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Applied and Environmental Microbiology, July 2002, p. 3300-3307, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3300-3307.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Tightly Bound Binary Toxin in the Cell Wall of Bacillus sphaericus

Daniela Klein, Igor Uspensky, and Sergei Braun*

Department of Biological Chemistry, A. Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Received 26 December 2001/ Accepted 11 April 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
A number of Bacillus sphaericus strains are toxic to mosquito larvae, especially to Culex spp. and to some species of Anopheles and Mansonia. Formulations of B. sphaericus are used as commercial mosquito control agents in field operational programs.

It has been established early in the research of entomopathogenic B. sphaericus that the bulk of toxicity of the most-potent strains resides in the parasporal crystals packaged with bacterial spores. The crystal, which becomes visible in the cytoplasm at about stage III of sporulation (4, 40), contains two polypeptides, a 51-kDa protein (P51) and a 42-kDa protein (P42), which together form the binary toxin (7, 16). P51 is the primary component of binding to the Culex midgut epithelium, while P42 binds efficiently only in the presence of P51 but is responsible for the larvicidal action (13). Binding of the binary toxin to midgut epithelium causes swelling of mitochondrial and endoplasmic reticula and enlargement of vacuoles, followed by lysis of epithelial cells, midgut perforation, and the death of larvae (9, 11, 14). Neither subunit alone is toxic (3, 12, 26).

Some slightly toxic B. sphaericus strains such as SSII-1 express during the vegetative stage of growth mtx, mtx2, and mtx3 genes (21, 36, 38), which share sequence homology with the family of bacterial adenosyl-ribosylating toxins. Many potent B. sphaericus strains contain the mtx genes. Expression of the toxins Mtx and Mtx2 has been demonstrated in quite a few such strains, including B. sphaericus 2297 (37, 38).

Another larvicidal activity has been observed in cell wall preparations obtained from stationary cells of B. sphaericus 1593 (25). This activity has remained unexplored ever since, while research priority was given to the binary toxin.

Though B. sphaericus has a narrower range of host species than the main mosquito control agent Bacillus thuringiensis subsp. israelensis, it is able to persist in the environment for a longer time than B. thuringiensis subsp. israelensis, especially in waters polluted with organic materials (11, 15, 28). However, populations of Culex mosquitoes resistant to the binary toxin of B. sphaericus have been selected under laboratory conditions (18, 27, 31). Field resistance, as a consequence of vector control programs based on B. sphaericus application, has also been reported in some countries, such as France (G. Sinègre, M. Babinot, J.-M. Quermel, and B. Gaven, Prog. Abstr. VIIIth Eur. Meet. Soc. Vector Ecol., p. 17, 1994), India (30), and Brazil (33). At the same time, in spite of massive field usage of B. thuringiensis subsp. israelensis in mosquito, chironomid midge, and black fly control, no resistance has been detected in field populations of these dipterans. This event has been explained by the presence of a set of toxic proteins of a different nature that interact synergistically, increasing larvicidal activity of B. thuringiensis subsp. israelensis and suppressing development of resistance (24). In this context, the understanding of larvicidal activities of B. sphaericus other than the binary toxin, and especially of the enigmatic cell wall toxicity, could lead to a B. sphaericus larvicide of improved quality. It could also reduce or, perhaps, even eliminate the development of resistance. Below, we describe the results of our study of the cell wall toxicity in B. sphaericus.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Bacterial strains and genetic manipulations.
B. sphaericus strains were supplied by the Pasteur Institute, Paris, France. Escherichia coli XL1-Blue cells were used as recipient strains for subcloning experiments. Plasmid vectors pMK31, which carries the p42 toxin gene, and pCK2297-R, which carries the p51 binary toxin gene (16), were kindly provided by A. P. Porter, National University of Singapore; pBEST501, which carries the neomycin resistance cassette (19), was kindly provided by M. Itaya, Mitsubishi Casei Institute of Life Sciences, Tokyo, Japan.

