USE OF THE CPCR REGULATOR GENE FOR OBTAINING NEW RECOMBINANT STRAINS OF BACILLUS THURINGIENSIS WITH REDUCED SPORULATION CAPACITY

Abstract

The present disclosure relates to the use of a cpcR regulator gene, which directs the expression of promoters of genes encoding Cry proteins, for reducing the sporulation of a strain of Bacillus thuringiensis, new recombinant strains of Bacillus thuringiensis and uses thereof as biopesticide.

Claims

1. A method of reducing sporulation of a recombinant strain of Bacillus thuringiensis, comprising the expression of a cpcR regulator gene of the sequence SEQ ID NO: 1 in said Bacillus thuringiensis.

2. The method of claim 1, wherein said CpcR regulator directs the expression of promoters of genes encoding Cry and Cyt proteins, said promoters having the sequence SEQ ID NO: 2 as follows: TABLE-US-00004 X.sub.1TGAAX.sub.2AAAAX.sub.3X.sub.4X.sub.5X.sub.6CAX.sub.7X.sub.8AX.sub.9ATTTX.sub.10CX.sub.11TCX.sub.12X.sub.13X.sub.14X.sub.15X.sub.16T X.sub.17X.sub.18AX.sub.19ATGTX.sub.20X.sub.21TX.sub.22GX.sub.23TAX.sub.24AX.sub.25TX.sub.26X.sub.27X.sub.28X.sub.29AX.sub.30X.sub.31TX.sub.32 X.sub.33,with: X.sub.1= AorG;X.sub.2= CorT;X.sub.3= AorT;X.sub.4= A,Tor G;X.sub.5= A,CorT;X.sub.6= AorG;X.sub.7= CorT;X.sub.8= A orCX.sub.9= A,TorG;X.sub.10= AorC;X.sub.11= AorC; X.sub.12= A,CorT;X.sub.13= A,GorT;X.sub.14= AorC;X.sub.15= A,GorT;X.sub.16= AorG;X.sub.17= AorT;X.sub.18= A,Cor T;X.sub.19= CorT;X.sub.20= AorC;X.sub.21= A,CorG;X.sub.22= AorT;X.sub.23= CorT;X.sub.24= GorT;X.sub.25= CorT; X.sub.26= GorT;X.sub.27= AorG;X.sub.28= AorT;X.sub.29= A,G orT;X.sub.30= C,GorT;X.sub.31= AorG;X.sub.32= AorG; X.sub.33= CorT.

3. The method of claim 2, wherein the promoter is selected from the group consisting of: P.sub.32 of the sequence SEQ ID NO: 3, P.sub.41 of the sequence SEQ ID NO: 4, P.sub.35 of the sequence SEQ ID NO: 5, P.sub.45 of the sequence SEQ ID NO: 6.

4. The method of claim 1, wherein the genes encoding toxins are cry genes or cyt genes.

5. The method of claim 1, wherein the strain of Bacillus comprises cry1, cry2, cry3, cry4, cry5, cry6, cry8, cry9, cry11, cry14, cry21, cyt1 or cyt2 genes.

6. A recombinant strain of Bacillus thuringiensis comprising: at least one gene encoding Cry and/or Cyt toxins, at least one promoter having the sequence SEQ ID NO: 2, allowing the expression of said at least one gene encoding Cry and/or Cyt toxins, and a cpcR regulator gene of sequence SEQ ID NO: 1, which directs the expression of said at least one promoter.

7. The recombinant strain of Bacillus thuringiensis as claimed in claim 6, wherein the promoter is selected from the group consisting of: P.sub.32 of the sequence SEQ ID NO: 3, P.sub.41 of the sequence SEQ ID NO: 4, P.sub.35 of the sequence SEQ ID NO: 5, P.sub.45 of the sequence SEQ ID NO: 6.

8. The recombinant strain of Bacillus thuringiensis as claimed in claim 6, wherein the promoter is cloned on the same plasmid as the cpcR regulator gene.

9. The recombinant strain of Bacillus thuringiensis as claimed in claim 6, wherein the genes encoding toxins are cry genes or cyt genes.

10. The recombinant strain of Bacillus thuringiensis as claimed in claim 6, wherein the strain of Bacillus comprises cry1, cry2, cry3, cry4, cry5, cry6, cry8, cry9, cry11, cry14, cry21, cyt1 or cyt2 genes.

