Molecular genetic manipulation of truncated Cry1C protein synthesis in Bacillus thuringiensis to improve stability and yield

Abstract

Cry1 protoxins of Bacillus thuringiensis are insecticidal 135-kDa proteins synthesized and assembled into parasporal crystals during sporulation. After ingestion, these crystals dissolve in the midgut and active toxins with molecular masses of about 65-kDa are released from the N-terminal half of the molecule by midgut proteases. Direct synthesis of the toxin-containing N-terminal half of Cry1 molecules using recombinant DNA techniques results in a low level of unstable truncated proteins that do not crystallize. In the present study, inclusions of truncated Cry1C (Cry1C-t) were obtained by combining genetic elements from other endotoxin genes and operons that enhance Cry protein synthesis and crystallization. Increased levels of Cry1C-t synthesis were achieved by using cyt1A promoters to drive expression of the 5' half of cry1C that included in the construct the 5' cry3A STAB-SD mRNA stabilizing sequence and the 3' stem-loop transcription terminator. RNA dot blot analysis showed that the STAB-SD and 3' transcriptional termination sequences were important for stabilization of truncated cry1C (cry1C-t) mRNA. A low level of cry1C-t mRNA was present when only the cyt1A promoters were used to express cry1C-t, but no accumulation of Cry1C-t was detected in Western blots. The orientation of the transcription terminator was important to enhancing Cry1C-t synthesis. Inclusion of the 20- and 29-kDa helper protein genes in cry1C-t constructs further enhanced synthesis. The Cry1C-t protein was toxic to Spodoptera exigua larvae, though the toxicity (50% lethal concentration [LC(50)] = 13.2 microg/ml) was lower than that of full-length Cry1C (LC(50) = 1.8 microg/ml). However, transformation of the HD1 isolate of B. thuringiensis subsp. kurstaki with the cry1C-t construct enhanced its toxicity to S. exigua as much as fourfold.

FIG. 1

Summary of vector construction for expression of full-length and truncated cry1C genes. (A) The vectors pPFT3As and pPF1C were used as templates for further construction. The full-length cry1C gene, including its promoter region, was obtained as a 4.8-kb fragment of plasmid 2-44 (see Materials and Methods) partially digested with EcoRI and HindIII. This fragment was treated with the Klenow fragment and inserted into pHT3101 to generate pPF1C. (B) The full-length and truncated cry1C ORFs beginning at the ATG codon were inserted into the SalI-SphI sites and SalI sites of pPF-CH (see Materials and Methods) to generate pPFT1Cs and pPFT1Cs-t, respectively. For cry1C-t without the STAB-SD sequence, the same fragment used for construction of pPFT1Cs-t was inserted into SalI sites of pHTCytA. To add the 3′ TTS to truncated cry1C-t, a 479-bp cry3A termination sequence was inserted into the SphI site of pPFT1Cs-t [pPFT1Cs-3t(+) and pPFT1Cs-3t(−)]. (C) The vectors pPFT2Asf and pPFT11Ast were used as templates for amplification of orf2 and the 20-kDa protein gene. (D) For the 20-kDa protein, a 1.5-kb fragment from pPFT11Ast was inserted into the SphI site of pPFT1Cs-t (pPFT1Cs-20k). For the ORF2 protein gene, a 850-bp fragment from pPFT2Asf was inserted into the XbaI-SalI site of pPF-CH (pPF-ORF2). Then 2.38- and 3.41-kb fragments, obtained from SalI-SphI partial digestion of pPFT1Cs-3t(+) and pPFT1Cs-20k, respectively, were inserted into the SalI-SphI site of pPF-ORF2 to generate pPFT1Csf-3t(+) and pPFT1Csf-20k.

FIG. 2

Transcript levels for different cry1C-t constructs. Lane 1, pPFT1C-t (cyt1A p + cry1C-t); lane 2, pPFT1Cs-t (cyt1A p + STAB-SD + cry1C-t); lane 3, pPFT1Cs-3t(+) (cyt1A p + STAB-SD + cry1C-t + stem-loop [5′-3′]); lane 4, pPFT1Cs-3t(−) (cyt1A p + STAB-SD + cry1C-t + stem-loop [3′-5′]); lane 5, pPFT1Csf-3t(+) (cyt1A p + STAB-SD + orf2 + cry1C-t + stem-loop [5′-3′]); lane 6, pPFT1Cs-20k (cyt1A p + STAB-SD + cry1C-t + 20-kDa protein gene); lane 7, pPFT1Csf-20k (cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene). The ratios shown for lanes 2 through 7 are relative to the value for the dot in lane 1, which was assigned a value of 1. Each value represents the average value (ratio) obtained from three separate experiments. Different letters beneath the ratios indicate that values were significantly different at P = 0.05.

FIG. 3

Synthesis of Cry1C-t by different constructs as determined by SDS-PAGE and Western blot analysis. (A and B) SDS–12% PAGE gel (A) and Western blot of the same gel (B). The relative amounts of Cry1C-t produced by the strains are indicated below the lanes in panel A. Lane 1, pPFT1C-t (cyt1A p + cry1C-t); lane 2, pPFT1Cs-t (cyt1A p + STAB-SD + cry1C-t); lane 3, pPFT1Cs-3t(+) (cyt1A p + STAB-SD + cry1C-t + stem-loop [5′-3′]); lane 4, pPFT1Cs-3t(−) (cyt1A p + STAB-SD + cry1C-t + stem-loop [3′-5′]); lane 5, pPFT1Csf-3t(+) (cyt1A p + STAB-SD + orf2 + cry1C-t + stem-loop [5′-3′]); lane 6, pPFT1Cs-20k (cyt1A p + STAB-SD + cry1C-t + 20-kDa protein gene); lane 7, pPFT1Csf-20k (cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene); lane M, molecular size marker. (C) Control for the Western blot analysis: B. thuringiensis subsp. israelensis, which produces Cry11A and Cyt1A. Lane 1, SDS-PAGE gel; lane 2, Western blot of the same gel. The ratios shown for lanes 3 through 7 are relative to the amount of the 58-kDa protein in lane 2, which was assigned a value of 1. Each value represents the average value (ratio) obtained from three separate experiments. Different letters beneath the ratios indicate that values were significantly different at P = 0.05.

FIG. 4

Phase-contrast micrographs of sporulated cells of B. thuringiensis subsp. israelensis 4Q7 that expressed representative constructs. (A) pPFT1C-t (cyt1A p + cry1C-t); (B) pPFT1Cs-3t(+) (cyt1A p + STAB-SD + cry1C-t + stem-loop [5′-3′]); (C) pPFT1Csf-20k (cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene). The arrows indicate inclusions formed by Cry1C-t.

FIG. 5

Purified inclusions of Cry1C-t as determined by SDS-PAGE and Western blot analysis. (A) SDS–10% PAGE gel; (B) Western blot of the same gel. Lane 1, molecular size marker; lanes 2 and 3, pPFT1Cs-3t(+) (cyt1A p + STAB-SD + cry1C-t + stem-loop [5′-3′]); lanes 4 and 5, pPFT1Csf-20k (cyt1A p + STAB-SD + orf2 + cry1C-t + 20-kDa protein gene). Five micrograms (lanes 3 and 5) or 10 μg (lanes 2 and 4) of protein was loaded and separated.