Y Shi1, W Xu, M Yuan, M Tang, J Chen, Y Pang. 1. State Key Laboratory for Biocontrol and Institute of Entomology, Zhongshan (Sun Yat-sen) University, Guangzhou, P.R. China.
Abstract
AIMS: To determine the expression time courses and high expression level of Vip2A(c) and Vip1A(c) in Bacillus thuringiensis, and survey their insecticidal toxicity and insecticidal spectrum. METHODS AND RESULTS: A kind of new vegetative insecticidal toxin genes encoded by a single operon from B. thuringiensis had been cloned and sequenced. The individual genes, 5-terminus truncated genes and the operon were respectively expressed in Escherichia coli. Only N-terminus deleted Vip2A(c) and Vip1A(c) proteins could be purified by Ni-NTA agarose, while others were processed and their N-terminal signal peptides were cleaved. The individual genes and the operon were also expressed in B. thuringiensis. Both proteins were mostly secreted into the cell supernatants. The expression level of Vip1A(c) was influenced because of the interruption of vip2A(c) gene on the operon. Bioassays showed that neither separate protein nor both performed any toxicity against tested lepidopteran and coleopteran insects. CONCLUSIONS: Vip2A(c) and Vip1A(c) have similar secretion mechanism in E. coli and B. thuringiensis. Vip1A(c) remained its high expression level only when being expressed with vip2A(c) gene as an operon in B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of vip2A(c) and vip1A(c) genes in E. coli and B. thuringiensis were investigated. This would help to make clear the secretion mechanism of VIP proteins and study the function of ADP-ribosyltransferase Vip2.
AIMS: To determine the expression time courses and high expression level of Vip2A(c) and Vip1A(c) in Bacillus thuringiensis, and survey their insecticidal toxicity and insecticidal spectrum. METHODS AND RESULTS: A kind of new vegetative insecticidal toxin genes encoded by a single operon from B. thuringiensis had been cloned and sequenced. The individual genes, 5-terminus truncated genes and the operon were respectively expressed in Escherichia coli. Only N-terminus deleted Vip2A(c) and Vip1A(c) proteins could be purified by Ni-NTAagarose, while others were processed and their N-terminal signal peptides were cleaved. The individual genes and the operon were also expressed in B. thuringiensis. Both proteins were mostly secreted into the cell supernatants. The expression level of Vip1A(c) was influenced because of the interruption of vip2A(c) gene on the operon. Bioassays showed that neither separate protein nor both performed any toxicity against tested lepidopteran and coleopteran insects. CONCLUSIONS: Vip2A(c) and Vip1A(c) have similar secretion mechanism in E. coli and B. thuringiensis. Vip1A(c) remained its high expression level only when being expressed with vip2A(c) gene as an operon in B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of vip2A(c) and vip1A(c) genes in E. coli and B. thuringiensis were investigated. This would help to make clear the secretion mechanism of VIP proteins and study the function of ADP-ribosyltransferase Vip2.
Authors: Cliff S Han; Gary Xie; Jean F Challacombe; Michael R Altherr; Smriti S Bhotika; Nancy Brown; David Bruce; Connie S Campbell; Mary L Campbell; Jin Chen; Olga Chertkov; Cathy Cleland; Mira Dimitrijevic; Norman A Doggett; John J Fawcett; Tijana Glavina; Lynne A Goodwin; Lance D Green; Karen K Hill; Penny Hitchcock; Paul J Jackson; Paul Keim; Avinash Ramesh Kewalramani; Jon Longmire; Susan Lucas; Stephanie Malfatti; Kim McMurry; Linda J Meincke; Monica Misra; Bernice L Moseman; Mark Mundt; A Christine Munk; Richard T Okinaka; B Parson-Quintana; Lee Philip Reilly; Paul Richardson; Donna L Robinson; Eddy Rubin; Elizabeth Saunders; Roxanne Tapia; Judith G Tesmer; Nina Thayer; Linda S Thompson; Hope Tice; Lawrence O Ticknor; Patti L Wills; Thomas S Brettin; Paul Gilna Journal: J Bacteriol Date: 2006-05 Impact factor: 3.490