The present invention relates to a novel regulator VipR able to regulate the expression of genes, and more specifically the expression of vip3A. It also relates to an expression system allowing the expression of a protein of interest through the VipR regulation. This expression system can be used to transform Bacillus strains.
The Bacillus thuringiensis species includes a large number of Gram-positive spore-forming bacterial strains belonging to the Bacillus cereus group and distinguished by the production of parasporal crystal inclusions (1). Many strains of B. thuringiensis are entomopathogenic and their insecticidal properties are primarily due to the crystal proteins that consist of Cry and Cyt toxins, encoded by plasmid genes (2-4). The presence of cry genes is the common feature of all strains of the B. thuringiensis species, and the expression of most cry genes is dependent on the sporulation-specific factors Sigma E and Sigma K, which are functional in the mother cell compartment during sporulation. However, a few cry genes do not follow the same regulation pathway (5). They are also expressed in the mother cell or in a non-sporulating subpopulation during the sporulation process, but independently of the sporulation sigma factors. These are notably the cry3 genes encoding toxins active against coleopteran insects (6) and the cry genes of strain LM1212 which are expressed under the control of a specific transcriptional activator, CpcR, encoded by a plasmid gene (7). Different combinations of toxins can be found in the crystal inclusions depending on the B. thuringiensis strains. As an example, the commercial strain kurstaki HD1 contains 6 cry genes: cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa and cry2Ab (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). All these cry genes encode proteins active against lepidopteran larvae and confer a broad insecticidal spectrum within this insect order (8). However, the pathogenicity of B. thuringiensis towards some insects and other arthropods also depends on various chromosomal factors including those belonging to the PIcR virulence regulon (9, 10). Moreover, several B. thuringiensis strains harbor plasmid genes encoding insecticidal proteins other than the Cry toxins. This is notably the case of the Vegetative insecticidal protein Vip3A toxins which contribute significantly to the overall insecticidal activity of the B. thuringiensis strains (11). This type of insecticidal toxin was first discovered in the culture supernatant of the B. thuringiensis strain AB88 and its production was detected from the vegetative growth phase to the end of the stationary phase and sporulation (12).
It has been shown that the vip3A gene is present in about 50% of the B. thuringiensis strains tested and is located on large plasmids also harboring cry1I and cry2A genes (13, 14). Complete sequencing of the B. thuringiensis strain kurstaki HD1 indicates that the vip3A gene is located on the large plasmid pBMB299 which also carries the cry1Aa, cry1Ia, cry2Aa and cry2Ab genes (15). The Vip3A toxins are specifically toxic against lepidopteran insects belonging to the Noctuidae family, including important agricultural pests like Spodoptera exigua and Spodoptera frugiperda which are poorly susceptible to the Cry1A and Cry2A toxins (16, 17). Moreover, the specific receptors recognized by Vip3A toxins in the insect midgut are different from those recognized by the Cry toxins (18-20). These properties mean that vip3A genes are very often used in combination with cry genes in plant transgenesis pyramid strategies to increase plant resistance to insect pests but also to bypass the resistance acquired by certain insects to Cry toxins (21).
Surprisingly, the Inventors have discovered that the upstream region of vip3A is not sufficient to induce the expression of gene fused with this region in the strain B. thuringiensis strain kurstaki HD73 Cry−. Then, the Inventors have shown that the expression of Vip3A in the HD73 strain containing pBMB299 is controlled by a regulator whose gene is located on this same plasmid. This regulator gene called vipR is responsible for vip3A gene transcription during the stationary phase. They also demonstrated that vipR transcription is positively autoregulated and the determination of the vipR and vip3A promoters pinpointed a putative VipR target upstream from the Sigma A-specific −10 region of the vip3A and vipR promoters. Surprisingly, this conserved sequence was also found upstream of cry1I and cry2 genes. Finally, they showed that vip3A and vipR expression is drastically increased in a Δspo0A mutant unable to initiate sporulation. In conclusion, a novel regulator involved in the entomopathogenic potency of B. thuringiensis through a sporulation-independent pathway has been characterized.
The Inventors have then developed an expression system comprising the regulator VipR and its promoter PvipR, a promoter regulated by VipR and a gene coding for a protein of interest. In such expression system, the promoter regulated by VipR allows the transcription of the gene encoding for the protein of interest. This expression system may be used to transform Bacillus strains allowing them to produce proteins of interest.
