The Cry48Aa-Cry49Aa binary toxin from Bacillus sphaericus exhibits highly restricted target specificity

The Cry48Aa/Cry49Aa binary toxin of Bacillus sphaericus was recently discovered by its ability to kill Culex quinquefasciatus mosquito larvae through a novel interaction between its two components. We have investigated the target specificity of this toxin and show it to be non-toxic to coleopteran, lepidopteran and other dipteran insects, including closely related Aedes and Anopheles mosquitoes. This represents an unusually restricted target range for crystal toxins from either B. sphaericus or Bacillus thuringiensis. Gut extracts from Culex and Aedes larvae show differential processing of the Cry48Aa protein, with the location of cleavage sites in Culex reflecting those previously shown for the activation of Cry4 toxins in mosquitoes. Pre-activation of Cry48Aa/Cry49Aa with Culex extracts, however, fails to induce toxicity to Aedes larvae. Co-administration of Cry49Aa with Cry4Aa gives higher than predicted toxicity, perhaps suggesting weak synergism against Culex larvae between Cry49Aa and other three-domain Cry toxins.     

球形芽孢杆菌杀蚊毒素蛋白及其 遗传操作研究进展

蚊虫是多种人类传染疾病的主要传播媒介,如疟疾、丝虫病、乙型脑炎、黄热病和登革热等,对人类的健康造成了极大的危害［１］。控制蚊虫被认为是消除这些蚊媒疾病的有效途径。在过去的４５年里,尽管化学杀虫剂和各种抗病药物的使用对降低疟疾和蚊媒疾病的发病率和死亡率...     

Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae.

In the quest for effective control of mosquitoes, attention has turned increasingly to strains of the bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce potent toxins with specific mosquitocidal activities. However, sedimentation of the bacterial spores limits the duration of effective control after field application of these bacilli. We describe here the cloning of genes encoding the 51.4- and 41.9-kDa toxins from B. sphaericus 2297, the 100-kDa toxin from B. sphaericus SSII-1, and the 130-kDa toxin from B. thuringiensis subsp. israelensis into the broad-host-range plasmid pRK248 and the transfer of these genes for expression in Caulobacter crescentus CB15. The recombinant C. crescentus cells were shown to be toxic to mosquito larvae. Caulobacter species are ubiquitous microorganisms residing in the upper regions of aquatic environments and therefore provide the potential for prolonged control by maintaining mosquitocidal toxins in larval feeding zones.     

Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae.

In the quest for effective control of mosquitoes, attention has turned increasingly to strains of the bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce potent toxins with specific mosquitocidal activities. However, sedimentation of the bacterial spores limits the duration of effective control after field application of these bacilli. We describe here the cloning of genes encoding the 51.4- and 41.9-kDa toxins from B. sphaericus 2297, the 100-kDa toxin from B. sphaericus SSII-1, and the 130-kDa toxin from B. thuringiensis subsp. israelensis into the broad-host-range plasmid pRK248 and the transfer of these genes for expression in Caulobacter crescentus CB15. The recombinant C. crescentus cells were shown to be toxic to mosquito larvae. Caulobacter species are ubiquitous microorganisms residing in the upper regions of aquatic environments and therefore provide the potential for prolonged control by maintaining mosquitocidal toxins in larval feeding zones.     

Mtx toxins synergize Bacillus sphaericus and Cry11Aa against susceptible and insecticide-resistant Culex quinquefasciatus larvae

Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC(50)) of Mtx-1 and Mtx-2 of 0.246 and 4.13 microg/ml, respectively. The LC(50)s were 0.406 to 0.430 microg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression.

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3, 600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

Tightly bound binary toxin in the cell wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.     

Expression of mosquitocidal toxin genes in a gas-vacuolated strain of Ancylobacter aquaticus.

A series of plasmids bearing the binary toxin genes of Bacillus sphaericus 2297 or 2317.3, the 100-kDa toxin gene of B. sphaericus SSII-1, or the 130-kDa (cryIVB) toxin gene of Bacillus thuringiensis subsp. israelensis were constructed and introduced into Ancylobacter aquaticus by electroporation. The transformed A. aquaticus cells exhibited significant toxicity towards mosquito larvae, demonstrating a potential use of recombinant A. aquaticus for biological control of mosquitoes.     

