JOURNAL

OF INVERTEBRATE

PATHOLOGY

57, 149-158 (1991)

Cloning and Expression of Bacillus thuringiensis S-Endotoxin DNA in B. sphaericus E. BAR,* J. LIEMAN-HURWITZ,* *Department flnterdepartmental

E. RAHAMIM,t

israelensis

A. KEYNAN,” AND N. SANDLER*

of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem Unit Electron Microscopy Laboratory, The Hebrew University-Hadassah School, Jerusalem, Israel

91904,

and Medical

Received February 5, 1990; accepted June 12, 1990 israelensis S-endotoxin genes were cloned into Bacillus sphaericus 2362, stable transformants reacting with antibody to the 28- and 65-kDa B. thuringiensis israelensis crystal proteins and approximately 10 times more toxic to Aedes mosquito larvae than the original host strain. The LC,, after 48 hr of exposure of Aedes larvae to the most active transformed clone was 0.19 k&ml, compared with an LC,, of 1.9 kg/ml for 8. sphaericus 2362 and less than 0.1 &ml for B. thuringiensis israelensis. The cloning vector, plasmid pPL603E, was also effective in transforming B. subtilis 1E20 with B. thuringiensis israelensis DNA, producing highly toxic clones with less stable gene expression than the clones of B. sphaericus. 8 IWI Academic Bacillus

thuringiensis

producing

Press. Inc. KEY

WORDS:

Bacillus

thuringiensis;

Bacillus

sphaericus;

Bacillus

subtilis;

Aedes

aegypti;

bio-

logical control; endotoxin.

INTRODUCTION Bacillus thuringiensis israelensis is a bacterium that produces a highly effective mosquito larval toxin during the sporulation segment of its life cycle. Larvae of all the major disease-bearing mosquito species are affected (Goldberg and Margalit, 1977), as are. larvae of Simulium, commonly called black flies (WHO, 1982). The toxin appears in the form of an amorphous parasporal crystal which is made up of several major proteins of approximate molecular sizes of 28,65, 128, and 135 kDa (Huber and Luthy, 1981; Tyrell et al., 1981; Armstrong et al., 1985). The crystals undergo proteolysis at the alkaline pH of the midgut of susceptible insect larvae to release active toxin, whose affects include hemolysis of mammalian red blood cells and destruction of larval gut epithelial cells. The presence of 28-kDa protein is associated with both hemolysis and toxicity to larvae (Thomas and Ellar, 1983; Yamamoto et al., 1983; Davidson and Yamamoto et al., 1984; Armstrong et al., 1985), although toxicity has also been attributed to other crystal proteins, either singly or in combination (Hurley et al., 1985;

Wu and Chang, 1985; Chilcott and Ellar, 1988; Delecluse et al., 1988). Bacillus sphaericus is another sporeforming bacterium that produces a mosquito larval toxin during sporulation. Although highly effective toward susceptible larvae, the target spectrum of B. sphaericus is more limited than that of B. thuringiensis israelensis and does not include either Aedes mosquitoes or Simulium. However, B. sphaericus exerts effective control in mosquito breeding sites longer than B. thuringiensis israelensis, possibly due to its superior ability to multiply and sporulate in the mosquito larvae or in the natural environment (Pantuwatana and Sattabongkot, 1990) so that a combination of the broad target spectrum of B. thuringiensis israelensis with the natural persistence characteristic of B. sphaericus could have practical importance. We report here the cloning of B. thuringiensis israelensis S-endotoxin DNA in B. sphaericus 2362 with plasmid pPL603E as vector, leading to the stable expression of moderate toxicity to Aedes mosquitoes by the recombinant. B. sphaericus 2362, a strain with high insecticidal activity against Culex larvae, dif149 0022-2011/91 $1.50 Copyright Q 1991 by Academic Press, Inc. AU rights of reproduction in any form reserved.

