAAGGAAGATGAAC

[0183] SEQ ID No. 4: Promoter consensus sequence

[0184] TTC-N-N-N-N-AT-N-G-N-N-GAA-N-N-TAT-N-T-N-T-N-CTTT

[0185] where N is A or T or C or G

[0186] SEQ ID No. 5: Promoter PvipR

[0187] TTTTGTACATGCTTAAACAAGCGAAAATGTACATGTTTATCTTGACATTTACAAAGAGATGAATATATTTGAATTTTATTAATTAAATCCAGAAAAAAATATCAGAATAGAAAATGCTTTAAGGCCTATTTGCAGTTGAATATTTATGGTATGAATATTGCCGTATTTCTTAGTATGCTAAAAATACAAGCCTGTAAATCACTTTGTTATGACTG TAAAGTAAAAGAAGGATATTGAACTATTTTATTATTTATAAGGTATTATATATACTTTGAGATTCAGAAGGTTTGAATAAGTTACGCTTTATATGCCGATATTTTGGTTCAACTAGAGTATCAAATGTGGAATTTCTTTGCTATATAAAACCTTTACTTCTTAATCTTATATAAGCACAAAGTAAAACATAAGGGGATTTTTGAAAAAAGAGTATTATGGTCTAGTATAAAACTTGTATTTTAGAACTTTAAATATATTAAGATAATTCAAAGGGGAAAATAAAATCCAATAATCATTAAGATGGTAGTTTCTTTGGATTCGTTTCTTAAAATATCTATTTTTGATTTTCCTTATCTATTTTGTAATAAAACTTTTCATTAATAGATGAAAATATGTATGCTTTATTAGGAAATAATTAAGG TATCTTTTAATTATCTATAATTATTTGTTATAGAGGAATTAGATAATTGAAGTTCTTTTCATTTGGGAATACGGTGATAAAAAAGGAGGGACGGAGTTATAAGGATCAATAAGAATACATAATTAAATTGTTTTACAATAAAACATTCCATTCTCATTAATATTCTTATAATCACAAAAACCAAATGCTATAAAATGGATTGTTTGAAAAAGTTGTGAATATGCATATTTTTGGATGCGTGTACAATTATAATCATCATGATATCACTTCTCTTTTAAGAATGCGAAGTTTTGTCGTTATGTAAACATGGGTGCTCAAAACTTTACATTTTTTGTACAATATAAAATATTAAAATACAGTTGTACAGATAATTGATATTTCTTTATATGAAAAAATGCTTATAAATACTATGCAAAGCTAACGTATAATTTTTTAGACCTTATAGAATCAAATAAGAATAAAGATAGAAAATAAAATTGAAATACTAAGGCTAGCAAAGTAACGTGAGGTAATAGCATCTATTCTGAGTTTTGTAGTTAAATGTTACCTAGTGATAAGTAATTCAGTATTAAGGGAGGAGAAATGTA

[0188] SEQ ID No. 6: Promoter P amidase 1

[0189] CCTAAAATTTCAAGGCATTTTAAATGTTTTGTCTAGAAAAGTATGGATAACTTCATGAATGGGTGAAAATATCTATGCTTTTTTATTACAAGACAAAGATATACTGTACTCATAA

[0190] SEQ ID No. 7: Promoter P amidase 2

[0191] GCTTTTAAGAACCCCTATTTTAAATGTTTTGTCTAGAAAAGTATGGATAACTTCATGAATGGGTGAAAATATCTATGCTTTTTTATTACAAGACAAAGATATACTGTACTCATAA

[0192] SEQ ID No. 8: Promoter Pcry1I

[0193] TATTTTCTAGTAATACATATGTATAGAGCAACTTAATCAAGCAGAGATATTTTCACCTATCGATGAAAATATCTCTGCTTTTTCTTTTTTTATTTGGTATATGCTTTACTTGTAA

[0194] SEQ ID No. 9: Promoter Pcry2Ab

[0195] CACTTCTAAATGAAGTGAAAGTGGGGGTAGTTCAAAAAAAGCATAGATATCTTCTTCTATAGGTGAAGATATCTATGCTTTTTCTTTTTAAATTAAAGATATACTTTACTCATAC

