Identification and Characterization of 33 Bacillus cereus sensu lato Isolates from Agricultural Fields from Eleven Widely Distributed Countries by Whole Genome Sequencing.

The phylogeny, identification, and characterization of 33 B. cereus sensu lato isolates originating from 17 agricultural soils from 11 countries were analyzed on the basis of whole genome sequencing. Phylogenetic analyses revealed all isolates are divided into six groups, which follows the generally accepted phylogenetic division of B. cereus sensu lato isolates. Four different identification methods resulted in a variation in the identity of the isolates, as none of the isolates were identified as the same species by all four methods-only the recent identification method proposed directly reflected the phylogeny of the isolates. This points to the importance of describing the basis and method used for the identification. The presence and percent identity of the protein product of 19 genes potentially involved in pathogenicity divided the 33 isolates into groups corresponding to phylogenetic division of the isolates. This suggests that different pathotypes exist and that it is possible to differentiate between them by comparing the percent identity of proteins potentially involved in pathogenicity. This also reveals that a basic link between phylogeny and pathogenicity is likely to exist. The geographical distribution of the isolates is not random: they are distributed in relation to their division into the six phylogenetic groups, which again relates to different ecotypes with different temperature growth ranges. This means that we find it easier to analyze and understand the results obtained from the 33 B. cereus sensu lato isolates in a phylogenetic, patho-type and ecotype-oriented context, than in a context based on uncertain identification at the species level.

Towards novel Cry toxins with enhanced toxicity/broader: a new chimeric Cry4Ba / Cry1Ac toxin.

Attempts have been made to express or to merge different Cry proteins in order to enhance toxic effects against various insects. Cry1A proteins of Bacillus thuringiensis form a typical bipyramidal parasporal crystal and their protoxins contain a highly conserved C-terminal region. A chimerical gene, called cry(4Ba-1Ac), formed by a fusion of the N-terminus part of cry4Ba and the C-terminus part of cry1Ac, was constructed. Its transformation to an acrystalliferous B. thuringiensis strain showed that it was expressed as a chimerical protein of 116 kDa, assembled in spherical to amorphous parasporal crystals. The chimerical gene cry(4Ba-1Ac) was introduced in a B. thuringiensis kurstaki strain. In the generated crystals of the recombinant strain, the presence of Cry(4Ba-1Ac) was evidenced by MALDI-TOF. The recombinant strain showed an important increase of the toxicity against Culex pipiens larvae (LC50 = 0.84 mg l-1 ± 0.08) compared to the wild type strain through the synergistic activity of Cry2Aa with Cry(4Ba-1Ac). The enhancement of toxicity of B. thuringiensis kurstaki expressing Cry(4Ba-1Ac) compared to that expressing the native toxin Cry4Ba, might be related to its a typical crystallization properties. The developed fusion protein could serve as a potent toxin against different pests of mosquitoes and major crop plants.

Cry4Ba and Cyt1Aa proteins from Bacillus thuringiensis israelensis: Interactions and toxicity mechanism against Aedes aegypti.

Individual crystal proteins from Bacillus thuringiensis israelensis exhibit variable levels of insecticidal activities against mosquito larvae. In all cases, they are much less active compared to the whole crystal proteins due to described complex synergistic interactions among them. In the present study we investigated the effects of Cyt1A98 (a Cyt1Aa type protein) on Cry4BLB (a Cry4Ba type toxin) insecticidal activity toward the dengue vector Aedes aegypti. The bioassay analyses demonstrated the ability of Cyt1A98 protein to enhance Cry4BLB toxin larvicidal activity even at a low proportion in the mixture (1%). In vitro interaction assays showed that Cyt1A98 provides supplementary binding sites for Cry4BLB in A. aegypti BBMVs. Moreover, it enhances the formation of Cry4BLB oligomeric structure. These results support that Cyt1A98 protein could act as a membrane-bound receptor fixing Cry4BLB δ-endotoxins and promoting its oligomerization.

Effects of the P20 protein from Bacillus thuringiensis israelensis on insecticidal crystal protein Cry4Ba.

The accessory protein P20 from Bacillus thuringiensis israelensis has been defined as an important molecular chaperone for forming crystal Cyt1Aa, and enhancing Cry11Aa and Cry4Aa expression. To investigate its putative role in Cry4Ba delta-endotoxin production and toxicity, a p20 gene was cloned and introduced into B. thuringiensis recombinant strain expressing cry4Ba type gene (cry4BLB). The delta-endotoxin synthesis was enhanced by 262%. The generated inclusions were assayed against third instar larvae of Aedes aegypti. The combination of P20 protein with Cry4BLB delta-endotoxin led to a stable mortality rate of 25% with doses ranging from 0.2 mg l(-1) to 0.6 mg l(-1). Cry4BLB crystals produced in the presence of P20 were much less soluble than those produced by the control strain lacking P20 at pH lower than or equal to 10.5. The observed toxicity perturbation correlates with a decrease of Cry4BLB inclusions solubility. The presence of P20 protein has affected Cry4BLB crystallization and altered greatly its solubility properties. Cry4Ba effectiveness against A. aegypti larvae is related to the solubilization step in larval guts environment.