Stock cultures of B. sphaericus were maintained on nutrient agar (Difco Laboratories, Detroit, Mich.). Synchronized cultures were obtained from starters transferred a few times from mid-logarithmic cultures in NYSM broth (25) grown at 28°C with high aeration. B. sphaericus 2297 was used as the recipient strain. The cells were transformed by electroporation using the procedure of Taylor and Burke (35), except that the cells were incubated at 37°C with shaking for 90 to 120 min prior to plating into selective medium containing neomycin (10 µg/ml). B. sphaericus recombinant cells were grown in Luria-Bertani (LB) broth (23) containing neomycin (5 µg/ml). E. coli cells were transformed either by electroporation or by heat shock and selected on LB agar medium containing ampicillin (100 µg/ml).

Plasmid pBB544 was constructed by inserting a 1.3-kb NotI-PstI fragment (neo gene from pBEST501) at the AflIII site of pBluescript SK vector. The integrative plasmid pBB544-trP42A for a single crossing over was constructed by inserting an internal 908-bp fragment of the p42 gene from B. sphaericus 2297 as follows. A 908-bp AccI-NcoI fragment of the p42 gene was excised from plasmid pMK31. This fragment was blunt ended with the Klenow fragment of DNA polymerase I and inserted into pBB544 digested with EcoRV, yielding pBB544-trP42A.

Analytic procedures.
Washed cells were either passed once through a French press cell at a pressure of 14,000 lb/in2 or broken using a sonicator (W-385; Heat Systems Ultrasonic, Inc.) operated continuously for 1 min at a power setting of 50 and an output of 3. Cell wall fractions were obtained by centrifugation of cell homogenates. For some experiments, cells or cell walls were suspended in Tris-HCl buffer (50 mM, pH 7.5) containing urea (7 M) and incubated for 2 h at room temperature with continuous mixing. The pellet was collected by centrifugation, and the extraction was repeated. Twice-extracted residues were washed several times in deionized water and freeze-dried.

Enzymatic treatment of dried spore-crystal complex (50 mg/ml) or of the urea-treated cell wall (50 mg/ml) was carried out as follows. Preparations were suspended in a suitable buffer and incubated with various enzymes (1 mg/ml) while under stirring for 18 h at 30°C. At the end of incubation, all samples were centrifuged, and the pellet was washed with water and freeze-dried. For the treatment with serine proteases, cells were suspended in Tris-HCl buffer (50 mM, pH 7.8) containing CaCl2 (1 mM). During treatment with lysozyme, phosphate buffer (50 mM, pH 8) was used.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Laemmli (20). The gels were stained for protein with Coomassie blue G 250 (E. Merck, Darmstadt, Germany) or with silver stain. For Western blot analysis, proteins resolved by SDS-PAGE were transferred onto nitrocellulose membrane with a Hoefer TE 22 Mighty Small Transphor Electrophoresis unit (Amersham Pharmacia Fine Chemicals, Inc.). Bands containing antigenic determinants of P42 or P51 were visualized by exposing the nitrocellulose membrane for 2 h to the appropriate serum. Subsequently, the membrane was exposed for 1 h to alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Sigma) at a 1:10,000 dilution. The detection was carried out using Sigma alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in conjunction with Nitro Blue Tetrazolium. M. M. Lecadet of the Pasteur Institute, Paris, kindly provided the antibodies against P42 and P51.

Total DNA from B. sphaericus 2297 was digested with the appropriate enzymes, subjected to electrophoresis in 0.8% agarose gels, and transferred onto nylon membranes by the capillary method (34) using 0.4 M NaOH as the solvent. DNA probes were labeled with digoxigenin-11-dUTP, using the standard random primed DNA labeling reaction (Boehringer Mannheim). The labeling yield was estimated as described by the manufacturer.