150

BAR

fers from other strains in its ability to grow under a broader range of conditions (Yousten, 1984). To the best of our knowledge, this strain has not been transformed previously, nor has pPL603E been used as a vector for the cloning of B. thuringiensis israelensis genes. In order to determine the effectiveness of our transforming system, the recombinant DNA that was used to transform B. sphaericus was previously inserted into B. subtilis. The expression of B. thuringiensis israelensis toxin genes in the two Bacilli was found to be different. MATERIALS Bacterial thuringiensis

AND METHODS

strains and plasmids. The B. israelensis strain was initially

isolated from a spore/crystal powder produced by Laboratoire Roger Bellon, S.A. B. sphaericus 2362 was obtained from Dr. Sergei Brown at The Hebrew University of Jerusalem. B. subtilis 1E20 was obtained from the Bacillus Genetic Stock Center, Ohio State University, Columbus. The Escherichia coli plasmid pUC 8 was used to transform E. coli JM83. B. subtilis containing the plasmid pPL603E was received from the laboratory of Dr. Paul Lovett, Department of Biological Sciences, University of Maryland, Baltimore County, Catonsville, Maryland. Plasmid pPL603E, the cloning vector for B. subtilis and B. sphaericus, is a derivative of PUB 110 coding for resistance to neomycin or kanamycin (Kn) and chloramphenico1 (Cm). It contains a single Hind111 site within the chloramphenicol acetyltransferase (cat) gene, allowing for insertional inactivation. The region preceding’the cat structural gene includes a promoter which is active during both vegetative growth and sporulation in B. subtilis and a segment conferring inducibility by Cm (Mongkolsuk et al., 1984; Duvall et al., 1984). Enzymes. Restriction enzymes were purchased from BRL, Biolabs, BoehringerMannheim and Anglican. Preparation of recombinant DNA. For

ET

AL.

transformation

of B. subtilis and B. sphaericus, B. thuringiensis israelensis DNA was

prepared by a method involving lysis at a high pH which yields preferentially the DNA of large plasmids (Casse et al., 1979), and both vector (pPL603E) and donor DNA were digested with Hind111 and ligated with T4 DNA ligase. For transformation of E. coli, the 72mDa B. thuringiensis israelensis plasmid which carries the toxin genes (Gonzalez and Carlton, 1984; Ward et al., 1984), isolated by cesium chloride gradient centrifugation of DNA prepared from a B. thuringiensis israelensis strain containing only this plasmid, was digested with Hind111 and the 9.7kb restriction fragment which contains the toxin genes (Ward et al., 1984; McLean and Whitely, 1987; Donovan et al., 1988), was electroeluted from a 1% agarose gel, and ligated to HindIII-cut pUC 8 with T4 DNA ligase . Transformation of B. subtilis and E. coli. Competent cells of E. coli were prepared for transformation by the standard procedure (Maniatis et al., 1982). B. subtilis was transformed by the standard procedure as described by Gryczan et al. (1978). Transformation of B. sphaericus. Transformation of B. sphaericus was by the protoplast transformation method originally developed by Chang and Cohen (1979). The method as used by McDonald and Burke (1984) on B. sphaericus 1593 was modified in the following ways to provide optimum results with strain 2362. The cells were grown to early exponential phase in MBS medium (Kalfon et al., 1983). Lysozyme (0.6 mg/ml) was added to the medium for protoplast formation, and, in contrast to the procedure of McDonald and Burke (1984), no bovine serum albumin was added during transformation. The regeneration medium included l-l. 1% agar. Hemolytic assay. The quantitative hemolytic assay was done as described by Sandler et al. (1985). Immunoassay. For immunoassay of transformed colonies, washed pellets taken

