Investigation of Bacillus Thuringiensis Plasmid Instability and its Effect on the Synthesis of Crystals

Abstract

In order to explore plasmid instability in Bt, four Bt strains belonging to two Bt subspecies were cultured at 42°C for 9 days. HD1 and QBT376 belong to subspecies kurstaki, while H14 and QBT218 belong to subspecies israelensis. Results showed 100% crystal loss for H14 and QBT218, while 76% and 90% crystal loss for HD1 and QBT376, respectively, showing that cry-carrying plasmids are more stable in Bt kurs. than in Bt isr.. HD1, QBT376, and QBT218 cured clones showed significant protease activity compared to their non-cured counterparts. Microscopic observation revealed the delay of sporulation for high number of HD1 and QBT376 cry- clones, while the absence of spores in several H14 and QBT218 cry- clones. Spo-cry- clones of Bti strains had irregular elongated cell shape. Kinetics/day of plasmid curing for H14 and QBT218 showed H14 to have higher pBtoxis plasmid stability. The number of vegetative cells in Bti strains increased with the increase of curing period. As an attempt to create hybrid Bt strains, cry1Aa gene was extracted to transform cured and non-cured strains.