11. Use of a recombinant strain of Bacillus thuringiensis as claimed in claim 6 as biopesticide.

12. The use of the recombinant strain of Bacillus thuringiensis as claimed in claim 11 for protecting cultures.

13. The use of the recombinant strain of Bacillus thuringiensis as claimed in claim 11 to control vectors.

14. The use of the recombinant strain of Bacillus thuringiensis as claimed in claim 10 to control nematodes.

15. A method for obtaining a recombinant strain of Bacillus thuringiensis, comprising the steps of introducing in said strain both: (1) a genetic construction comprising at least one gene encoding a toxin under the control of a promoter having the sequence SEQ ID NO: 2, and (2) an expression system comprising the CpcR regulator of the sequence SEQ ID NO: 1.

16. The method for obtaining a recombinant strain of Bacillus thuringiensis, as claimed in claim 15, wherein the genetic construction as defined in step 1 of claim 15 and the expression system as defined in step 2 of claim 15 are on the same plasmid.

17. The use of the recombinant strain of Bacillus thuringiensis as claimed in claim 13, wherein the vectors transmit pathogens responsible for mammalian diseases.

18. The use of the recombinant strain of Bacillus thuringiensis as claimed in claim 17, wherein the vectors are mosquitos.

19. The use of the recombinant strain of Bacillus thuringiensis as claimed in claim 14, wherein the nematodes cause mammalian disease.

Description

[0071] In addition to the above features described, the invention further comprises other features which will emerge from the following description, which refers to examples illustrating the present invention, as well as to the appended figures.

[0072] FIG. 1 shows the construction of a Bacillus thuringiensis strain.

[0073] FIG. 2 shows the characterization of the LM1212 DNA fragment carrying the cpcR gene activating transcription from the P.sub.35 promoter.

[0074] FIG. 3 shows the expression of cry1Ab under the control of CpcR and P.sub.35 in a kurstaki strain. A) Plasmids pHT16-18P35-cry1Ab and pHT-1c. B) Phase-contrast microscopy of the strain kurstaki HD73 transformed with plasmids pHT16-18P35-cry1Ab and pHT-1c. The arrows indicate some crystal inclusions.

[0075] FIG. 4 shows the analysis of a Cry1 Ab toxin produced in a kurstaki strain under the control of CpcR and P.sub.35. A) SDS-Page. M corresponds to molecular weight markers. 1. 20 L of crude extract from colonies of the strain kurstaki HD73 (pHT16-18, pHT304). 2. 20 L of crude extract from colonies of the strain kurstaki HD73 (pHT16-18P35-cry1Ab, pHT304). 3. 20 L of crude extract from colonies of the strain kurstaki HD73 (pHT16-18P35-cry1Ab, pHT-1c). The crude extracts were prepared in the same way and the use of 20 L of crude extract means that the same amount of each preparation is loaded on the SDS-Page. B) Western blotting of lanes 1, 2 and 3 with antisera against Cry1Ab.

[0076] FIG. 5 shows the transcriptional analysis of the P35lacZ fusion. A. Detail of the constructs. The upper panel shows the DNA fragment 5a carrying the cpcR gene.

[0077] The two lines below the schematic representation of the cpcR locus indicates the DNA regions used to create fragment 5a. The middle panel highlighted in dark grey shows the organization of plasmid (pHT-5a-P35Z) and the lower panel highlighted in light grey shows the organization of plasmids (pHT1618-5a) and (pHT-P35Z). B. -galactosidase assay of strains HD (pHT-5a-P35Z) in dark grey lozenges and HD (pHT1618-5a) (pHT-P35Z) in light grey squares. The time indicated is relative to t0 which indicates the beginning of the transition between exponential and stationary phase.

Example 1: Construction of a Bacillus Thuringiensis Strain to Screen for the Regulator of the Expression from the P.SUB.35 .Promoter

[0078] The previous results obtained by the inventors suggested that, in the LM1212 strain, a regulation system limits the production of toxins to the subpopulation of non-sporulating cells. Thus, to screen the LM1212 genes responsible for the regulation of the P.sub.35 promoter, the promoter directing the expression of the cry gene, the inventors introduced a reporting system into a Bacillus thuringiensis strain, as described thereafter.

1) Materials and Methods

[0079] The Bt strain kurstaki HD73 Cry.sup. is chosen to clone the LM1212 genes responsible for the activation of the P.sub.35 promoter and for the negative effect on sporulation.