Bioassays involving mosquito larvae.
Freeze-dried samples were generally used for bioassays involving mosquito larvae. The samples were suspended in deionized water (5 mg/ml), homogenized on the Vortex mixer for 7 min with six to seven glass beads (diameter, 3 mm), and diluted in deionized water to the desired concentration. Each concentration of toxin was tested at 26 to 28°C, in three to four replicates, in petri dishes (diameter, 7 cm) each containing 20 second-instar larvae of Culex pipiens of the laboratory colony in 20 ml of deionized water. Twenty-four hours after toxin introduction, a drop of 10% Saccharomyces cerevisiae suspension was added to each dish. Larval mortality was recorded after 48 h. If larval mortality in the absence of larvicide was between 2.5 and 10%, the real mortality was recalculated according to Abbott's (1) formula. Bioassays in which mortality in the control dishes exceeded 10% were not used. Concentrations of samples that kill 50% of the test larvae (50% inhibitory concentration [LC50]) after 48 h of exposure were calculated by the log-probit analysis method (17). For each determination of LC50, the standard deviation (SD) was calculated (results are presented as means ± SD). Larvicidal activity was estimated in comparison with the B. sphaericus standard SPH 88 (1,700 international toxic units [ITU] of B. sphaericus 2362/mg; Pasteur Institute, Paris).

Samples were tested for synergism in mixture with SPH 88 standard, or with other preparations, as described. The synergistic ratio (SR) was estimated as the ratio of observed to calculated specific larvicide toxicity according to the following formula: SR = Aobs/(Axrx + Astrst), where Aobs, Ax, and Ast are specific activities (in ITU per milligram) of the respective mixed larvicide and its sample and standard component, while rx and rst are proportions (by weight) of sample and standard components in the mixture. Synergism is proven when the SR is significantly greater than 1.0. The significance of the difference between SR values and between the indices of toxicity (LC50 or ITU) was estimated according to Student's t test.

Electron microscopy.
Cell pellets from a 1-ml cell suspension of B. sphaericus were fixed in 1.5% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) and postfixed in 1% osmium tetraoxide in the same buffer. The fixed cells were embedded in Epon resin according to the method of Luft (22), sectioned to 70 to 90 nm, and stained with uranyl acetate and lead citrate. The sections were viewed with a Philips EM 300 electron microscope operating at 60 kV.


   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Cell wall toxicity of B. sphaericus.
Myers and Yousten (25) have shown the presence of larvicidal activity in purified cell wall of B. sphaericus 1593. Since this highly toxic strain produces binary toxin crystals, imperfect separation between cell wall and the crystals could account for this phenomenon.

We have observed the same phenomenon in B. sphaericus 2297. Trying to separate the activities, we attempted to remove the binary toxin by the extraction of cells or cell wall of B. sphaericus 2297 with 7 M urea, which solubilizes toxin crystal. Indeed, all but traces of binary toxin was thus removed from the sporulating cultures of B. sphaericus 2297, lowering their toxicity from an LC50 of 10 ng/ml to 10 µg/ml. However, the toxicity of cell wall prepared from stationary cells sampled prior to cell lysis was not affected.

Urea extraction became a useful tool for discrimination between the crystal and cell wall toxins. Thus, the combined activity of both sources of toxin was slight (LC50 = 1.2 ± 0.3 µg/ml) in early exponential cultures (3 to 4 h). It did not change considerably till 8 h, and then it dramatically increased, reaching an LC50 of 10 ± 2.5 ng/ml at 12 h, and remained stable thereafter.

Cell wall activity measured in urea-extracted cell debris was not detectable in 4-h cultures (Fig. 1). Eight-hour cells were slightly toxic to C. pipiens larvae (LC50 = 0.15 ± 0.05 µg/ml). The toxicity peaked in the second half of the logarithmic phase (after 12 h of growth) reaching an LC50 of 12 ± 2.0 ng/ml, an activity nearly that of the binary toxin standard. At this culture age, only 0.001% of heat-resistant spores were present. Binary toxin polypeptides P51 and P42 just began to be detectable on SDS-PAGE (not shown). Unlike crystal-associated activity, which remained stable in senescent lysed cultures (24 to 48 h), cell wall activity in B. sphaericus 2297 decreased to about 30% of its peak value (LC50 = 36 ± 3.0 ng/ml) in 16-h culture. It was dramatically lower in 24-h culture (LC50 = 0.21 ± 0.04 µg/ml) and disappeared almost completely in later cultures. The difference between toxicity measurements of sequential samples was highly significant (t = 7.9 to 18.5; P < 0.001 in all cases). Reduction of the cell wall component coincided with progressive lysis of B. sphaericus cells and the emergence of temperature-resistant spores.



