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Applied and Environmental Microbiology, May 2007, p. 3404-3411, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.00021-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cloning, Functional Characterization, and Mode of Action of a Novel Insecticidal Pore-Forming Toxin, Sphaericolysin, Produced by Bacillus sphaericus{triangledown}

Hisashi Nishiwaki, Kenta Nakashima, Chiharu Ishida, Tadayuki Kawamura, and Kazuhiko Matsuda*

Department of Applied Biological Chemistry, School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan

Received 5 January 2007/ Accepted 20 March 2007

An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.

* Corresponding author. Mailing address: Department of Applied Biological Chemistry, School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan. Phone: 81-742-43-7153. Fax: 81-742-43-1445. E-mail: kmatsuda{at}nara.kindai.ac.jp

{triangledown} Published ahead of print on 30 March 2007.

Applied and Environmental Microbiology, May 2007, p. 3404-3411, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.00021-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hu, X., Fan, W., Han, B., Liu, H., Zheng, D., Li, Q., Dong, W., Yan, J., Gao, M., Berry, C., Yuan, Z. (2008). Complete Genome Sequence of the Mosquitocidal Bacterium Bacillus sphaericus C3-41 and Comparison with Those of Closely Related Bacillus Species. J. Bacteriol. 190: 2892-2902 [Abstract] [Full Text]  


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