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 with the sequence SEQ ID No1 for directing the expression of at least one promoter comprising the sequence:
5′ TTC-N-N-N-N-AT-N-G-N-N-GAA-N-N-TAT-N-T-N-T-N-CTTT 3′ (SEQ ID No4),
According to a specific embodiment, the vipR regulator gene is used together with the promoter PvipR (SEQ ID No5) which directs the expression of said VipR regulator. In such embodiment, the vipR regulator gene and its promoter PvipR (SEQ ID No5) may be combined in an expression system. The Sigma A-specific −10 box has the following sequence: TNNNNT with 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.
Preferably the SEQ ID No4 is the 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 No4: 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′ X2-TTC-X1-X2-X3-X4-AT-X5-G-X6-X7-GAA-X6-X4-TAT-X8-T-X9-T-X8-CTTT-X4 3′.
The transcriptional VipR regulator has at least 90%, preferably 95%, and more preferably at least 98% or 100% identity with the sequence SEQ ID No2.
The promoters regulated by transcriptional VipR regulator may be selected in the group consisting of PvipR (SEQ ID No5), Pvip3 (SEQ ID No15 or SEQ ID No3), Pamidase-1 (SEQ ID No6), Pamidase-2 (SEQ ID No7), Pcry1I (SEQ ID No8 or SEQ ID No76), Pcry2Ab (SEQ ID No9) PamidasepBMB65 (SEQ ID No72 or SEQ ID No73), PamidasepBMB95 (SEQ ID No65 or SEQ ID No74) and PamidasepHT73 (SEQ ID No66 or SEQ ID No75), preferably the promoters are selected in the group consisting of PvipR, Pvip3 and Pcry1I and more preferably the promoter is Pvip3.
The term “protein of interest” means an endogenous protein or a protein which is not naturally expressed by the bacterial strain according to the invention, also referred to as a heterologous protein. Preferably, the protein of interest is a cytotoxic protein naturally expressed by a strain of the Bacillus genus or a protein of industrial interest such as enzymes, such as proteases, lipases, amylases; hormones; antigens, for example, usable as immunogens, peptides or proteins for therapeutic use; the protein of interest can thus find application in the field of crop protection, vector control, the commercial production of enzymes and the pharmaceutical industry, in particular for the production of vaccines.
In a particular embodiment, the transcriptional VipR regulator activates expression of genes encoding proteins of interest selected in the group consisting of Vip3A (SEQ ID No10), Cry1I (SEQ ID No11) and Cry2Ab (SEQ ID No12), preferably the protein of interest is selected in the group consisting of Vip3A and Cry1I and more preferably the protein of interest is Vip3A.
The transcriptional VipR regulator activates the expression of genes during the stationary phase, continues for several hours at a high level when bacteria are grown in a rich medium such as LB and preferably the activation occurs at the onset of the stationary phase.
Preferably the Bacillus strain is chosen among Bacillus thuringiensis, Bacillus cereus, Bacillus weihenstephanensis, Bacillus subtilis, Bacillus megaterium, Bacillus brevis, and more preferably Bacillus thuringiensis.
The present invention thus also relates to an expression system of a protein of interest. This expression system is understood as a system comprising several components (sequences) allowing the activation of the expression of a protein of interest by a regulator.
The present invention further relates to an expression system of a protein of interest in a Bacillus strain comprising:
The Sigma A-specific −10 box and preferred embodiments of first promoter of SEQ ID No4 are as previously defined.
Preferably the Bacillus strain is chosen among Bacillus thuringiensis, Bacillus cereus, Bacillus weihenstephanensis, Bacillus subtilis, Bacillus megaterium, Bacillus brevis, and more preferably Bacillus thuringiensis.
In a preferred embodiment, the protein of interest may be selected in the group consisting of Vip3A (SEQ ID No10), Cry1I (SEQ ID No11) and Cry2Ab (SEQ ID No12), preferably the protein of interest is selected in the group consisting of Vip3A and Cry1I and more preferably the protein of interest is Vip3A.
The first promoter may be selected in the group consisting of Pvip3 (SEQ ID No15 or SEQ ID No3), Pamidase-1 (SEQ ID No6), Pamidase-2 (SEQ ID No7), Pcry1I (SEQ ID No8 or SEQ ID No76), Pcry2Ab (SEQ ID No9), PamidasepBMB65 (SEQ ID No72 or SEQ ID No73), PamidasepBMB95 (SEQ ID No65 or SEQ ID No74) and PamidasepHT73 (SEQ ID No66 or SEQ ID No75), preferably the promoters are selected in the group consisting of Pvip3 and Pcry1I and more preferably the promoter is Pvip3.
