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Journal of Bacteriology, October 2004, p. 6465-6476, Vol. 186, No. 19
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.19.6465-6476.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

Puneet Khandelwal,1 Devapriya Choudhury,1 Ajanta Birah,2 M. K. Reddy,3 Gorakh Prasad Gupta,2 and Nirupama Banerjee3*

Centre for Biotechnology, Jawaharlal Nehru University,1 Division of Entomology, IARI,2 International Centre for Genetic Engineering and Biotechnology, New Delhi, India3

Received 11 June 2004/ Accepted 23 June 2004

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.

* Corresponding author. Mailing address: International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. Phone: 91-11-26181242. Fax: 91-11-26162316. E-mail: nirupama{at}icgeb.res.in.

Journal of Bacteriology, October 2004, p. 6465-6476, Vol. 186, No. 19
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.19.6465-6476.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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