Vol. 56, No. 2

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1990, p. 340-344

0099-2240/90/020340-05$02.00/0 Copyright C 1990, American Society for Microbiology

Transfer of the Toxin Protein Genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and Their Expression CATHERINE BOURGOUIN,lt* ARMELLE DELECLUSE,1 FRANGOISE DE LA TORRE,2 JESIKIEL SZULMAJSTER2 Unite de Biochimie Microbienne, Institut Pasteur, 75724 Paris Cedex 15,1 and Laboratoire d'Enzymologie, AND

Centre National de la Recherche Scientifique, 91190 Gif sur Yvette,2 France Received 12 June 1989/Accepted 1 November 1989

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.

Ohio State University, Columbus) were used as recipient strains in the transformation experiments. Strain 4Q2-81 is a crystal-minus strain cured of all resident plasmids, and strain 4Q2-72 is a crystal-producing strain harboring only the 72-MDa plasmid, which encodes all the B. thuringiensis subsp. israelensis crystal proteins (10, 20). For cloning experiments, Escherichia coli JM103 was used as a recipient strain. The recombinant plasmid pGSP04, previously described (19), was the source of the toxin genes of B. sphaericus. Plasmids pHV33 (18), pBC16-1 (17), and pBU4 were used in the transformation experiments. The bifunctional vector pBU4 (Fig. 1) was obtained by ligation of EcoO109-restricted pUC19 and EcoRI-restricted pBC16-1 after the cohesive ends were filled in. The relative orientations of pUC19 and pBC16-1 were determined by restriction enzyme mapping. General procedures. Plasmids were isolated from E. coli by the alkaline lysis procedure described by Birnboim and Doly (4). This procedure was slightly modified as previously described (6) for the purification of plasmids from B. thuringiensis. Cloning experiments and restriction enzyme analysis were carried out as described by Maniatis et al. (15). For protein analysis, transformants were allowed to sporulate at 30°C in the presence of 30 [Lg of tetracycline per ml. Bacterial protein extracts, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting were carried out as previously described (6). Transformation procedure. Transformation of B. thuringiensis subsp. israelensis was performed essentially as described by Heierson et al. (11). Bacteria were grown in Min 3 medium and harvested at an optical density at 650 nm of 2. Cells were washed in Tris hydrochloride (50 mM, pH 7.5) and suspended in Tris hydrochloride-sucrose (optical density at 650 nm, 0.8). For each transformation experiment, a cell suspension of 5 ml of bacteria in Tris hydrochloridesucrose (optical density at 650 nm, 0.8) and 5 ,ug of plasmid DNA were used. After incubation with DNA, bacteria were diluted in 5 ml of Luria broth and incubated for 1.5 h at 37°C. Samples (200 jxl each) of bacteria were then plated in soft agar on Luria agar plates supplemented with 30 pg of tetracycline per ml for direct selection of transformants. Biological assay. Larvicidal activities of transformants and wild-type strains were determined for three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens. Sporulated bacterial cells were harvested and washed in

Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus are two aerobic sporeforming microorganisms which produce proteinaceous crystalline inclusions at the onset of sporulation. These inclusions are toxic for larvae of a number of mosquito species which are vectors of severe tropical diseases. The intact crystalline inclusions of B. thuringiensis subsp. israelensis are composed of at least four polypeptides of 28, 68, 125, and 135 kilodaltons (kDa) (9, 16) which are all toxic, although to different extents (7). These polypeptides present different specificities and could also act in synergism (6, 8, 21). The genes encoding these polypeptides have been cloned and sequenced in several laboratories (for a review, see reference 14). The toxin of B. sphaericus consists of two polypeptides with apparent molecular sizes of 43 and 56 kDa (measured by polyacrylamide gel electrophoresis) (5). However, after the two genes specifying these two polypeptides were cloned and sequenced, the molecular sizes deduced from the sequence were found to be 41.9 and 51.4 kDa, respectively (1, 2). Both polypeptides seem to be required for larvicidal activity (3). The toxins produced by each of these microorganisms have different host range specificities. B. sphaericus toxin is toxic mainly to the larvae of Culex and Anopheles species, while the B. thuringiensis subsp. israelensis inclusions are more active against Culex and Aedes species (13). B. sphaericus also has the benefit of longer persistence in polluted aquatic ponds. It is therefore of great advantage for mosquito control to combine, by genetic engineering, the toxin genes from the two microorganisms in one microorganism in order to widen the host range spectrum. In this paper we report for the first time the transfer of the toxin genes of B. sphaericus 1593 into B. thuringiensis subsp. israelensis and their expression and describe some properties of the transformants. MATERIALS AND METHODS Strains and plasmids. Strains 4Q2-81 and 4Q2-72 of B. thuringiensis subsp. israelensis (a gift from H. D. Dean, * Corresponding author. t Present address: Molecular Neurobiology Laboratory, The Salk Institute, P.O. Box 85800, San Diego, CA 92138.

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VOL. 56, 1990

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TRANSFER AND EXPRESSION OF B. SPHAERICUS TOXIN GENES

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distilled water, and the optical density at 650 nm was adjusted to 3. Protein content of these stock suspensions was further measured by the BioRad assay after overnight incubation of the suspensions in NaOH (0.05 N). Five serial dilutions of each suspension were tested against secondinstar mosquito larvae, and mortality was scored at 24, 48, and 72 h. Each dilution was tested in duplicate against 20 larvae in glass petri dishes containing a final volume of 6 ml. LC50s (concentrations of cell protein leading to 50% mortality) were determined on log-Probit paper.

