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The Bacillus thuringiensis PlcR-Regulated Gene inhA2 Is Necessary, but Not Sufficient, for Virulence

Unité Génétique Microbienne et Environnement, 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, France,³ Bayer CropSciences, Ghent B-9000, Belgium²

Received 1 November 2002/ Accepted 20 February 2003

We previously reported that Bacillus thuringiensis strain 407 Cry 32^- secretes a zinc-requiring metalloprotease, InhA2, that is essential for virulence in orally infected insects. Analysis of the inhA2-lacZ transcriptional fusion showed that inhA2 expression is repressed in a PlcR^- background. Using DNase I footprinting experiments, we demonstrated that PlcR activates inhA2 transcription directly by binding to a DNA sequence showing a one-residue mismatch with the previously reported PlcR box. It was previously reported that PlcR is essential for B. thuringiensis virulence in oral infection by contributing to the synergistic properties of the spores on the insecticidal activity of the Cry1C protein. We used complementation experiments to investigate whether the PlcR^- phenotype was due to the absence of InhA2. The results indicated that overexpression of inhA2 in the plcR strain did not restore the wild-type phenotype. However, virulence was fully restored in the inhA2 complemented mutant. Thus, inhA2 is the first example of a PlcR-regulated gene found to be directly involved in virulence. However, it is not sufficient for pathogenicity when the other members of the PlcR regulon are lacking. This suggests that InhA2 may act in concert with other PlcR-regulated gene products to provide virulence.

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