The cytA gene encoding the 28-kDa polypeptide of Bacillus thuringiensis subsp. israelensis Bacillus thuringiensis subsp. israelensis produces crystalline inclusions during sporulation that are toxic to mosquito and blackfly larvae (15,35). These inclusions are, when solubilized, cytolytic for various mammalian cells, including erythrocytes (33). The crystals are composed of at least four polypeptides of 135, 125, 68, and 28 kDa. It has been shown by electron microscopic studies that these crystals are composite and consist of three major inclusion types differentiated on the basis of electron opacity, size, and shape. Purification and protein analysis of one of three types of component crystal revealed that it contained only the 68-kDa polypeptide (21). It was therefore suggested that the 28-kDa and 125-135-kDa polypeptides could be assembled separately in the two other inclusions (21).The diversity of the polypeptides has complicated the identification of the protein(s) responsible for larvicidal activity. The major difficulty in identifying the toxic polypeptide(s) was the purification of the different polypeptides. One solution to this problem was the cloning of the different genes. It has previously been shown that the toxin genes of B. thuringiensis subsp. israelensis were located on a 72-MDa resident plasmid (16,37). No copy of the toxin gene was found on the chromosomal DNA. Cloning experiments with the 72-MDa plasmid and analysis of the cloned products clearly indicated that the 135-, 125-, and 68-kDa proteins were involved in the toxicity to mosquitoes, alone or in combination (for a review, see reference 13).There is still controversy concerning the activity of the 28-kDa protein. Although biochemical and cloning experiments revealed that this polypeptide is responsible for the in vitro cytolytic activity of the crystals (4,7,18,20,27,40), its contribution to the mosquitocidal activity remains unclear. Earlier biochemical studies suggested that the 28-kDa polypeptide was not toxic to Aedes aegypti larvae (9,18,20,30,36). However, several groups found that the purified protein was toxic for this insect, although the 50% lethal concentration (LC50) observed was much higher than that obtained for Chilcott and Ellar [10]), then the study of the cloned 28-kDa product alone does not reflect the involvement of this protein in the overall toxicity of the crystals. This paper reports a novel approach to the study of the role of the 28-kDa polypeptide in the toxicity of the parasporal body. The experimental approach involved the disruption of the cytA gene present on the 72-MDa resident plasmid by in vivo recombination in B. thuringiensis subsp. israelensis. The toxicity of crystals depleted of the 28-kDa protein was determined on three mosquito species and compared with that obtained with native crystals. The results obtained indicated that the 28-kDa polypeptide has only a minor effect on the mosquitocidal activity and that that effect is restricted to Anopheles stephensi.
MATERIALS AND METHODSBacterial str...