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  		FIG. 1. Dynamics of cell wall toxicity in a growing culture of B. sphaericus 2297. The toxicity against Culex larvae was measured on the insoluble residue after sonication and urea extraction. Error bars, SDs.

 
Distribution of cell wall toxicity in B. sphaericus strains.
Cell wall larvicide was found in a number of strains containing binary toxin genes but not in all of them (Table 1). The cells of B. sphaericus 2297, 2362, 1593, and 1691 harvested after 12 h of culture and extracted with urea were as toxic as the best preparations of the binary larvicide. B. sphaericus LP1-G was less potent. Cell wall toxicity was not detected in the strain SSII-1, a low-potency strain which expresses mtx genes (11, 37, 38) but lacks the binary toxin gene (btx). Whole cells and urea-extracted cell wall of SSII-1 were inactive, indicating that cell wall activity is not related to Mtx proteins. Finally, the presence of cell wall toxicity in B. sphaericus LP1-G, a strain that does not carry the mtx gene but produces binary toxin, corroborates the notion that Mtx is not responsible for cell wall toxicity.


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  		TABLE 1. Cell wall-associated larvicide activities in various strains of B. sphaericus

 
Several other strains lacking btx genes were surveyed. Most were devoid of larvicidal activity, though cell walls of two strains, B. sphaericus 2377 and 2173 of the flagellar serotype 26a, 26b, were active against C. pipiens larvae (Table 1).

Synergism between urea-extracted cell wall and the binary toxin.
Cell wall larvicide interacted synergistically with the B. sphaericus binary toxin. The Pasteur Institute standard of binary toxin larvicide (SPH 88, used in these experiments) is prepared from B. sphaericus cultures that contain only spores and crystals and, thus, are devoid of cell wall toxicity. The synergistic effect was observed in all tested ratios of toxicities (Table 2), with the maximal SR of 2.3 observed at a 1:1 ratio. Cell wall samples prepared from the cultures of B. sphaericus 2297 were synergistic to the same extent, not only with the binary toxin standard, SPH 88, but also with similar preparations made from B. sphaericus strains 2297 and 1593 (data not shown). The Mtx-containing strain SSII-1 produced no synergism when mixed with binary larvicide.


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  		TABLE 2. Synergism between binary toxin and cell wall toxin from cultures of different ages

 
The cell wall toxins of B. sphaericus 2377 and 2173, lacking btx genes, did not act synergistically with the binary toxin and, thus, differed from the phenomenon under investigation. It is possible that this form of cell wall toxicity also exists in some strains that contain the binary toxin genes. It has been reported recently (32) that B. sphaericus IAB872 is active against the binary toxin-resistant Culex species, while its btx gene product is active only against susceptible species. Shi et al. (32) have, indeed, surmised the existence of an unknown toxin.

Effect of enzymes and of heat treatment on the binary toxin and cell wall larvicide.
Serine proteases, trypsin and subtilisin, considerably reduced the activity of the binary toxin preparations to 21 and 12% of the original activity, respectively (Table 3). The treatment of cell wall larvicide with the same enzymes produced completely different results. While half of the weight of the urea-treated cell walls prepared from B. sphaericus 2297 was lost, all the activity remained in the insoluble material (25). Obviously, the cell wall toxin is well protected against proteolysis. In fact, trypsin was obviously activating. However puzzling this effect was, the very nature of biological assay precludes the differentiation between the effects of trypsinization either on the intrinsic quality of the toxin, on the size of toxin particles, or on its efficacy in the mosquito midgut.


