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Journal of Bacteriology, June 2002, p. 3296-3304, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3296-3304.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route

Sinda Fedhila,1* Patricia Nel,1 and Didier Lereclus1,2

Unité de Recherches de Lutte Biologique, Institut National de la Recherche Agronomique, La Minière, 78285 Guyancourt Cedex,1 Unité de Biochimie Microbienne, Centre National de la Recherche Scientifique (URA2172), Institut Pasteur, 75724 Paris Cedex 15, France2

Received 27 November 2001/ Accepted 26 March 2002

The entomopathogenic bacterium Bacillus thuringiensis is known to secrete a zinc metalloprotease (InhA) that specifically cleaves antibacterial peptides produced by insect hosts. We identified a second copy of the inhA gene, named inhA2, in B. thuringiensis strain 407 Cry-. The inhA2 gene encodes a putative polypeptide showing 66.2% overall identity with the InhA protein and harboring the zinc-binding domain (HEXXH), which is characteristic of the zinc-requiring metalloproteases. We used a transcriptional inhA2'-lacZ fusion to show that inhA2 expression is induced at the onset of the stationary phase and is overexpressed in a Spo0A minus background. The presence of a reverse Spo0A box in the promoter region of inhA2 suggests that Spo0A directly regulates the transcription of inhA2. To determine the role of the InhA and InhA2 metalloproteases in pathogenesis, we used allelic exchange to isolate single and double mutant strains for the two genes. Spores and vegetative cells of the mutant strains were as virulent as those of the parental strain in immunized Bombyx mori larvae infected by the intrahemocoelic route. Exponential phase cells of all the strains displayed the same in vitro potential for colonizing the vaccinated hemocoel. We investigated the synergistic effect of the mutant strain spores on the toxicity of Cry1C proteins against Galleria mellonella larvae infected via the oral pathway. The spores of {Delta}inhA2 mutant strain were ineffective in providing synergism whereas those of the {Delta}inhA mutant strain were not. These results indicate that the B. thuringiensis InhA2 zinc metalloprotease has a vital role in virulence when the host is infected via the oral route.

* Corresponding author. Mailing address: Unité de Recherches de Lutte Biologique, Institut National de la Recherche Agronomique, La Minière, 78285 Guyancourt Cedex, France. Phone: 33 1 30 83 36 36. Fax: 33 1 30 43 80 97. E-mail: sindah{at}jouy.inra.fr.

Journal of Bacteriology, June 2002, p. 3296-3304, Vol. 184, No. 12
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.12.3296-3304.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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