[0196] SEQ ID No. 10: Vip3A protein

[0197] MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDEILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLDAINTMLRVYLPKITSMLSDVMKQNYALSLQIEYLSKQLQEISDKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDGSPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALKTASELITKENVKTSGSEVGNVYNFLIVLTALQAKAFLTLTTCRKLLGLADIDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVEAKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYG DMDKLLCPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEIDLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENSRLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLEPWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYTVKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTTGTDLKGVYLILKSQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLYQGGRGILKQNLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAKDVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK

[0198] SEQ ID N°11: cry1I protein

[0199] MKLKNQDKHQSFSSNAKVDKISTDSLKNETDIELQNINHEDCLKMSEYENVEPFVSASTIQTGIGIAGKILGTLGVPFAGQVASLYSFILGELWPKGKNQWEIFMEHVEEIINQKISTYARNKALTDLKGLGDALAVYHDSLESWVGNRNNTRARSVVKSQYIALELMFVQKLPSFAVSGEEVPLLPIYAQAANLHLLLLRDASIFGKEWGLSSSEISTFYNRQVERAGDYSDHCVKWYSTGLNNLRGTNAESWVRYNQFRRDMTLMVLDLVALFPSYDTQMYPIKTTAQLTREVYTDAIGTVHPHPSFTSTTWYNNNAPSFSAIEAAVVRNPHLLDFLEQVTIYSLLSRWSNTQYMNMWGGHKLEFRTIGGTLNISTQGSTNTSINPVTLPFTSRDVYRTESLAGLNLFLTQPVNGVPRVDFHWKFVTHPIASDNFYYPGYAGIGTQLQDSENELPPEATGQPNYESYSHRLSHIGLISASHVKALVYSWTHRSADRTNTIEPNSITQIPLVKAFNLSSGAAVVRGPGFTGGDILRRTNTGTFGDIRVNINPPFAQRYRVRIRYASTTDLQFHTSINGKAINQGNFSATMNRGEDLDYKTFRTVGFTTPFSFLDVQSTFTIGAWNFSSGNEVYIDRIEFVPVEVTYEAEYDFEKAQEKVTALFTSTNPRGLKTDVKDYHIDQVSNLVESLSDEFYLDEKRELFEIVKYAKQLHIERNM

[0200] SEQ ID No. 12: cry2Ab protein

[0201] MNSVLNSGRTTICDAYNVAAHDPFSFQHKSLDTVQKEWTEWKKNNHSLYLDPIVGTVASFLLKKVGSLVGKRILSELRNLIFPSGSTNLMQDILRETEKFLNQRLNTDTLARVNAELTGLQANVEEFNRQVDNFLNPNRNAVPLSITSSVNTMQQLFL NRLPQFQIQGYQLLLLPLFAQAANLHLSFIRDVILNADEWGISAATLRTYRDYLKNYTRDYSNYCINTYQSAFKGNLNTRLHDMLEFRTYMFLNVFEYVSIWSLFKYQSLLVSSGANLYASGSGPQQTQSFTSQDWPFLYSLFQVNSNYVLNGFSGARL SNTFPNIVGLPGSTTHALLAARVNYSGGISSGDIGASPFNQNFNCSTFLPPLLTPFVRSWLDSGSDREGVATVTNWQTESFETTLGLRSGAFTARGNSNYFPDYFIRNISGVPLVVRNEDLRRPLHYNEIRNIASPSGTPGGARAYMVSVHNRKNNI HAVHENGSMIHLAPNDYTGFTISPIHATQVNNQTRTFISEKFGNQGDSLRFEQNNTTARYTLRGNGNSYNLYLRVSSIGNSTIRVTINGRVYTATNVNTTTNNDGVNDNGARFSDINIGNVVASSNSDVPLDINVTLNSGTQFDLMNIMLVPTNISPLY

[0202] SEQ ID N°13: STAB SD

[0203] gaaaggaggg atgcc

[0204] SEQ ID N°14: Tcry

[0205] aaactcaggt ttaaatatcg ttttcaaatc aattgtccaa gagcagcatt acaaatagat

[0206] aagtaatttg ttgtaatgaa aaacggacat cacctccatt gaaacggagt gatgtccgtt

[0207] ttactatgtt attttctagt aatacatatg tatagagcaa cttaatcaag cagagatatt

[0208] SEQ ID N° 15: Promoter Pvip3

[0209] AAGGGTATTTCACAAATGATCGATTATATTTATAGGAAAAGGATAGATCACTTCATCTATAGATGAAATTATCTATCCTTTTATTTGTTAGATAGGAGATATAATTTACTCATAG