Preferably, the expression system of a protein of interest also comprises:
However the choice of these sequences should not be considered as limiting because person skilled in the art to can substitute these sequences with sequences with equivalent functions.
In specific embodiments, the expression system of a protein of interest comprises:
The components of the expression system and of the expression system of a protein of interest may be inserted into at least one vector such as a plasmid.
Preferably, the plasmids exhibit a high segregational stability (for example, the vector must stain in the bacterial strain for at least 25 generations) and/or a structural stability (the vector does not recombine with the chromosomal DNA of the bacteria into which it is incorporated); it may be chosen for example among a high copy number vector pHT304, pHT315 and pHT370 or a low copy number vector chosen for example among pHT73 or pBMB299.
The several components of the expression systems do not need to be harbored by the same vector. Accordingly, the VipR regulator and its promoter may be located on a first vector and the protein of interest and the promoter regulated by VipR may be located on a second vector.
In another embodiment, the expression system of a protein of interest may be used to produce at least two proteins of interest. Here again, the components of the expression system can be located on the same vector or on different vectors. Preferably, the expression system of a protein of interest comprises sequences of several genes encoding cytotoxic proteins, and more preferably this expression system comprises the gene encoding Vip3A and the genes encoding at least one Cry toxin.
The vectors according to the invention can be introduced into the host bacterium according to techniques known to those skilled in the art; in particular, the transformation of the host bacterium can be carried out by electroporation (Lereclus et al., 1989) or by heterogramic conjugation (Tieu-Cuot et al., 1987). The expression system can also be introduced on the bacterial chromosome or on a resident plasmid by homologous recombination (Lereclus et al., 1992).
The present invention further relates to a recombinant strain of Bacillus, preferably the strain of Bacillus is non sporulating.
In order to suppress the sporulation activity of a strain of the genus Bacillus, it is possible to inactivate gene essential to sporulation such as the genes involved in the expression of the transcriptional regulator Spo0A responsible for the initiation of sporulation or in expression of the sigma sporulation factors SigE, SigF, SigH and SigK; preferably, the inactivated gene is 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 the adjacent regions located upstream and downstream of the gene, using plasmids whose replication is heat-sensitive, for example the plasmids pRN5101 (Lereclus et al., 1992) or pMAD (Arnaud et al., 2004), and using the protocols described in these articles. The deletion of the spo0A gene (designated Δspo0A) has a very early effect, as soon as the bacteria enter stationary phase, in particular preventing the bacteria from engaging in the sporulation process (Lereclus et al., 1995). Deletion of the sigE gene (designated ΔsigE) has a later effect, blocking the progression of the sporulation process (Bravo et al., 1996). In both cases, the Bacillus strain can not sporulate, dies and contains almost only the protein of interest.
Preferably, the strain used is Bt kurstaki HD73 Δspo0A or Bt 407 ΔsigE.
The protein of interest produced in the non sporulating Bacillus strain are encapsulated in the dead bacteria, facilitating the recovery of this protein.
According to another embodiment of the invention, the Bacillus strain of the invention does not express amidase 1 or amidase 2 for example by deleting corresponding genes, or expresses inactive Amidase 1 or Amidase 2 proteins.
The present invention also relates to a method for preparing a recombinant strain of Bacillus comprising the steps of:
The culture of the bacterial strain is carried out on a culture medium containing at least one source of nitrogen and glucose in suitable concentrations at a temperature preferably between 25 and 35° C., preferably the temperature is about 30° C.; for example, the culture medium is LB medium.
The present invention also relates to a method for producing a protein of interest comprising the steps of:
Purification of the protein of interest can be achieved by centrifugation; in addition, the methods of exclusion chromatography, ion exchange chromatography or even affinity chromatography can be implemented.
According to one embodiment, the protein of interest produced is an insecticidal protein of B. thuringiensis.
It is known that such proteins can be used as a biopesticide in preparations conventionally containing the insecticidal protein in crystal form and bacterial spores, to control crop pests as well as disease vectors such as mosquitoes. In particular, they may be proteins of the Cry family (crystal proteins) or proteins of the Cyt family (cytolytic insecticidal toxins) or Vip3 proteins (toxins active against lepidopteran insects) and more preferentially the proteins of Vip3 proteins or of the Cry family.
The present invention thus relates to the use of a recombinant strain of Bacillus expressing insecticidal proteins according to the invention as a biopesticide.