CLONING

AND

EXPRESSION

from cultures at various stages of growth were lyophilyzed, then suspended in Na&O,, pH 10.5, at a concentration of 2 mg/ml and incubated for 30 min at 37°C to solubilize the crystal proteins. Portions (5 ~1) of the extracts were spotted on nitrocellulose filter strips and incubated for 2 hr at 37°C in a solution of 5% low-fat milk powder in 50 mM Tris-HCl, pH 7.5. Following overnight exposure to rabbit antibody to the desired isolated protein, the strips were incubated first with goat anti-rabbit IgG bound to alkaline phosphatase (from BioYeda, Israel) and subsequently with nitro blue tetrazolium and 5-bromo-4-chloro3-indoyl phosphate for color development. For preparation of antibodies, the 28 and 65kDa toxin proteins were isolated from solubilized B. thuringiensis israelensis crystals by chromatography on a Whatman DE-52 ion-exchange column followed by gel filtration on Sephacryl S-200 and G-75 columns and a final additional ion-exchange separation on DE-52 for purification of the 28-kDa protein. The appropriate bands were electroeluted from a preparative polyacrylamide gel. Antibodies to B. sphaericus crystal proteins were prepared from the 20% ammonium sulfate precipitate of a lysed sporulating culture, which contained three main peptide bands on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE). The individual bands were electroeluted from an 8% SDS-PAGE gel, and rabbit antibodies to the 43-kDa protein, which represents the toxin (Baumann et al., 1988), were prepared for use in these experiments. Toxin bioassay. To test toxicity toward mosquito larvae, triplicate samples of 10 third instar larvae of either Culex pipiens or A. aegypti were placed in plastic cups containing 50 ml of water. Suspensions of lyophilyzed powders of bacterial cultures were added at the desired concentration, and the cups were checked for the presence of dead larvae after standing at 27°C for 24 and 48 hr. The LD,, was

OF

151

S-ENDOTOXIN

calculated (Rishikesh

as recommended by the WHO and Quennelec, 1983). DNA hybridization. Hybridization of HindIII-digested recombinant DNA with 32P-labeled DNA of the 9.7-kb B. thuringiensis israelensis toxin genes reisolated from an E. coli transformant was done according to Maniatis et al. (1982). Electron microscopy. Specimens of B. subtilis and B. thuringiensis israelensis were fixed with 2% glutaraldehyde and postfixed with 0~0,. The specimens were dehydrated and embedded in Epon. Thin sections cut with a diamond knife were picked up on copper grids and poststained with 2% uranyl acetate followed by 0.2% lead citrate. Samples were viewed with a Philips 300 TEM. RESULTS Selection of Transformed B. sphaericus Clones and Stability of the Transformed Phenotype

Protoplasts of B. sphaericus 2362 were transformed with Hind111 cut B. thuringiensis israelensis DNA ligated to pPL603E as described above. Transformed clones containing B. thuringiensis israelensis DNA were identified by the Kn resistance derived from the presence of plasmid pPL603E, together with sensitivity to Cm due to the insertion of foreign DNA at the Hind111 site within the cat gene of the plasmid. Five hundred colonies from plates of protoplast regeneration medium were transferred in parallel to plates of nutrient agar containing either Kn (10 Kg/ml) or Cm (50 kg/ml). Kn resistant, Cm sensitive clones constituted approximately 60% of the total colonies. About 8% of the transformants containing recombinant DNA were found to be very stable, in that they did not lose the transformed phenotype after 20 passages of culture in the absence of Kn. An additional 20% were moderately stable and could be transferred five times before losing Kn resistance. The remaining

152

BAR ET AL.

approximately 72% lost Kn resistance immediately on being grown on plates not containing Kn. Growth and Sporulafion of B. sphaericus Transformants Rates of growth and sporulation of transformants were compared with the wild-type B. sphaericus 2362. While the growth rates of cultures of transformed clones were similar to those of the wild-type strain, sporulation was delayed in transformants containing recombinant DNA, although not in transformants which had received nonrecombinant pPL603E (Fig. 1). The transformed strain did not contain typical B. thuringiensis israelensis crystals. Identification

