title: Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity ofBacillus thuringiensisToxin in Cotton Bollworm,Helicoverpa armigera.
abstract: Bacillus thuringiensistoxin proteins are deployed in transgenic plants for pest management. The present studies were aimed at characterization of gut bacterial proteases involved in activation of inactive Cry1Ac protoxin (pro-Cry1Ac) to active toxin inHelicoverpa armigera.Bacterial strains were isolated fromH. armigeramidgut and screened for their proteolytic activation toward pro-Cry1Ac. Among 12 gut bacterial isolates seven isolates showed proteolytic activity, and proteases from three isolates (IVS1, IVS2, and IVS3) were found to be involved in the proteolytic conversion of pro-Cry1Ac into active toxin. The proteases from IVS1, IVS2, and IVS3 isolates were purified to 11.90-, 15.50-, and 17.20-fold, respectively. The optimum pH and temperature for gut bacterial protease activity was 8.0 and 40°C. Maximum inhibition of total proteolytic activity was exerted by phenylmethane sulfonyl fluoride followed by EDTA. Fluorescence zymography revealed that proteases from IVS1, IVS2, and IVS3 were chymotrypsin-like and showing protease band at ~15, 65, and 15 kDa, respectively. Active Cry1Ac formed from processing pro-Cry1Ac by gut bacterial proteases exhibited toxicity towardH. armigera. The gut bacterial isolates IVS1, IVS2, and IVS3 showed homology withB. thuringiensis(CP003763.1),Vibrio fischeri(CP000020.2), andEscherichia coli(CP011342.1), respectively. Proteases produced by midgut bacteria are involved in proteolytic processing ofB. thuringiensisprotoxin and play a major role in inducing pathogenicity ofB. thuringiensistoxins inH. armigera.
authors.affiliation: Department of Entomology, International Crops Research Institute for the Semi-Arid Tropics, PatancheruIndia; Department of Biochemistry, Gulbarga University, KalaburagiIndia.
authors.forename: Visweshwar
authors.initials: V
authors.lastname: Regode
authors.affiliation: Department of Biochemistry, Gulbarga University, Kalaburagi India.
authors.forename: Sreeramulu
authors.initials: S
authors.lastname: Kuruba
authors.affiliation: Department of Entomology, International Crops Research Institute for the Semi-Arid Tropics, Patancheru India.
authors.forename: Akbar S
authors.initials: AS
authors.lastname: Mohammad
authors.affiliation: Department of Entomology, International Crops Research Institute for the Semi-Arid Tropics, Patancheru India.
authors.forename: Hari C
authors.initials: HC
authors.lastname: Sharma
keywords: Cry1Ac proteins; Helicoverpa armigera; midgut bacteria; proteases; transgenics