The bacterium, Lysinibacillus sphaericus, as an insect pathogen

Since the first bacteria with insecticidal activity against mosquito larvae were reported in the 1960s, many have been described, with the most potent being isolates of Bacillus thuringiensis or Lysinibacillus sphaericus (formerly and best known as Bacillus sphaericus). Given environmental concerns over the use of broad spectrum synthetic chemical insecticides and the evolution of resistance to these, industry placed emphasis on the development of bacteria as alternative control agents. To date, numerous commercial formulations of B. thuringiensis subsp. israelensis (Bti) are available in many countries for control of nuisance and vector mosquitoes. Within the past few years, commercial formulations of L. sphaericus (Ls) have become available. Because Bti has been in use for more than 30 years, its properties are well know, more so than those of Ls. Thus, the purpose of this review is to summarise the most critical aspects of Ls and the various proteins that account for its insecticidal properties, especially the mosquitocidal activity of the most common isolates studied. Data are reviewed for the binary toxin, which accounts for the activity of sporulated cells, as well as for other toxins produced during vegetative growth, including sphaericolysin (active against cockroaches and caterpillars) and the different mosquitocidal Mtx and Cry toxins. Future studies of these could well lead to novel potent and environmentally compatible insecticidal products for controlling a range of insect pests and vectors of disease.     

Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control.     

Production of Cry11A and Cry11Ba toxins in Bacillus sphaericus confers toxicity towards Aedes aegypti and resistant Culex populations.

Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297.     

Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae

Larvae of Culex quinquefasciatus are much more susceptible to the toxin of Bacillus sphaericus than are larvae of Aedes aegypti. In the present study, the rate of ingestion, dissolution, and the cleavage by midgut proteases of the B. sphaericus toxin were compared in larvae of these species to determine whether these factors account for the differences in susceptibility. During filter feeding, larvae of both species removed significant quantities of B. sphaericus toxin from suspensions. Filtration rates for 1 hr, the time at which C. quinquefasciatus exhibited marked intoxication, were higher for A. aegypti (576-713 microliters/larva/hr) than for C. quinquefasciatus (446-544 microliters/larva/hr). Within 24 hr of exposure, A. aegypti larvae ingested 97-99% of the toxin particulates and suffered not more than 10% mortality in suspensions which induced complete mortality in C. quinquefasciatus within 2 hr of exposure. Quantification of the particulate toxin present in larvae after exposure to B. sphaericus suspensions revealed that larvae of both species contained only minor amounts of the toxin, suggesting the larvae had been able to solubilize the toxin after ingestion. Proteases recovered from the feces of larvae cleaved at 43-kDa protein isolated from B. sphaericus toxin extract to 40 kDa in both species. Thus, differences in susceptibility to the B. sphaericus toxin between A. aegypti and C. quinquefasciatus are not due to differences in rates of ingestion, dissolution, or the specificity of proteases.     

Expression of mosquito active toxin genes by a Colombian native strain of the gram-negative bacterium Asticcacaulis excentricus

Mosquito control with biological insecticides, such as Bacillus sp. toxins, has been used widely in many countries. However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect. In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column. Two strains of Asticcacaulis excentricus were chosen to introduce the B. sphaericus binary toxin gene and B. thuringiensis subsp. medellin cry11Bb gene cloned in suitable vectors. In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatus, Aedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium. A. excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B. sphaericus binary toxin was toxic to mosquito larvae. Crude protease A. excentricus extracts did not degrade the Cry11Bb toxin. The flotability studies indicated that the recombinant A. excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.     

A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria

A recombinant plasmid harboring cry4A, cry4B and cry11A from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus has been constructed. The three cry genes were placed under the control of the cry4B promoter whereas the binary toxin gene was controlled by its native promoter. The expression of toxins in Escherichia coli harboring the resulting plasmid, p4BDA-5142, was investigated. Cry4B expression was highest compared to other toxins. Although the level of toxin expression was low compared with E. coli expressing single toxins, the recombinant E. coli strain harboring p4BDA-5142 exhibited broad range mosquito-larvicidal activity against all Aedes, Culex and Anopheles larvae. This work has shown that the development of the recombinant plasmid can be used to broaden the host range spectrum of the appropriate bacterial host for mosquito control.     

Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin.