[0080] The thermosensitive plasmid pRN5101-AmyE-HD73::tet is first constructed following the same procedure as described in Verplaetse et al., 2015, except that the antibiotic resistance cassette confers resistance to tetracycline instead of spectinomycin and that the amyE flanking regions used for homologous recombination were amplified using Bt HD73 genomic DNA instead of Bt 407. A P.sub.35-lacZ transcriptional fusion is then cloned between the amy up and amy down fragments cloned from the kurstaki strain. The P.sub.35-lacZ fusion is then introduced at the amyE locus of the kurstaki HD73 chromosome by homologous recombination (FIG. 1).

2) Results

[0081] The resulting strain is designated HD73 Cry.sup. [amyE::P.sub.35-lacZ].

Example 2: Characterization of the LM1212 Genes Activating Transcription from the P.SUB.35 .Promoter

[0082] i. Localization of the Genes Responsible for the Expression from the P.sub.35 Promoter on the DNA of LM1212 Strain

1) Materials and Methods

[0083] The DNA fragment ORF28-ORF32 from plasmid PLM248 of strain LM1212 is cloned into plasmid pHT304 (Arantes, O. et al., 1991) and the resulting plasmid is called pHT-1a. pHT-1a is then introduced into the Bt strain HD73 Cry.sup. [amyE::P.sub.35-lacZ] and the recombinant clones are isolated on HCT plates (0.7% casein hydrolysate, 0.5% tryptone, 0.68% KH2 PO4, 0.012% MgSO4_7H2O, 0.00022% MnSO4_4H2O, 0.0014% ZnSO4_7H2O, 0.008% ferric ammonium citrate, 0.018% CaCl.sub.2_4H2O, 0.3% glucose, pH 7.2) (Lecadet et al., 1980; Verplaetse et al., 2016) containing yeast extract (0.05%), glucose 0.3% and X-Gal (100 g/mL).

2) Results

[0084] The parental strain gives white colonies. In sharp contrast, the strain harboring plasmid pHT-1a gives blue colonies (FIG. 2). This result indicates that the genes responsible for the expression from the P.sub.35 promoter are located on the DNA fragment 1a corresponding to LM1212 ORF28 to 32.

ii. Identification of the Genes Involved in the Transcriptional Activity

1) Materials and Methods

[0085] The DNA region ORF28 to 32 is sub-cloned into the pHT304 plasmid and the resulting plasmids are transformed into the Bt strain HD73 Cry.sup. [amyE::P.sub.35-lacZ](FIG. 2).

2) Results

[0086] It appears that the ORF28 encoding a transcriptional regulator is sufficient to activate P.sub.35-directed transcription. However, this activity is obtained if a short DNA fragment located just upstream of the ORF29 is fused upstream from ORF28 (Fragment 1e, FIG. 2). ORF28 encodes a transcriptional regulator belonging to the family of response regulators, suggesting that it should act as a two-component system in association with a histidine kinase. Such a kinase may be encoded by ORF32.

[0087] However, results show that ORF28 alone is able to activate the P.sub.35 promoter. In silico analysis of these genes (ORF28 and 32) indicates that ORF28 is unique and is not found in the available sequences of the bacteria of the B. cereus group (about 100 genomes).

[0088] In contrast, ORF32 is highly conserved among all the bacteria of the B. cereus group, including the LM1212.

[0089] Thus, ORF28 may function as a response regulator activated by the histidine kinase naturally present in the kurstaki HD73 strain.

iii. Effect of the Plasmid pHT-1c on the Sporulation of the Bacteria

1) Materials and Methods

[0090] pHT-1c, which corresponds to the plasmid pHT304 carrying the gene ORF28 and a promoter region, is introduced in Bt strain HD73 Cry.sup. [amyE::P.sub.35-lacZ].

2) Results

[0091] The introduction of pHT-1c in Bt strain HD73 Cry.sup. [amyE::P.sub.35-lacZ] negatively affects sporulation by a 10-fold factor (Table 1).