[0210] SEQ ID N° 72: Promoter P Amidase P bmb65

[0211] GCTTTTAAGAACCCCTATTTTAAATGTTTTGTCTAGAAAAGTATGGATAACTTCATGAATGGGTGAAAATATCTATGCTTTTTATTACAAGACAAAGATATACTGTACTCATAA

[0212] SEQ ID N° 73: Promoter P amidase pBMB65 (Intergenic sequence)

[0213] ACTTAGGACATTGAACTACTCACCACTTAACGTCCTTACAGACTTTTTGAAGTGGGAGATTCCTGCTAAGATAACCAATGTATCCACCGGTTATCTTAGCAGGCGCTACCCGTAGTCCCTACAGTGAGAACATAAGTATCTTGTACATTGACGCACCACACTCCCTACTTTAGTTTGGTTTTCGCAACTTCGGCAAGCATGGGGCGAAAGAACGTTGAAGATTTTACAGTTGCAACGTTTTTCTGAAACCAACCTCATAACTTCAGTTTTCGAAGAGCAATCTGTATAAGTAAATGATAGCAAATATGTTGGCTTATGCCTATCGATATTCATATCCCACCTAAAATTGGTGTTTCACACCTTGACATTTTTGAGGAAGGAGTCTTCTGTTGGAAAACGATAAAATCTTTTTAATTACTTGAATTCTAAGTGTAGAAACTTTGTAATTTGTATGAATTGAGGGATTGAATTCCTTGTGTTAATTAACATATATATCGATTTGATATAAAGATAGGATCTAAAAGGAGCTAACAAACTGTTAGCTCCTTTTAGATCCTTTTTAAGTATATCGCACAAAAATCAAATCATACATCACGAAAAGTAGCTTTTAAGAACCCCTATTTTAAATGTTTTGTCTAGAAAAGTATGGATAACTTCATGAATGGGTGAAAATATCTATGCTTTTTTATTACAAGACAAAGATATACTGTACTCATAAATGTAGAAATATCTATTTAAGAAAGATTGAAATCATATCCAATAATCTGATAGG

[0214] SEQ ID No. 74: Promoter P amidase pBMB95

[0215] CCTAAAATTTCAAGGCATTTTAGATGTTTTATGTGGAAAAGCATGGTTAACTTCATCAATAGGTGAAAATATCTATGCTTTTTTATTTCAGGACAGAGATATACTGTACTCATAA

[0216] SEQ ID N° 65: Promoter P amidase pBMB95 (Intergenic sequence)

[0217] ATCACGAAAAGAAGTTTTTAAGAACCATTCCTAAAATTTCAAGGCATTTTAGATGTTTTATGTGGAAAAGCATGGTTAACTTCATCAATAGGTGAAAATATCTATGCTTTTTATTTCAGGACAGAGATATACTGTACTCATAAATATAGAAATATGTATTTAAGAAAGATTGAAAGCGTATTAAATAATCTGATAGGA

[0218] SEQ ID N° 75: Promoter P amidase pHT73 GCTTTTAAGAACCCCTATTTTAAATGTTTTGTCTAGAAAAGTATGGATAACTTCATGAATGGGTGAAAATATCTATGCTTTTTATTACAAGACAAAGATATACTGTACTCATAA

[0219] SEQ ID N° 66: Promoter P amidase pHT73 (Intergenic sequence)

[0220] TGTAGAAACTTTGTAATTTGTATGAATTGAGGGATTGAATTCCTTGTGTTAATTAACATATATATCGATTTGATATAAAGATAGGATCTAAAAGGAGCTAACAAACTGTTAGCTCCTTTTAGATCCTTTTTAAGTATATCGCACAAAAATCAAATCATACATCACGAAAAG TAGCTTTTAAAGAACCCCTATTTTAAATGTTTTGTCTAGAAAAGTATGGATAACTTCATGAATGGGTGAAAATATCTATGCTTTTTATTACAAGACAAAGATATACTGTACTCATAAATGTAGAAATATCTATTTAAGAAAGATTGAAATCATATCCAATAATCTGATAGGA

[0221] SEQ ID N°76: Promoter Pcry1I (intergenic sequence)