of B. thuringiensis

israelensis Toxin DNA in Stably Transformed Clones of B. sphaericus DNA isolated from four clones each of the moderately stable and highly stable groups of B. sphaericus transformants was tested for hybridization with the 9.7-kb Hind111 fragment of B. thuringiensis israelensis DNA. The fragment did not hybridize detectably with DNA from B. sphaericus 2362 or with DNA from the four moderately stable transformed colonies which had been selected at random for this test. However, the four clones of the highly stable group contained DNA which hybridized with the fragment (Fig. 2). No plasmid DNA was detected after agarose gel electrophoresis of DNA prepared from the transformants and the probe hybridized with material which migrated with the chromosomal DNA. Expression of B. thuringiensis israelensis Toxin Genes in the Stable Transformants of B. sphaericus Immunoassay. Enzyme-linked immunoassay was performed as described under Materials and Methods. Cell extracts and culture supernatants were exposed to antibodies to the 28- and 65kDa B. thuringiensis isruelensis crystal proteins and to the

0 2

6

‘0

20

24 Hours

28

38

42

46

FIG. 1. Growth and sporulation of Bacillus @men’cus 2362 cells, regenerated protoplasts, and representative transformed clones. B. sphaericus 2362 (0) and regenerated protoplasts of B. sphacricus 2342 (0) were grown .in MBS medium. B. sphaericus 2362 transformed with pPL603E (A) was grown in MBS with kanamycin. B. sphaericus 2362 transformed with a ligation mixture of pPL603E and HindHI-cut B. rhuringiensis isruelensis DNA was grown either in MBS with kanamycin (5 kg/ml) (A) or in brain heart infusion broth (BHI) to mid-exponential stage, when the culture was centrifuged and washed with fresh BHI. and the pellet was suspended in the original volume of MBS (D). The cells were grown at 32°C with vigorous shaking. The rate of growth was measured by reading the optical density of the cultures at 620 nm. At various times, culture aliquots were plated with or without heat treatment (incubation at 80°C for 10 min). The percentage sporulation at each time is defined as the percentage of heat-resistant colonies relative to the total colony count.

43-kDa B. sphaericus toxin protein. As shown in Table la, the B. sphaericus 43kDa protein is present at maximum concentration after 12 hr of culture, whereas the B. thuringiensis israelensis 28- and 65kDa proteins require at least 24 hr to reach full strength. Bioassay. Suspensions of lyophilyzed powers prepared from cultures at different times were assayed as described under Materials and Methods for toxicity toward Culex pipiens, which is sensitive to both B. thuringiensis israelensis and B. sphaericus

CLONING

AND

EXPRESSION

2. Hybridization of HindHI-restricted DNA of sphaericus 2362 and of recombinant clones with the 9.7-kb Hind111 fragment coding for B. thuringiensis israelensis toxin. The experiment was performed as described under Materials and Methods. The autoradiogram was prepared from an agarose gel containing the following samples: Lane A, B. sphaericus 2362; lane B, the 9.7-kb Hind111 fragment; lane C, clone 4; lane d, clone 17; lane e, clone 13; lane f, clone 8; lanes 1, 2, 3, and 4, representative clones of the moderately stable group of transformants; lane 5. HindIII-digested DNA (not visible on autoradiogram). FIG.

Bckillus

toxins, and A. aegypti, which is sensitive only to B. thuringiensis israelensis toxin. All clones were toxic to C. pipens. The LC,, after 48 hr of exposure of A&es larvae to the most active transformed clone, clone 62, was 0.19 p,g/ml when the recombinant was grown in the absence of Kn and 0.30 t&ml when Kn was included in the culture medium. These values may be compared with a minimum LC,, of 1.9 t&ml for B. sphaericus 2362 and a usual LCS, of less than 0.1 pg/ml for B. thuringiensis israelensis (Table lb; Fig. 3). It may be noted that toxicity to Aedes larvae is definitely not dependent on expression of the 65-kDa B. thuringiensis israelensis crystal protein gene in the transformants (Table lb). Hemolysis. Hemolytic assay results with the transformants were negative compared with B. thuringiensis israelensis controls (data not shown). Expression of B. thuringiensis toxin DNA in B. subtilis

