Overproduction of Encapsulated Insecticidal Crystal Proteins in a Bacillus thuringiensis spoOA Mutant

Abstract

The spo0A gene of Bacillus subtilis encodes the key factor involved in the initiation of sporulation. It was previously shown that the B. thuringiensis (Bt) cryIIIA gene, encoding a toxin active against coleopteran larvae, is overexpressed in an spo0A mutant of B. subtilis. In this paper we describe the construction of a Bt spo0A mutant strain and its use to produce insecticidal crystal proteins. The spo0A gene of Bt was cloned and identified by its ability to transform a B. subtilis spo0A mutant to prototrophy. Its nucleotide sequence is homologous to the B. subtilis gene. The spo0A gene was replaced in the Bt genome with a disrupted copy to give an Spo- strain unable to initiate sporulation. When the cryIIIA gene was cloned in the Bt spo0A mutant, large amounts of toxins were produced and accumulated to form a large crystal inclusion which remained encapsulated within the ghost cell. These encapsulated toxins were highly active against coleopteran larvae. We anticipate that the cryIIIA expression system and the Bt spo0A mutant will provide a convenient process to generate novel formulations of stabilized and environmentally safe Bt-based biopesticides.

The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects

“…The B. thuringiensis 407 Cry − ∆spo0A mutant strain has been described elsewhere (Lereclus et al, 1995). This strain is an asporogenic acrystalliferous mutant.…”

“…The disrupted plcR gene was then inserted between the BamHI and HindIII sites of the thermosensitive plasmid pRN5101, conferring resistance to erythromycin (Em, 5 µg ml − "). The resulting plasmid, pRNB1Km R , was introduced into B. thuringiensis and B. cereus by electroporation (Lereclus et al, 1989) and the chromosomal wild-type copy of plcR was replaced by the disrupted copy as previously described (Lereclus et al, 1995). Mutant colonies had an Em S , Km R phenotype.…”

Regulation of insecticidal crystal protein production in Bacillus thuringiensis

“…The cry40 and cry34 genes of B. thuringiensis subsp, thompsoni also comprise an operon (Brown and Whiteley, 1992;Brown, 1993). In contrast, the crylllA, crylllB, and crylllB2 genes are each associated with downstream orfs that encode crystal proteins of 28-32 kDa, but the orf genes associated with crylllB and crylllB2 appear to be independently transcribed (Donovan et aL, 1992 (Adams etaL, 1991), SpoOF , KinNHknA , and Spo0A Lereclus et aL, 1995). The primary means of temporally and spatially regulating gene expression in B. subtilis is by the transitory expression of vegetative-and sporulation-specific-sigma factor subunits of RNA polymerase (Errington, 1993;Hoch, 1993;and references therein).…”

“…Several studies have shown that B. thuringiensis strains that harbour mutations affecting the spoOA phosphorylation cascade overproduce CrylllA protein Lereclus et aL, 1995) as well as other members of the Crylll class of ICPs (T. Malvar and J. A. Baum, unpublished data).…”

“…Based on these observations and other results, it was proposed that any mutation that blocks the phosphorylation of Spo0A would result in CrylllA overproduction . Subsequently, a spoOA mutant of B. thuringiensis was shown to overproduce CrylllA (Lereclus et aL, 1995 (Agaisse and Lereclus, 1994a). The failure to initiate sporulation via Spo0A-P may affect the down regulation of these regulators which, in turn, prolongs cryltlA transcription.…”