High production of CryIVB protein does not lead to formation of crystalline inclusions in Bacillus thuringiensis subsp. israelensis

• Main Authors: S. Panjaisee, S. Charoenpornwatana, S. Pantuwatana, A. Bhumiratana, W. Panbangred
• Other Authors: Chiang Mai University
• Format: Article
• Published: 2018
• Subjects: Biochemistry, Genetics and Molecular Biology; Immunology and Microbiology
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/17918
• Institution: Mahidol University

In this study, we used a 3.7 kb XbaI fragment harbouring the cryIVB gene (with its own promoter) which encodes the 130 kDa (CryIVB) larvicidal protein from B. thuringiensis subsp. israelensis. This was placed downstream to either the cat-86 gene promoter (P(cat-86)), a spore-stage-specific expression promoter) or the bgaB gene promoter (P(bgaB1)) a vegetative-stage-specific expression promoter). These two constructs were then subcloned into pBC16 to obtain pBTC3 and pBTC6. Alternatively, the promoter region of the cryIVB gene was deleted, and truncated genes with 35 and 20 nucleotides remaining upstream from the start codon were obtained. These were transcriptionally fused downstream to P(cat-86)) or P(bgaB) yielding pBTC35L and pBTC20L, or pBTC35F and pBTC20F, respectively. All plasmids constructed were successfully transferred into acrystalliferous B. thuringiensis subsp. israelensis (B.t.i.) strain c4Q2-72 by electroporation. None of the B.t.i. c4Q2-72 transformants containing the above recombinant plasmids, whether vegetative or sporulating, could produce observable crystal structures when cells were observed under the electron microscope. This finding indicated that CryIVB protein alone could not form crystals even when it was synthesized in large quantity. The difference in strength between double promoters or the P(cryIVB) promoter alone in controlling the cryIVB gene was also examined by transcriptional fusion with cat-86 reporter gene in promoter probe cloning vector, pPL703 and transformed into B.t.i. c4Q2-72. The specific activity of chloramphenicol acetyltransferase (CAT) at 6, 12, 24, 36 and 48 h of growth was then compared. It was found that clones harbouring pBTPF6 which contained P(bgaB) together with P(cryIVB) could produce much more CAT (60 to 200-fold) than pBTP603 (P(cat-86)) with P(cryIVB)) and also much more CAT than pPLC1 in which the cat-86 gene was under the control of P(cryIVB) alone. pBTPF6 produced high CAT activity at early stages of growth (15.91 U/mg protein at 6 h) whereas pBTP603 and pPLC1 produced less CAT near sporulation (0.07 to 0.15 U/rag protein). Hence, the construct with P(bgaB) inserted upstream of P(cryIVB) led to high downstream expression during vegetative stages of growth, and crystal formation by CrylVB protein did not occur even though large amounts of protein were produced and accumulated in cells which harboured pBTC6. Although it was a strong promoter, the recombinant plasmid, pBTC6 was not stably maintained in the B.t.i. 4Q2-72 host. By contrast, pBTC1 and pBTC3 seemed to be stable in the B.t.i. host for at least 4 weeks upon daily subculture in media without tetracycline. This result suggests that plasmid stability was affected by overexpression of the gene during early stages of growth.