After their expression, the proteins are protected from degradation by the bacterial membrane which constitutes the envelope of the bacterial sac. In addition, the non-sporulation of the bacterium gives the invention the advantage of not disseminating the spores in the environment.
The acrystalliferous strain B. thuringiensis HD73 Cry− belonging to serotype 3 (22) was used as a heterologous host throughout this study and was designated as HD73−. Escherichia coli strain DH5α was used as the host strain for plasmid construction. E. coli strain BL21 λDE3 (Invitrogen) was used to produce the Vip3Aa protein. E. coli strain ET12567 (51) was used to prepare demethylated DNA prior to electroporation in B. thuringiensis (52, 53). The plasmids and bacterial strains used in the study are listed in Table 1 and 2, respectively. Bacteria were routinely grown in LB medium at 37° C., and B. thuringiensis cells were cultured at 30° C. when indicated. Time 0 was defined as the beginning of the transition phase between the exponential and stationary phases of B. thuringiensis growth. The following concentrations of antibiotics were used for B. thuringiensis selection: erythromycin 10 μg/mL, streptomycin 200 μg/mL and kanamycin 200 μg/mL. For E. coli selection, kanamycin 20 μg/mL and ampicillin 100 μg/mL were used. When needed, 20 mM of xylose was added to the culture.
E. coli/B. thuringiensis high-copy number shuttle vector
B. thuringiensis kurstaki HD73 cured of the plasmid pHT73
B. thuringiensis kurstaki HD1 strain. The strain harbors the
mut)
E. coli BL21(DE3) strain harboring the pET28aΩvip3A plasmid,
Plasmids were extracted from E. coli cells by the alkaline lysis method using the Promega DNA Extraction Kit. DNA fragments were purified using Promega Gel and PCR Clean-Up System. Chromosomal DNA was extracted from exponentially growing B. thuringiensis HD1 cells using the Qiagen Puregene Yeast/Bacteria kit. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs and used following the manufacturer's protocol. The primers used in the study (Table 3) were synthesized by Eurofins Genomics. PCR was performed with a 2720 Thermal cycler (Applied Biosystems) or a Master cycler Nexus X2 (Eppendorf). All the constructs were verified by PCR and sequencing. Sequencing was performed by Eurofins genomics.
The DNA sequences of the vip3A plasmid of nine B. thuringiensis strains were compared using blast. 17 genes, among them the vip3A and 3 cry genes, all encoded in the same direction, were defined as an island of insecticidal toxins on the B. thuringiensis HD1 pBMB299 plasmid (NZ_CP004876.1) used as the reference. Each gene of the insecticidal toxins island of the pBMB299 was compared using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with the corresponding gene on the vip3A plasmid of the following strains: B. thuringiensis BGSC4C1 (CP015177), CT-43 (CP001910), HD12 (CP014853), HD29 (CP010091), IS5056 (CP004136), L7601 (CP020005), YBT-1520 (CP004861) and YC-10 (CP011350). The sequence of the orf-HTH-encoded protein (WP_000357137.1) was subjected to structural prediction using the Phyre2 software (27) available online and the HHpred server (54) that detects structural homologues. The intergenic region upstream from the vip3A coding sequence was analyzed for the presence of secondary structures in nucleic acid sequences using the Mfold web server (28). The same sequence was also analyzed for the presence of a potential Rho-independent transcription terminator using the ARNold web server (http://rssf.i2bc.paris-saclay.fr/toolbox/arnold/).
The BL21 (vip3) strain was grown in LB medium supplemented with 20 μg/ml of kanamycin at 37° C. to an OD600 nm of 0.6. The expression of vip3A was induced by adding 1 mM IPTG and growth was continued for 4 h at 37° C. Bacterial cells were collected by centrifugation and resuspended in 5% of the initial culture volume in the lysis buffer (50 mM Tris, 300 mM NaCl, 7.5% glycerol, pH8.0). The bacteria were treated with 1 mg/ml of lysozyme on ice for 60 min and then lysed by sonication. The suspension was centrifuged at 5095×g for 10 min to remove bacterial debris. The supernatant that contains the Vip3A protein was loaded onto 1 ml of Ni-NTA agarose resin previously equilibrated with the lysis buffer. The resin with bound Vip3A proteins was successively washed with 4 mL of lysis buffer containing 25- and 50-mM imidazole. The Vip3A protein was eluted with 1.5 mL of lysis buffer containing 250- and 500-mM imidazole. To remove the imidazole, the buffer of the protein was exchanged against PBS using a PD-10 column (Sigma). The purified protein was stored at −80° C. before use.