The operon containing the genes encoding the subunits of the binary crystal toxin of Bacillus sphaericus strain LP1-G, BinA and BinB (41.9 kDa and 51.4 kDa, respectively), was cloned and sequenced. Purified crystals were not toxic to Culex pipiens larvae. Comparison of the amino-acid sequences of this strain (Bin4) with those of the three other known toxin types (Bin1, Bin2 and Bin3) revealed mutations at six positions, including a serine at position 93 of BinA4, whereas all other types of BinA toxin from B. sphaericus had a leucine at this position. Reciprocal site-directed mutagenesis was performed to replace this serine in BinA4 from LP1-G with a leucine and the leucine in the BinA2 protein from strain 1593 with a serine. Native and mutated genes were cloned and overexpressed. Inclusion bodies were tested on C. pipiens larvae. Unlike the native Bin4 toxin, the mutated protein was toxic, and the reciprocal mutation in Bin2 led to a significant loss of toxicity. In vitro receptor-binding studies showed similar binding behaviour for native and mutated toxins. In the absence of any experimental data on the 3D structure of these proteins, sequence analysis and secondary-structure predictions were performed. Amino acid 93 of the BinA polypeptide probably belongs to an alpha helix that is sensitive to amino-acid modifications. Position 93 may be a key element in the formation of the BinA-BinB complex responsible for the toxicity and stability of B. sphaericus Bin toxins.     

Differential effects of Bacillus sphaericus strain 2362 on Culex quinquefasciatus and its competitor Culex cinereus in West Africa

The larval susceptibility to Bacillus sphaericus strain 2362 of the non-man-biting mosquito Culex cinereus and the urban filariasis vector Culex quinquefasciatus, two competitor mosquitoes in polluted habitats, was compared. In the laboratory, both species ingested a similar amount of B. sphaericus spores when fed c. 2 x 10(5) spores per ml for 30 min. However, in the same experiment, third-instar larvae of Cx quinquefasciatus were reduced by 98% at 24 h exposure while Cx cinereus larvae were only reduced by 6% at 72 h. In the field, preimaginal populations of Cx cinereus ingested, within a week, more than 99% of the applied spores, but showed no significant decrease through 14 days in cesspools treated at 10 g/m2 of a flowable concentrate of B. sphaericus 2362, containing 2 x 10(10) spores/g. It is proposed that specific biological control of Cx quinquefasciatus could result from appropriate treatment of breeding-sites with larvicidal B. sphaericus and competitive displacement by Cx cinereus or other mosquitoes with larvae that are more tolerant of B. sphaericus.     

Cloning, sequencing, and expression of a gene encoding a 100-kilodalton mosquitocidal toxin from Bacillus sphaericus SSII-1.

A cosmid library was prepared from a partial BamHI digest of total DNA from Bacillus sphaericus SSII-1. Two hundred fifty Escherichia coli clones were screened for toxicity against larvae of the mosquito Culex quinquefasciatus. One toxic clone, designated pKF2, was chosen for further study. Two toxic subclones, designated pXP33 and pXP34, obtained by ligating PstI-derived fragments of pKF2 into pUC18, contained the same 3.8-kb fragment, but in opposite orientations. Sequence analysis revealed the presence of an open reading frame corresponding to a 100-kDa protein and the 3' end of a further open reading frame having significant homology to open reading frames of transposons Tn501 and Tn21. The sequence of the SSII-1 toxin was compared with those of known toxins and was found to show regional homology to those of ADP-ribosyltransferase toxins. The distribution of the toxin gene among other B. sphaericus strains was examined.     

Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush-border membranes from mosquito larvae.

The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from Culex pipiens larvae midgut cells was demonstrated by an in vitro binding assay. Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B. sphaericus 1593 specifically bound to BBMF. Direct binding and homologous competition experiments indicated a single class of B. sphaericus toxin receptors, with a dissociation constant (Kd) of approximately 20 nM and a maximum binding capacity (Bmax) of approximately 7 pmol/mg BBMF protein. The sugars GalNAc, GlcNAc and N-acetyl neuraminic acid had no detectable inhibitory effect on toxin binding to C. pipiens BBMF. Binding experiments with the non-susceptible mosquito species Aedes aegypti failed to detect significant binding of B. sphaericus binary toxin to A. aegypti BBMF.