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RESULTS AND DISCUSSION Using the procedure of Heierson et al. (11) for genetic transformation of B. thuringiensis, we were able to transform B. thuringiensis subsp. israelensis 4Q2-81 with each of the vectors pHV33 and pBC16-1, with frequencies ranging from 0.2 x 10-7 to 0.2 x 10-6 per ,ug of plasmid DNA. Analysis of the transformants indicated that clones harboring pHV33 did not sporulate, whereas clones carrying pBC16-1 were sporulating normally. On the basis of these results, we constructed the bifunctional vector pBU4 from pBC16-1 and pUC19 (see Materials and Methods and Fig. 1), thus enabling genes of interest to be first cloned in E. coli. Furthermore, in B. thuringiensis subsp. israelensis transformants containing pBU4, expression of the B. thuringiensis subsp. israelensis crystal proteins and sporulation took place normally (see below).

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FIG. 2. Protein analysis of the transformants of B. thuringiensis expressing the B. sphaericus crystal proteins. Samples of bacterial extracts corresponding to 500 ,ul of spore-crystal suspension (optical density at 650 nm, 3) were subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis followed by staining with Coomassie blue. Lanes 2 to 4, strain 4Q2-81 containing pBU4, pGSplO, and pGSpll, respectively; lanes 5 to 7, strain 4Q2-72 containing pBU4, pGSplO, and pGSpll, respectively; lane 1, purified crystals of B. thuringiensis subsp. israelensis; lane 8, purified crystals of B. sphaericus; lanes S, molecular weight markers. Sizes of the markers are indicated in the right margin. In the left margin, small numbers indicate the molecular weights of the B. thuringiensis subsp. israelensis crystal proteins and large numbers indicate the molecular weights of the B. sphaericus crystal proteins.

BOURGOUIN ET AL.

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APPL. ENVIRON. MICROBIOL.

EX/71 FIG. 3. Electron micrographs of B. thuringiensis subsp. israelensis 4Q2-81 containing the plasmid vector pBU4 (a) or recombinant plasmid pGSP1O harboring the B. sphaericus toxin genes (b and c). EX, Exosporium; SP, spore; S, crystal.

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For the expression of the B. sphaericus toxin genes in B. thuringiensis subsp. israelensis, the Hindlll DNA fragment of pGSP04, which carries the 41.9- and 51.4-kDa protein genes of B. sphaericus, was inserted in both orientations into pBU4, and the resulting recombinant plasmids, pGSP10 and pGSP11 (Fig. 1), were isolated from E. coli recombinant clones. Plasmids pGSP10 and pGSP11 were introduced into the crystal-minus strain 4Q2-81 and the crystal-plus strain 4Q2-72 of B. thuringiensis subsp. israelensis by transformation. In all cases, the recombinant plasmids were stably maintained. Protein analysis of sporulated cultures of B. thuringiensis subsp. israelensis transformants indicated that crystal proteins either of B. sphaericus in 4Q2-81 transformants or of both B. sphaericus and B. thuringiensis subsp. israelensis in transformants of strain 4Q2-72 are the main proteins synthesized during sporulation (Fig. 2, lanes 3, 4, 6, and 7). The presence of the B. sphaericus crystal proteins in the B. thuringiensis subsp. israelensis transformants was confirmed by immunodetection using an antiserum directed

against an alkaline extract of B. sphaericus spores (data not shown). As expected, no B. sphaericus cross-reacting proteins were detected in B. thuringiensis subsp. israelensis transformants containing only the vector pBU4. Furthermore, the synthesis of the B. thuringiensis subsp. israelensis resident toxins was not altered by the expression of the B. sphaericus proteins (Fig. 2, lanes 6 and 7). Also evident was the higher level of expression of the B. sphaericus toxin genes in transformants containing plasmid pGSP10, in which the transcription of the B. sphaericus genes is in the same direction as the direction of transcription from the lacZ promoter of the vector (Fig. 2). There is no obvious explanation for that observation, since it seems unlikely that the lacZ promoter would be functional during sporulation of gram-positive bacteria. B. thuringiensis subsp. israelensis transformed with the genes specifying the B. sphaericus proteins shows crystalline structures. Indeed, electron microscopic observation of transformant 4Q2-81(pGSP10) showed that this clone produced inclusions with a regular crystalline lattice (Fig. 3). However, these inclusions are smaller than those detected in B. sphaericus (12). In transformant 4Q2-72(pGSP10), similar small inclusions were detected in addition to the typical large, irregular crystals of B. thuringiensis subsp. israelensis (Fig. 4). The components of the irregular crystals of B. thuringiensis subsp. israelensis fit together and are enclosed in a membrane, whereas the small inclusions are not found closely associated with these B. thuringiensis subsp. israelensis crystals (Fig. 4b). However, it is also possible that the large inclusions observed in transformant 4Q2-72(pGSP10) could contain B. sphaericus crystal proteins. In all cases, inclusions were located outside the exosporium, as the crystals of B. thuringiensis subsp. israelensis normally are, whereas in B. sphaericus the inclusions are located between the two layers of the exosporium. Biological activities of the transformants were determined for three mosquito species: A. aegypti, A.- stephensi, and C. pipiens. Transformant 4Q2-81(pGSP10), which expressed only the B. sphaericus proteins, was very active against C. pipiens, the level of activity being almost identical to that of the reference B. thuringiensis subsp. israelensis strain (Ta-

TRANSFER AND EXPRESSION OF B. SPHAERICUS TOXIN GENES

VOL. 56, 1990

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