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Applied and Environmental Microbiology, December 2005, p. 8903-8910, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8903-8910.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

FlhA Influences Bacillus thuringiensis PlcR-Regulated Gene Transcription, Protein Production, and Virulence

Laurent Bouillaut,¹ Nalini Ramarao,¹ Christophe Buisson,¹ Nathalie Gilois,¹ Michel Gohar,^1, Didier Lereclus,¹ and Christina Nielsen-LeRoux^1,2*

Unité Génétique Microbienne et Environnement, Institut National de Recherche Agronomique, La Minière, 78285 Guyancourt Cedex, France,¹ Institut Pasteur, Département de Microbiologie Fondamentale et Médicale, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France²

Received 29 August 2005/ Accepted 31 August 2005

Bacillus thuringiensis and Bacillus cereus are closely related. B. thuringiensis is well known for its entomopathogenic properties, principally due to the synthesis of plasmid-encoded crystal toxins. B. cereus appears to be an emerging opportunistic human pathogen. B. thuringiensis and B. cereus produce many putative virulence factors which are positively controlled by the pleiotropic transcriptional regulator PlcR. The inactivation of plcR decreases but does not abolish virulence, indicating that additional factors like flagella may contribute to pathogenicity. Therefore, we further analyzed a mutant (B. thuringiensis 407 Cry^– flhA) previously described as being defective in flagellar apparatus assembly and in motility as well as in the production of hemolysin BL and phospholipases. A large picture of secreted proteins was obtained by two-dimensional electrophoresis analysis, which revealed that flagellar proteins are not secreted and that production of several virulence-associated factors is reduced in the flhA mutant. Moreover, we quantified the effect of FlhA on plcA and hblC gene transcription. The results show that the flhA mutation results in a significant reduction of plcA and hblC transcription. These results indicate that the transcription of several PlcR-regulated virulence factors is coordinated with the flagellar apparatus. Consistently, the flhA mutant also shows a strong decrease in cytotoxicity towards HeLa cells and in virulence against Galleria mellonella larvae following oral and intrahemocoelic inoculation. The decrease in virulence may be due to both a lack of flagella and a lower production of secreted factors. Hence, FlhA appears to be an essential virulence factor with a pleiotropic role.

Present address: Unité Microbiologie et Génétique Moléculaire, Institut National de Recherche Agronomique, 78850 Thiverval-Grignon, France.

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