Conjugative Transfer of the vip3A-Encoding Plasmid pBMB299.
The B. thuringiensis HD1 (pHT1618K) strain was used as a pBMB299 plasmid donor strain, and the streptomycin resistant HD73− SmR was used as the recipient strain. The donor and recipient strains were grown in LB at 37° C. until OD600 nm 0.7. Then 5×106 cells of the donor and recipient strain were mixed in 2 ml BHI broth. The mixed bacteria were transferred on a 0.45 am membrane by passing the bacterial suspension through the Swinnex© filter holder. The membrane was then put onto a BHI plate and incubated at 37° C. overnight. The bacteria were collected by scrapping and resuspended in physiological water. The suspension was then diluted and plated on LB plates containing 200 μg/mL kanamycin and streptomycin 200 μg/mL to select the exconjugant bacteria. The presence of the pBMB299 plasmid in colonies that have received the pHT1618K was confirmed by PCR using the primer pair vip3-fw/vip3-rev targeting the vip3A gene. The strain HD73− SmR (pHT1618K, pBMB299) was finally cured of the pHT1618K as described (25). Briefly, the bacterial strain was grown on HCT medium for 3 days at 30° C. until sporulation and spores were plated on LB medium containing 200 μg/mL of streptomycin. Isolated colonies were screened for the loss of kanamycin resistance on plates. The presence of the pBMB299 in HD73-SmR KanS clones was confirmed by PCR using the primer pair vip3-fw/vip3-rev.
For western blot, B. thuringiensis HD1 and HD73− SmR (pBMB299) strains were grown in LB medium at 37° C. in agitated cultures. For each time point, a 50 mL volume 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 the lysis buffer (50 mM Tris, 300 mM NaCl, 7.5% glycerol, pH 8.0). The suspension was then treated with 1 mg/mL of lysozyme on ice for 1 h, and sonicated. The proteins of the culture medium were precipitated according to the following steps: 100 mM of dithiothreitol was added to the suspension to prevent protein oxidation, then 19.62 g of (NH4)2SO4 was slowly added to 45 ml of sample to reach 70% saturation (0° C.) and the suspension was incubated overnight with slow agitation at 4° C. The precipitated proteins were collected by centrifugation at 5095×g for 10 min, and resuspended in 200 μL of lysis buffer. The protein concentration of the pellets and of the precipitated supernatant samples was determined using the Bradford method (55). 20 μg of proteins of each sample and 0.05 μg of purified Vip3A were separated using SDS-PAGE on a 7.5% polyacrylamide gel. Gels were either stained with Coomassie brilliant blue or subjected to Western blot assays. For Western blot assays, the proteins were electro-transferred to a PVDF membrane (Immun-Blot© PVDF Membrane, Bio-Rad). The membrane was blocked for 1 h in 5% skimmed milk dissolved in TBS-T buffer (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 8.0), then treated with anti-Vip3Aa11 polyclonal antibodies (56) diluted 1:100 000 in 5% skimmed milk TBS-T buffer for 1 h at room temperature. The membrane was washed three times with 15 ml of TBS-T buffer, and then treated with 1:20 000 diluted Goat anti-rabbit antibodies (Invitrogen G21234) for 1 h. Membranes were washed three times with TBS-T buffer before being revealed using the SuperSignal™ West PicoChemiluminescent substrate (ThermoScientific) according to the manufacturer's instructions, and imaged using the Chemidoc system (Bio-Rad).
β-galactosidase assay.
The β-galactosidase activity was monitored using a qualitative and quantitative method. For the qualitative assay, cells were streaked on LB plates containing X-gal at a concentration of 50 μg/mL and incubated at 37° C. for the indicated periods of time. The blue coloration reflects the activity of the β-galactosidase. For the quantitative method, strains were precultured in 10 ml of LB medium until OD600 nm 0.6-1.0 from freshly isolated strains on plates. Then, the preculture was used to inoculate 50 ml of LB medium at an OD600 nm=0.005 and bacteria were allowed to grow until being harvested by centrifugation at the indicated time points. The activity was measured as previously described and expressed as units per milligram of protein (57).
mRNA Extraction and Determination of the vip3a and vipR Transcriptional Start Sites.