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  		TABLE 3. Effect of enzymes on the activity of B. sphaericus binary toxin and cell wall-associated larvicide

 
Lysozyme caused a large decrease in toxicity of the binary toxin (71%) and even more pronounced loss in the cell wall (91%), presumably by affecting in the former the spore cortex or glycoproteins and/or polysaccharides of the exosporium, as well as a part of the cell wall matrix in the latter. However, the inactivation of toxins could also result from the release of latent proteases by the action of lysozyme. To exclude such inactivation by a serine protease present in the lysozyme preparation itself, we have preincubated it with the irreversible inhibitor phenylmethylsulfonyl fluoride without changing the outcome of the experiment (not shown).

Heating the binary toxin or the cell wall preparations for 15 min at 100°C completely inactivated both. Thus, heat treatment was not discriminatory.

Disruption of the p42 gene.
Despite the usefulness of urea in extraction of the binary toxin, one could not rule out the possibility that the elimination of the binary toxin background activity was complete. Thus, we decided to disrupt the p42 gene of B. sphaericus 2297. The remaining P51 protein of the binary toxin is not toxic by itself (3, 12, 26); thus, selective disruption of the p42 gene should eliminate cell wall toxicity, if attributed to binary toxin.

A plasmid, pBB544-trP42A, harboring neo and amp resistance genes was constructed that contained in the cloning site a truncated p42 gene (an internal 908-bp AccI-NcoI fragment); thus, ca. 100 bp was deleted from both the 3' and 5' termini. The plasmid pBB544-trP42A was introduced into B. sphaericus 2297 by electroporation as described in Materials and Methods. Colonies resistant to neomycin (Neor) were obtained. Since the plasmid pBB544-trP42A did not contain a gram-positive replicon, resistance could only stem from the integration of the neo gene into B. sphaericus 2297 chromosome via a Campbell-type recombination event (8). This insertion resulted in two incomplete copies of the p42 gene separated by the vector DNA harboring neo and amp resistance genes (Fig. 2). One of the copies encoding P42 lacked more than 30 amino acids from the N terminus, while the second lacked the same length at its C terminus. It has been shown previously that each of such deletions renders the P42 inactive (7).



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  		FIG. 2. Construction of B. sphaericus 2297(::pBB544-trP42A) by homologous recombination with integration of the nonreplicative plasmid pBB544-trP42A onto the chromosomal DNA of B. sphaericus 2297. Abbreviations for restriction sites: A, AccI; B, BamHI; EI, EcoRI; H, HindIII; N, NotI.

 
Tests were carried out on total chromosomal DNA from B. sphaericus 2297 and recombinant strains of B. sphaericus 2297(::pBB544-trP42) using probes corresponding to the entire pBB544-trP42A, to the p42 gene, and to the neo resistance gene. The resulting hybridization patterns (Fig. 3) were as expected from the integration scheme (Fig. 2). Thus, homologous recombination took place between the p42 fragment inserted in the pBB544-trP42A and the chromosomal p42 gene. Consequently, the gene disruption of the p42 was carried out successfully.



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  		FIG. 3. Restriction analysis of total DNA from B. sphaericus 2297 and from B. sphaericus 2297(::pBB544-trP42A). Total DNA was digested with EcoRI, HindIII, or BamHI-HindIII and subjected to electrophoresis in a 0.5% agarose gel. The DNA fragments were transferred onto Nylon+ membranes (Boehringer) and hybridized with digoxigenin-labeled probes corresponding to the entire plasmid pBB544-trP42A (A), to the p42 gene (B), or to the neo resistance gene (C). Lanes 1, 3, 5, 7, 9, 11, 13, 15, and 17 contain DNA from B. sphaericus 2297. Lanes 2, 4, 6, 8, 10, 12, 14, 16, and 18 contain DNA from B. sphaericus 2297(::pBB544-trP42A). The size of the reactive fragments was as expected from the scheme (Fig. 2).