[0222] GTGATGTCCGTTTTACTATGTTATTTTCTAGTAATACATATGTATAGAGCAACTTAATCAAGCAGAGATATTTTCACCTATCGATGAAAATATCTCTGCTTTTTCTTTTTTTATTTGGTATATGCTTTACTTGTAATCGAAAATAAAGCACTAATAAGAGTATTTATAGGTGTTTGAAGTTATTTCAGTTCATTTTTAAAGAAGGTTTAAAGACGTTAGAAAGTTATTAAGGAATAATATTTATTAGTAAATTCCACATATATTATATAATTAATTATGAAATATATGTATAAATTGAAAATGCTTTATTTGACATTACAGCTAAGTATAATTTTGTATGAATAAAATTATATCTGAAAATTAAATAATAGTATAAGTGGA

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Claims

1. Use of a vipR regulator gene having at least 90%, preferably at least 95% and more preferably at least 98% or 100% identity to the sequence SEQ ID N° 1 for directing the expression of at least one promoter comprising the following sequence: 5'TTC-NNNN-AT-NGNN-GAA-NN-TAT-NTNTN-CTTT 3'(SEQ ID N°4), in, N is A or T or C or G; and Therein, the sequence SEQ ID N°4 is located at least 15 to 21 nucleotides upstream of the σA-specific-10 box before the start codon of the gene encoding the target protein.

2. The use of the vipR regulator gene according to claim 1, wherein The promoter is selected from the group consisting of: PvipR (SEQ ID N°5), Pvip3 (SEQ ID N°15 or SEQ ID N°3), Pamidase-1 (SEQ ID N°6), Pamidase-2 (SEQ ID N°7), -Pcry1I (SEQ ID N°8 or SEQ ID N°76), Pcry2Ab (SEQ ID N°9), Pamidase pBMB65 (SEQ ID N°72 or SEQ ID N°73), P amidase pBMB95 (SEQ ID N°65 or SEQ ID N°74) and P amidase pHT73 (SEQ ID N°66 or SEQ ID N°75), preferably, the promoter is Pvip3 of SEQ ID N°3 and Pcry1I of SEQ ID N°8.

3. The use of the vipR regulator gene according to claim 1 or 2, wherein The target protein is selected from the group consisting of Vip3A and Cry1I. Preferably, the target protein is Vip3A.

4. An expression system for a target protein in a Bacillus strain, the expression system comprising: - at least one polynucleotide sequence encoding the protein of interest; - at least one first promoter comprising the following sequence: 5'TTC-NNNN-AT-NGNN-GAA-NN-TAT-NTNTN-CTTT 3'(SEQ ID N°4), wherein N is A or T or C or G; and wherein the sequence SEQ ID N°4 is located at least 14 nucleotides upstream of the σA-specific-10 box before the start codon of the gene encoding the target protein, and the sequence SEQ ID N°4 allows the expression of the polynucleotide sequence encoding the target protein; and - a vipR regulator gene having at least 90%, preferably at least 95% and more preferably at least 98% or 100% identity to SEQ ID N°1 directing the expression of said at least one first promoter, and a second promoter of SEQ ID N°5 directing the expression of said vipR regulator.

5. The expression system according to claim 4, wherein The first promoter is Pvip3 of SEQ ID N°3.

6. The expression system according to claim 4 or 5, wherein The target protein is selected from the group consisting of Vip3A and Cry1I. Preferably, the target protein is Vip3A.

7. The expression system according to any one of claims 4 to 6, wherein The expression system comprises a stabilizing sequence of the mRNA and / or a terminator sequence of the cry1Ac gene.

8. The expression system according to any one of claims 4 to 7, wherein The expression system is included in at least one vector.

9. The expression system according to claim 8, wherein The vector is a high copy number vector selected from pHT304, pHT315 and pHT370, or a low copy number vector selected from pHT73 or pBMB299.

10. A vector comprising the expression system according to any one of claims 4 to 9.

11. A recombinant strain of Bacillus, comprising the expression system according to any one of claims 4 to 9.

12. The recombinant strain according to claim 11, wherein The recombinant strain is a strain of Bacillus thuringiensis.

13. The recombinant strain of Bacillus according to claim 11 or 12, wherein The strain is a non-spore-forming strain. 14 . The recombinant strain of Bacillus according to claim 11 , which expresses Vip3A and at least one Cry toxin.

15. Use of the recombinant strain of Bacillus according to claim 14 as a biological insecticide.

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