TABLE-US-00003 TABLE 1 Effect of the DNA fragment 1c carrying the cpcR gene on the sporulation of B. thuringiensis stain HD73. The results are the mean of two independent experiments. Heat-resistant Strains spores (CFU/ml).sup.a HD73 amyE::Pcry35-lacZ (pHT304) 5.25E+08 (5.25E+07) HD73 amyE::Pcry35-lacZ (pHT304-1c) 4.99E+07 (9.42E+06) .sup.aThe bacteria were grown in liquid HCT medium (containing Yeast Extract 0.05% and Glucose 0.3%) at 30 C. 48 h after inoculation, aliquots were heated at 80 C. for 12 mins. The cells were plated and CFUs corresponding to heat-resistant spores were then counted.
iv. Conclusion

[0092] These data indicate that the gene corresponding to ORF28 encodes the regulator (designated as CpcR) responsible for two functions: i) the activation of cry gene expression in the LM1212 strain, and ii) the reduction of the sporulation rate.

[0093] The results also suggest that ORF28 functions as a two-component system associated with a kinase existing in all the Bt strains. This gene is responsible for the specific phenotype of the LM1212 strain, which is to differentiate into crystal-producers or spore-formers.

Example 3: Use of the CpcR Regulator to Produce Cry1Ab in Non-Sporulating Bt Cells

[0094] In order to validate the use of CpcR in a Bt strain for reducing its sporulation while maintaining toxin production, the inventors produced recombinant strains of Bt and analyzed the sporulation rate of this recombinant strain and the production of the toxin Cry1Ab.

1) Materials and Methods

[0095] a) The cry1Ab gene, a typical sporulation-dependent cry gene is PCR amplified from strain kurstaki HD1 (the biopesticide kurstaki HD1 Dipel) and is cloned downstream from the P.sub.35 promoter into plasmid pHT16-18 (Lereclus et al., 1992). The resulting plasmid, pHT16-18 P35-cry1Ab (i.e. the plasmid pHT16-18 carrying the cry1Ab gene from the kurstaki HD1 Dipel strain under the control of the P.sub.35 promoter) is transformed into Bt strain HD73 Cry.sup.. The resulting strain was then transformed with the plasmid pHT-1c carrying the cpcR gene. The bacteria were plated on HCT agar plates for 48 h at 30 C. and examined in phase-contrast microscopy (FIG. 3).

[0096] b) The proteins produced by these recombinant strains are analyzed by western blotting with antisera against the Cry1Ab toxin (FIG. 4).

2) Results

[0097] a) The results show the production of typical crystal inclusion in non-sporulating cells of the kurstaki strain harboring plasmids pHT16-18P.sub.35-cry1Ab and pHT-1c. Moreover, the sporulation rate of the recombinant strain carrying pHT16-18P.sub.35-cry1Ab and pHT-1c is significantly lower than that of the wild-type strain.

[0098] b) The production of Cry1Ab is strongly increased in the kurstaki strain harboring the cpcR gene and the P.sub.35-cry1Ab fusion (Panels A and B, Lanes 3).

[0099] The faint band of Cry1Ab observed in lanes 2 results from a low CpcR-independent expression of cry1Ab.

[0100] These results show that the invention provides a new range of biopesticides by the way of production of insecticidal Cry toxins in non-sporulating Bt strains.

Example 4: Transcription of the P.SUB.35 .Promoter is Higher when Cloned on the Same Plasmid as CpcR

1) Materials and Methods

[0101] The DNA fragment designated 5a and comprised of ORF28, the intergenic region between ORF28 and ORF29 and the intergenic region between ORF29 and ORF30, was either cloned upstream from a transcriptional fusion between P.sub.35 and the reporter gene lacZ on vector pHT304.18 (which has the same skeleton as vector pHT304, but the cloning site is in the opposite orientation to that of vector pHT304), giving plasmid pHT-5a-P35Z, or alone on vector pHT1618, giving plasmid pHT1618-5a. The latter was transformed in Bt strain HD73 Cry.sup. along with pHT-P35Z, giving strain HD (pHT1618-5a) (pHT-P35Z). Plasmid pHT-5a-P35Z was also transformed in Bt strain HD73 Cry.sup., giving strain HD (pHT-5a-P35Z). The cells were grown in liquid HCT containing 0.05% yeast extract and 0.3% glucose. Samples were harvested and assayed for -galactosidase activity (Perchat et al., 2011).

2) Results

[0102] FIG. 5 shows that even though P35 is active in both strain HD (pHT1618-5a) (pHT-P35Z) (light grey squares) and HD (pHT-5a-P35Z) (dark grey lozenges), its activity is higher in strain HD (pHT-5a-P35Z), in particular it is 14-fold higher after 4 hours (1200 units versus 17000 units, respectively).

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