Characterisation of novel Bacillus thuringiensis isolates against Aedes aegypti (Diptera: Culicidae) and Ceratitis capitata (Diptera: Tephridae).

Bacillus thuringiensis is successfully used in pest management strategies as an eco-friendly bioinsecticide. Isolation and identification of new strains with a wide variety of target pests is an ever growing field. In this paper, new B. thuringiensis isolates were investigated to search for original strains active against diptera and able to produce novel toxins that could be used as an alternative for the commercial H14 strain. Biochemical and molecular characterization revealed a remarkable diversity among the studied strains. Using the PCR method, cry4C/Da1, cry30Ea, cry39A, cry40 and cry54 genes were detected in four isolates. Three strains, BLB355, BLB196 and BUPM109, showed feeble activities against Aedes aegypti larvae. Interestingly, spore-crystal mixtures of BLB361, BLB30 and BLB237 were found to be active against Ceratitis capitata with an LC50 value of about 65.375, 51.735 and 42.972 µg cm(-2), respectively. All the studied strains exhibited important mortality levels using culture supernatants against C. capitata larvae. This suggests that these strains produce a wide range of soluble factors active against C. capitata larvae.

New Bacillus thuringiensis toxin combinations for biological control of lepidopteran larvae.

Cyt1Aa from Bacillus thuringiensis israelensis is known by its synergistical activity with B. thuringiensis and Bacillus sphaericus toxins. It is able to improve dipteran specific toxins activity and can prevent or overcome larval resistance to those proteins. The objective of the current study was to investigate the possible improvement of larvicidal activity of B. thuringiensis kurstaki expressing heterogeneous proteins Cyt1A and P20. cyt1A98 and p20 genes encoding the cytolytic protein (Cyt1A98) and the accessory protein (P20), respectively, were introduced individually and in combination into B. thuringiensis kurstaki strain BNS3. Immunoblot analysis evidenced the expression of these genes in the recombinant strains and hinted that P20 acts as molecular chaperone protecting Cyt1A98 from proteolytic attack in BNS3. The toxicities of recombinant strains were studied and revealed that BNS3pHTp20 exhibited higher activity than that of the negative control (BNS3pHTBlue) toward Ephestia kuehniella, but not toward Spodoptera littoralis. When expressed in combination with P20, Cyt1A98 enhanced BNS3 activity against E. kuehniella and S. littoralis. Thus, Cyt1Aa protein could enhance lepidopteran Cry insecticidal activity and would prevent larval resistance to the most commercialized B. thuringiensis kurstaki toxins.

Evidence of the importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa protein cytolytic activity in Escherichia coli.

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix alpha C corresponding to a substitution of Met(115) by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.

Evidence of DNA rearrangements in the 128-kilobase pBtoxis plasmid of Bacillus thuringiensis israelensis.

BUPM97 is a novel Tunisian isolate of Bacillus thuringiensis israelensis presenting insecticidal activity against Culex pipiens larvae. The delta-endotoxins pattern of this strain was different from that of the reference strain B. thuringiensis israelensis H14. Therefore, the study of its cry genes content was carried out by restriction-fragment-length-polymorphism (RFLP) using specific cry genes probes and by DNA sequencing. It was clearly demonstrated that in the strain BUPM97 the cry4A and cry10A genes were deleted from the B. thuringiensis israelensis 128-kb pBtoxis plasmid. In addition, a strong DNA sequence polymorphism was evidenced in the same plasmid downstream from the cry4B gene. This very particular DNA dynamic evidenced in this new strain of B. thuringiensis israelensis should be taken into consideration, regarding the strain stability during the industrial production of B. thuringiensis bioinsecticides.

Characterization of a cry4Ba-type gene of Bacillus thuringiensis israelensis and evidence of the synergistic larvicidal activity of its encoded protein with Cry2A delta-endotoxin of B. thuringiensis kurstaki on Culex pipiens (common house mosquito).

A newly isolated strain of Bacillus thuringiensis, named BUPM97, was identified as affiliated to the israelensis subspecies. This strain was selected for its insecticidal activity against larvae of several dipteran insects, such as the common house mosquito (Culex pipiens). A 5 kb EcoRI fragment, containing a cry4Ba-type gene, named cry4BLB, was cloned from BUPM97. The sequencing of this gene revealed an open reading frame of 3411 bp encoding a protein of 1136 amino acid residues. Similarity analysis of both nucleotide and amino acid sequences revealed that cry4BLB presents several differences from the other previously described cry4Ba-type genes. Particular attention was paid to a two-amino-acid substitution located in domain III of the N-terminal moiety of this protein, which is very important for both toxicity and specificity of the toxin. The transfer of cry4BLB to an acrystalliferous B. thuringiensis kurstaki strain, HD1CryB, showed that it was expressed, resulting in the production of the typical parasporal crystal inclusions. On the other hand, its transfer to another B. thuringiensis kurstaki strain synthesizing Cry2A endotoxin known to be weakly toxic to the dipteran insect C. pipiens, resulted in the clear increase in the insecticidal activity of the transformed strain. Hence, in the present paper, we demonstrate for the first time that Cry2A and Cry4B act synergistically towards C. pipiens.