 
Expression of binary toxin genes and crystal formation in B. sphaericus 2297(::pBB544-trP42A).
A polypeptide of 38 kDa immunologically related to P42 was detected by the Western blot analysis of the protein extract from the late stationary cell wall of the recombinant strain (compare the lanes b and e in Fig. 4), confirming that the truncated p42 genes of the recombinant strain express this protein. The amount of P51 in SDS sample buffer-extracted cell wall was minute compared to the same protein removed by lysozyme (compare lanes e and f in Fig. 4). Thus, the recombinant strain produced the P51, but evidently, most of it was covalently bound or otherwise strongly associated with the cell wall.



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  		FIG. 4. Molecular size and location of binary toxin-related polypeptides in B. sphaericus strain 2297 and B. sphaericus strain 2297(::pBB544-trP42A). In lanes a to c, the polypeptides were detected with antibodies against the P51 protein. In lanes d to f, the same samples were detected with antibodies against both P51 and P42. In lanes a and d, a spore-crystal mixture of B. sphaericus 2297 (0.01 mg) was extracted for 20 min at 80°C with 8% SDS. The soluble material was then subjected to PAGE. In lanes b and e, urea-treated cell wall (0.2 mg) of B. sphaericus 2297(::pBB544-trP42A) was extracted with 8% SDS at 80°C for 20 min. The soluble material was then subjected to PAGE. In lanes c and f, urea-treated cell wall (0.2 mg) of B. sphaericus 2297(::pBB544-trP42A) was suspended in phosphate buffer (50 mM, pH 7) containing EDTA (1 mM) and treated overnight at ambient temperature with lysozyme (0.01 mg per mg of cell wall preparation). The soluble material was mixed with sampling buffer and subjected to PAGE.

 
Examination under electron microscope of ca. 100 longitudinal thin sections of sporulated cultures from the recombinant strain revealed the presence of spores; however, no crystal inclusion could be identified, while in the longitudinal sections of the wild-type cells, both spore and crystal were always present (Fig. 5).



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  		FIG. 5. Typical electron micrographs of ultrathin sections of B. sphaericus 2297 and B. sphaericus 2297(::pBB544-trP42A). In the wild-type strain (left panel), spore (S) and crystal (C) are enclosed within the exosporium (E). In the recombinant strain (right panel), no crystal was observed.

 
Toxicity of the recombinant strain to mosquito larvae.
Samples from a fully sporulated culture of the strain 2297(::pBB544-trP42A) were not toxic to C. pipiens larvae (ca. 30% mortality at a dose of 0.25 mg/ml). These results confirmed that, as expected, both products of the truncated p42, the 38-kDa proteins, were not toxic (Table 4).


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  		TABLE 4. Larvicidal activities of toxin component mixturesa

 
In addition, in the urea-treated cell wall prepared from stationary or late logarithmic cultures of the recombinant B. sphaericus strain 2297(::pBB544-trP42A), no larvicidal activity was detected. Thus, we could conclude that the binary toxin is responsible for the cell wall toxicity of the parental B. sphaericus 2297. Finally, since this cell wall fraction of binary toxin was not extracted by concentrated urea solution, it has to be tightly, perhaps, covalently bound to cell wall.

Although no toxicity (LC50 of 50 ± 4.5 µg/ml) could be detected in cell wall of the recombinant B. sphaericus strain 2297(::pBB544-trP42A), an activation effect with the binary toxin (SPH 88) was observed. The synergistic effect (SR = 3.8 ± 0.27) was observed when both components were mixed at a ratio of 1:1 by weight (Table 4). The tightly bound P51, which is present in the cell wall of the recombinant strain (Fig. 4, lane f), should be responsible for synergism. This thesis was confirmed using E. coli cells harboring plasmid pCK2297R that expresses only P51 and E. coli cells harboring plasmid pMK31 that expresses only P42. When both E. coli strains were combined to a ratio of 1:1 (by weight) they were toxic to mosquito larvae (LC50 of 0.550 ± 0.005 µg/ml [Table 4]). E. coli cells harboring plasmid pCK2297R containing the p51 gene were slightly synergistic with the SPH 88 binary toxin (SR = 1.45 ± 0.17). No synergism was found between the binary toxin standard and E. coli cells harboring plasmid pMK31 containing only the p42 gene (SR = 1.01 ± 0.2). These results led us to conclude that the ratio of P51 and P42 is not optimal in the binary toxin standard. The slight deficiency of P51 in the crystal and the same of P42 in the cell wall may explain the small but significant synergism we observed in our study. The addition of P51 enhances the toxicity by combining with the excess of P42 in the crystal. Thus, the synergism observed between urea-treated cell wall and the SPH 88 binary toxin standard is due to the P51 present in the cell wall.