The ligation

mixture

israelensis

of HindIII-cut

OF

8-ENDOTOXIN

153

pPL603E and HindIII-cut DNA isolated from large B. thuringiensis israelensis plasmids was used to transform competent cells of B. subtilis, in addition to being used to transform B. sphaericus 2362 protoplasts. This plasmid has reportedly been stably inserted into B. subtilis previously (Mongkolsuk et al., 1984). The cloning host, B. subtilis 1E20 carries a plasmid (pBD6) related to pPL603E and including the same Kn site, making it a suitable host for transformation with pPL603E. However, pBD6 is lost at temperatures above 35°C (Dubnau et al., 1980), so that Kn resistant, Cm sensitive transformants could be identified on plates incubated at 42°C. The approximately 1000 resultant colonies were patched onto nutrient agar plates containing 4% whole human blood and either 0.1 or 10 kg/ml Cm. Approximately 7% of the transformants were hemolytic, producing circles of hemolyzed blood around the colonies after 1-2 days of incubation of 35°C. The host strain produced no hemolytic colonies. The hemolytic colonies, representing transformants containing recombinant DNA in which the insert was in expressible form, comprised less than 10% of the total Kn resistant, Cm sensitive colonies, which represented 94% of the total transformants. The hemolytic activity of the colonies which were positive on blood agar plates was quantified by assay of alkaline spore/ crystal extracts as described under Materials and Methods to identify the most active clones (data not shown). In contrast to the B. sphaericus transformants, crystalline inclusions were noted in several of the hemolytic colonies of transformed B. subtilis. Electron microscopy showed that the inclusions resembled those of B. thuringiensis israelensis (Fig. 4). However, in at least one transformant the crystals were present for only a few hours during sporulation, after which they were no longer detectable by electron microscopy. In cultures of other transformed colonies, observation by phase contrast micro-

154

BAR

ET

AL.

TABLE RESPONSE

OF Bacillus

sphuericus B. thuringiemis

CRYSTAL

israrlensis

Antibody

B. thuringiensis B. thuringiensis B. sphaericus B. thuringiensis B. thuringiensis B. sphaericus

B

israelensis israelensis

hr

12

TOXIN

ANTIBODY

24

AND

ANTIBODY

Supernatant

hr

48

hr

12

hr

24

hr

48

hr

28 kDa 65 kDa

++

+++ ++ +++

+ +++ iii-

+ -

++ it

+++ + -

28 kDa 65 kDa

+ + +++

+++ +++ +++

++ + +++

+ ++ +++

++ +++ +++

+++ +++ +++

43 kDa israelensis israelensis

sphaericus

PROTEIN

Cell pellet

Duration of culture: A

la TO Bacillus

TRANSFORMANTS

43 kDa

Note. A representative colony of the moderately stable group of transformants (A) and of the stable group of transformants (B) were grown in MBS with the addition of Kn (5 &ml) after innoculation from a starter culture in brain heart infusion broth (Difco), Samples of the culture were removed at various times following innoculation for immunoassay as described under Materials and Methods.

scope also indicated that the presence of crystals during sporulation was transient. Several hemolytically active colonies reacted strongly with antibody to the 28-kDa B. thuringiensis israelensis crystal protein. However, reaction with antibody to the 65 kDA protein could not be correlated with expression of toxin genes since the host

RESPONSEOF Bacillus

sphaericus B. thuringiensis

strain lE20, unlike the nonplasmid bearing B. subtilis 168, reacted with this antibody. Several hemolytic colonies resulting from the separate experiment in which B. subtilis lE20 was transformed with pPL603E containing the 9.7-kb Hind111 fragment reisolated from E. coli also reacted strongly with antibody to the 28-kDa protein.

