Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera
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Visweshwar Regode, Kuruba Sreeramulu, Mohammad S. Akbar, Hari C. Sharma. (6/10/2016). Isolation and Characterization of Gut Bacterial Proteases Involved in Inducing Pathogenicity of Bacillus thuringiensis Toxin in Cotton Bollworm, Helicoverpa armigera. Frontiers in Microbiology, 7: 1567.
Abstract
Bacillus thuringiensis toxin 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
in Helicoverpa armigera. Bacterial strains were isolated from H. armigera midgut 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 proCry1Ac
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 chymotrypsinlike
and showing protease band at ∼15, 65, and 15 kDa, respectively. Active Cry1Ac
formed from processing pro-Cry1Ac by gut bacterial proteases exhibited toxicity toward
H. armigera. The gut bacterial isolates IVS1, IVS2, and IVS3 showed homology
with B. thuringiensis (CP003763.1), Vibrio fischeri (CP000020.2), and Escherichia coli
(CP011342.1), respectively. Proteases produced by midgut bacteria are involved in
proteolytic processing of B. thuringiensis protoxin and play a major role in inducing
pathogenicity of B. thuringiensis toxins in H. armigera.