The addition of E. coli cells harboring the plasmid pCK2297R which produce only P51 to the urea-treated cell wall from the recombinant B. sphaericus 2297(::pBB544-trP42) does not influence the toxicity, whereas mixing of urea-treated cell wall from this recombinant strain with the P42-producing E. coli cells harboring the plasmid pMK31 largely reconstituted the toxicity to mosquito larvae (LC50 of 0.10 ± 0.03 µg/ml).

Conclusions.
The following conclusions can be drawn. (i) In many binary toxin-producing strains, P42 and P51 proteins are immobilized at the structure copurifying with the cell wall fraction. (ii) Binary toxin proteins are not completely removed from the cell wall by 7 M urea, though the solubilization of cell wall by lysozyme treatment renders these proteins soluble. (iii) The deposition of binary toxin protein at the cell wall precedes parasporal crystal formation and reaches maximum in late exponential cultures. (iv) The stoichiometry of binary toxin proteins in the two locations differs: in the cell wall fraction, P51 slightly predominates, while in the crystal, it is P42. (v) A different type of wall toxicity exists in the two B. sphaericus strains, 2173 and 2377. The species specificity and the nature of this toxicity are currently under investigation.

The presence of cell wall-bound binary toxin in presporulating cultures of entomopathogenic B. sphaericus raises interesting questions about its physiological role. B. sphaericus strains isolated from a variety of habitats all over the globe contain binary toxin genes differing only by a few bases (6, 28). The very conservative nature of the toxin as well as its complexity argues against its being a plain nutrient deposit for the germinating spore. The cell wall-bound toxin is certainly lost for the spore by the lysis.

The activity of binary toxin is strictly limited to mosquitoes, mainly of the genus Culex. Despite a large environmental survey, neither other susceptible species nor mosquito larvae that prey on species harboring the toxin were ever found. Nevertheless, B. sphaericus is not an obligatory pathogen of C. pipiens or other mosquito species. Although recycling of B. sphaericus in mosquito cadavers has been demonstrated under laboratory conditions (5, 10, 39), such conditions may rarely exist in the field, e.g., where the environment supports dense populations of mosquito larvae. B. sphaericus is an obligatory aerobic organism, while mosquito larvae cadavers drown within 48 h (39), limiting the ability of B. sphaericus to compete with naturally occurring anaerobic organisms, e.g., other bacilli. The cell wall deposit of the toxin would be useless in such recycling, anywhere.

In laboratory cultivation we have found but one difference between the physiology of B. sphaericus 2297 and its recombinant variant 2297(::pBB544-trP42A). After 42 h of cultivation, both strains reached a density of 3 x 108 to 6 x 108 cells per ml. However, these cultures of the parental strain yielded 100% heat-resistant spores, while the recombinant organism lacking the functional binary toxin yielded only 2 x 107 (less than 10%) heat-resistant spores per ml.


   ACKNOWLEDGMENTS
 
We are grateful to H. Pener and her staff (Laboratory of Entomology, Ministry of Health, Jerusalem, Israel) for helping us to start and to maintain the mosquito colony and for the permission to use some of her laboratory facilities. We thank the four anonymous reviewers for the attentive reading of our manuscript and very useful comments. The excellent linguistic assistance by H. J. Bromley-Schnur is acknowledged.

This research was supported in part by a grant from the Ministry of Sciences and Technologies of Israel.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biological Chemistry, A. Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel. Phone: 972-2-6585412. Fax: 972-2-6519597. E-mail: sergei{at}vms.huji.ac.il. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Applied and Environmental Microbiology, July 2002, p. 3300-3307, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3300-3307.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




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