TABLE lb TRANSFORMANTS TO Bacillus sphaericus TOXIN ANT~ODY AND israelensis CRYSTAL PROTEIN ANTIBODY Immunological

Kn (5 t-&N Strain

during sporulation

B. thuringiensis israelensis

B. sphaericus

proteins 28 kDa

response

65 kDa

B. sphaericus 2362

protein 43 kDa

LC,, OLdml)

Sporulation time (hr)”

+++

1.9 2 0.26

ca. 24

Clones 62 5 13 96

Note.

presence of Aedes estimated 0 Time

+++ ++ + ++ ++ + -

+ + +t +

+++ +++ +++ +++ +++ +++ +++ +++

0.19 0.30 0.57 1.16 0.21 0.53 1.7 2.1

+ * It f '2 2 "

0.05 0.12 0.07 0.14 0.03 0.13 0.50 0.76

ca. 48 >48 >48 >48 >48 >48

ca. 24 ca. 24

Four colonies of the stable group of transformants were grown to stationary phase in MBS in the of Kn (5 &ml) and resuspended in fresh MBS medium with or without Kn for sporulation. Mortality larvae after 48 hr of exposure to powders prepared from sporulated cultures is compared with the intensity of the immunological responses to the same powders (see Materials and Methods). required after resuspension for maximum sporulation.

CLONING

AND

EXPRESSION

I ,,,,, 0.1

05

1.0 ,w/ml

5.0

J 10.0

FIG. 3. Larvicidal activity of Bacillus sphaericus 2362 and of recombinant clones toward Aedes aegypri larvae. Cells from sporulated cultures were pelleted and lyophilized for bioassay as described under Materials and Methods. Mortality was determined after 24 hr (B. sphaericus 2362) or 48 hr (recombinant clones) of exposure of the larvae to 0.1, 0.25, 0.5, I, 2.5, 5, or 10 &ml of powder. The points represent the average of triplicate samples. Open symbols represent cultures grown in the presence of Kn (5 &ml). 0, clone 62; n , clone 13; A. clone 5; +, B. sphaericus 2362.

Several colonies resulting from both types of cloning in B. subtilis were highly larvicidal toward C., pipiens. In addition, expression of toxin genes could be induced by the presence of Cm (0.1 pgiml) in the growth medium. For example, comparing growth in the presence or absence,of Cm, there was an increase from 0 to 90% mortality of clone l-16 (0.4 p&ml), measured after 2 hr of exposure of the larvae. The presence of Kn in the medium, however, prevented sporulation and thus prevented the development of larvicidal activity. Colonies which developed improved resistance to Kn had apparently lost the inserted DNA in pPL603E since they had no larvicidal activity. Although the stability of the transformed phenotype in B. subtilis was not studied extensively, the disappearance of parasporal crystals, hemolytic or larvicidal activity, and reactivity with antibody to the 28-kDa B. thuringiensis israelensis crystal protein on retesting of a number of previously active recombinant clones suggested that the B. thuringiensis israelensis genes were less stable in B. subtilis than in B. sphaericus. DISCUSSION

The plasmid pPL603E

was very efficient

OF

GENDOTOXIN

155

at transforming both B. subtilis and B. sphaericus, with significant differences between the results in the two cases. The transformed phenotype expressing B. thuringiensis israelensis genes seemed to be unstable in B. subtilis, although the picture is complicated by the transient nature.of the B. thuringiensis israelensis-like crystals in this strain. The proteases associated with sporulation may be responsible for the disappearance of the crystals and toxicity, a phenomenon which has not been noted in previous reports of cloning of B. thuringiensis israelensis toxin in other B. subtilis strains (Sekar and Carlton, 1985; Ward et al., 1986; Ward and Ellar, 1988). The induction of toxin gene expression by Cm is also of interest and is consistent with the evidence that the inducer acts on sequences preceding the cat structural gene (Duval et al., 1987). A significant percentage of the B. sphaericus transformants were quite stable. The relative stability of the B. sphaericus transformants compared with those of B. subtilis may be due to incorporation of the plasmid (or at least the segment carrying the kanamycin resistance gene) into the bacterial chromosome, together with the inserted B. thuringiensis israelensis DNA. This explanation is consistent with the fact that we were unable to isolate the recombinant plasmid from the transformants on agarose gels, although it may be that the difftculty was due to the formation of large oligomeric plasmid forms and perhaps also strong binding of such forms to the cell membrane, as has been observed in B. subtilis in the case of the parent pUB110 by Nyberg (1985).

