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We report the complete sequence and analysis of pFR260, a novel megaplasmid of 260,595 bp from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. It carries 7 insecticidal genes: 3 cry8 copies previously reported, 2 vip1, and 2 vip2. Also, it carries a gene encoding a putative atypical Cry protein. These genes are arranged in a region of approximately 105 kbp in size with characteristics of a pathogenicity island with a potential coleopteran-specific insecticide profile. DNA strand composition asymmetry, as determined by GC skew analysis, and the presence of a Rep protein involved in the initiation of replication suggest a bidirectional theta mechanism of replication. In addition, many genes involved in conjugation and a CRISPR-Cas system were detected. The pFR260 sequence was deposited in GenBank under accession number KX258624.

We found and characterized two cry8 genes from the Bacillus thuringiensis strain INTA Fr7-4 isolated in Argentina. These genes, cry8Kb3 and cry8Pa3, are located in a tandem array within a 13,200-bp DNA segment sequenced from a preparation of total DNA. They encode 1,169- and 1,176-amino-acid proteins, respectively. Both genes were cloned with their promoter sequences and the proteins were expressed separately in an acrystalliferous strain of B. thuringiensis leading to the formation of ovoid crystals in the recombinant strains. The toxicity against larvae of Anthonomus grandis Bh. (Coleoptera: Curculionidae) of a spore-crystal suspension from the recombinant strain containing cry8Pa3 was similar to that of the parent strain INTA Fr7-4.

Insecticidal activity of Bacillus thuringiensis is attributed largely to the crystal proteins. These were found with specific toxic activity against insects in different orders. A novel cry8 gene from B. thuringiensis strain INTA Fr7-4 from Argentina was characterized. The encoded protein, Cry8Qa2, is 1184 amino acids long and its sequence is more similar to those of Cry8F class. We cloned and expressed the protein in an acrystalliferous strain of B. thuringiensis using two differentially regulated promoters. The recombinant strains produced ovoid crystals with low toxicity against larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae). The morphology and insecticidal properties of these crystals resembled those produced by the native strain INTA Fr7-4.