RNA was extracted from the B. thuringiensis HD1 strain grown at 37° C. in LB medium and harvested at T2. RNA samples were prepared as described (7) and stored at −80° C. before use. RACE-PCR was performed using the 5′ RACE System (Invitrogen, cat: 18374058) according to the instruction manual. The cDNA was generated using the specific primer GSP1. The subsequent PCR steps were realized with the nested gene-specific GSP2 and GSP3 primers. The transcription start site of vip3A was determined by sequencing the PCR product using GSP3. For vipR transcription start sites determination, RNA was extracted from the B. thuringiensis HD73− strain grown at 37° C. in LB medium and harvested at T2. The cDNA was generated using the specific primer vipR-GSP1. The subsequent PCR steps were realized with the nested gene-specific vipR-GSP2 and vipR-GSP3 primers. The P1 transcription start site of vipR was determined by sequencing the PCR product using the primer vipR-GSP3 and the P2 was identified by sequencing the PCR product using the primer midvipR-GSP3.
Expression of the vip3A Gene Requires the Presence of the Plasmid pBMB299.
To study the regulation of vip3A gene expression, the Inventors used the B. thuringiensis kurstaki HD73 strain as a heterologous and naive host which does not carry naturally the vip3A gene (22). Specifically, they have used a kurstaki strain designated HD73− which was cured of the plasmid pHT73 carrying the cry1Ac gene (23). The transcriptional activity of the DNA fragment located upstream from the vip3A gene present on the pBMB299 plasmid (NZ_CP004876.1) of the B. thuringiensis kurstaki HD1 strain was analyzed. This DNA fragment of 709 bp was fused to the lacZ gene in the pHT304.18Z (24) and the resulting plasmid pHT-Pvip3 (
Identification of a Gene Involved in vip3A Expression.
To identify the pBMB299 gene(s) involved in vip3A expression, the Inventors performed a bioinformatic analysis on the vip3A-bearing plasmids from different B. thuringiensis strains (HD1, BGSC4C1, CT-43, HD12, HD29, IS5056, L7601, YBT-1520, YC-10). All of those are large plasmids with sizes ranging from 267 to 299 kb. In the HD1 strain, vip3A is located in a genetic environment containing multiple insertion sequences or transposase pseudogenes. Notably, vip3A is surrounded by two truncated DNA sequences showing similarities with the tnpB gene of the IS1341 family. An IS232 mobile element and a gene encoding a protein containing an N-terminal helix-turn-helix (HTH) domain putatively involved in DNA binding and annotated as a transcriptional antiterminator, are located upstream from vip3A (
Analysis of the vip3A Promoter Region.
The Inventors determined the transcription start site (TSS) of the vip3A gene using total RNA samples from HD1 cells harvested at T2. The 5′ end of the vip3A transcript (designated as the putative TSS) was identified 403 bp upstream from the vip3A start codon (
Characterization of the of the Regulator of vip3A Expression.
The structure prediction of the protein encoded by the orf-HTH gene indicated a similarity with Mga, the virulence regulator of S. pneumonia. The alanine replacement of two amino acids in the HTH domain of Mga abolished its DNA-binding activity and regulation of its target genes (29). To demonstrate that the protein (SEQ ID No45) encoded by the orf-HTH gene (SEQ ID No44) is responsible for the activation of the vip3A promoter, its HTH domain was modified based on its alignment with the HTH domain of the protein Mga (
The vipR Gene is Autoregulated.
The expression kinetics of vipR was studied using a 1581 bp DNA fragment (PvipR) including the end of the IS232 and the first 355 bp of the vipR coding sequences (
The Inventors searched for DNA sequences showing similarities with the putative VipR binding site in the plasmids pBMB299, pBMB65 and pBMB95 of the HD1 strain and in the plasmid pHT73 of the HD73 strain and identified a total of 10 conserved sequences located in the 5′ untranslated region of coding sequences (
vip3A and vipR Expression is Strongly Increased in a Δspo0A Genetic Background.
The activation of vipR and vip3A expression at the onset of stationary phase led the Inventors to address the role of a key regulator of the transition phase in Bacilli, Spo0A (33). vipR expression in the spo0A mutant was studied using a 2644-bp DNA fragment that includes the 5′ untranslated region upstream from 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 (
GAAGGATATTGAACTATTTTATTATTTATAAGGTATTATATATAC
Genomic and transcriptomic insights into the efficient entomopathogenicity of Bacillus thuringiensis. Sci Rep 5:14129.
Number | Date | Country | Kind |
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22305443.8 | Apr 2022 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2023/058561 | 3/31/2023 | WO |