The biological

activity of the transformed

B. sphaericus clones was lower than the activity measured in B. subtilis. If the mea-

sured toxicity was the maximum obtainable with these recombinants, it may mean either that the cat promoter of pPL603E is less efficient in B. sphaericus than in B. subtilis or that the inserted DNA in B. sphaericus has in fact been transposed to another location, where it lacks a strong

156

BAR

FIG. 4. thuringiensis

Electron micrographs of sporulated isruelensis. (a and b) Sporulating israelensis DNA. containing B. thuringiensis thuringiensis isruelensis x 30,195. The arrows sions (cl. Samples were prepared as described

ET AL.

cultures of recombinant Bacillus subrilis and of B. cells of B. subrilis lE20 transformed with pPL603E a x 32,765, b x 40,800. (c) Sporulating cultures B. indicate either spores (s) or crytaline parasporal incluunder Materials and Methods.

promoter. The ineffectiveness of low levels of chloramphenicol in inducing expression of the B. thuringiensis israefensis DNA in B. sphaericus, as opposed to the possibility of induction in the B. subtilis transformants, supports the latter suggestion. It is also possible that expression of the native B. sphaericus toxin genes interferes in some way with expression of the B. thuringiensis israefensis toxin genes. Both types of genes are expressed during sporulation, and it is of interest to note that the presence of the B. thuringiensis israelensis

toxin DNA in B. sphaericus interfered with the sporulation process. In considering the differences in levels of toxicity among the various clones, it should be kept in mind that we did not assay all the B. thuringiensis israelensis crystal proteins and that the clones may differ in expression of a protein not measured by us. In conclusion, toxicity toward Aedes mosquito larvae has been conferred on B. with sphaericus 2362 by transformation plasmid pPL603E ligated to HindIIIdigested B. thuringiensis israelensis plas-

CLONING

AND

EXPRESSION

mid DNA. The biological activity of the transformants was lower than that of B. thuringiensis israelensis, but greater than that of the parent B. sphaericus strain, and was stably transmitted in at least some of the clones. In contrast, B. subtilis lE20 transformed with the same ligation mixture yielded colonies with typical B. thuringiensis israelensis type parasporal crystal and high biological activity, but apparently of less stability than the B. sphaericus transformants. ACKNOWLEDGMENTS This research was supported by Grant PDC5544~G-SS-509801 from the U.S.-Israel Cooperative Development Research Program (CDR) of the U.S. Agency for International Development and by a donation from Ann and Paul Sperry. J. Lieman-Hurwitz was supported by a Levi Eshkol postdoctoral fellowship from the Israeli National Council for Research and Development. We are most grateful to Bet-told Fridlender and Raphael Hofstein of FRM Agricultural Sciences Partnership and Alex Honigman of the Institute of Microbiology of The Hebrew UniversityHadassah Medical School for their constant help and constructive criticism during the course of the project. We also thank Elizabeth Davidson of Arizona State University for stimulating discussions. We thank Pnina Bashkin for preparation of antibodies and Anita Kol for expert technical assistance.

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J. M.. JR., AND CARLTON, B.‘C. 1984. A large transmissable plasmid is required for crystal toxin production in Bacillus thuringiensis variety is;

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Cloning and expression of Bacillus thuringiensis israelensis delta-endotoxin DNA in B. sphaericus.

Bacillus thuringiensis israelensis delta-endotoxin genes were cloned into Bacillus sphaericus 2362, producing stable transformants reacting with antib...
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