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Journal of Bacteriology, April 2006, p. 3024-3036, Vol. 188, No. 8
0021-9193/06/$08.00+0     doi:10.1128/JB.188.8.3024-3036.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Functional Domains of the Bacillus subtilis Transcription Factor AraR and Identification of Amino Acids Important for Nucleoprotein Complex Assembly and Effector Binding

Irina Saraiva Franco,¹ Luís Jaime Mota,^1, Cláudio Manuel Soares,² and Isabel de Sá-Nogueira^1,3*

Laboratory of Microbial Genetics, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal,¹ Laboratory of Protein Modeling, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal,² Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal³

Received 28 December 2005/ Accepted 8 February 2006

The Bacillus subtilis AraR transcription factor represses at least 13 genes required for the extracellular degradation of arabinose-containing polysaccharides, transport of arabinose, arabinose oligomers, xylose, and galactose, intracellular degradation of arabinose oligomers, and further catabolism of this sugar. AraR exhibits a chimeric organization comprising a small N-terminal DNA-binding domain that contains a winged helix-turn-helix motif similar to that seen with the GntR family and a larger C-terminal domain homologous to that of the LacI/GalR family. Here, a model for AraR was derived based on the known crystal structures of the FadR and PurR regulators from Escherichia coli. We have used random mutagenesis, deletion, and construction of chimeric LexA-AraR fusion proteins to map the functional domains of AraR required for DNA binding, dimerization, and effector binding. Moreover, predictions for the functional role of specific residues were tested by site-directed mutagenesis. In vivo analysis identified particular amino acids required for dimer assembly, formation of the nucleoprotein complex, and composition of the sugar-binding cleft. This work presents a structural framework for the function of AraR and provides insight into the mechanistic mode of action of this modular repressor.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Imperial College London, Center for Molecular Microbiology and Infection, Armstrong Road, Flowers Building—2nd floor, London SW7 2AZ, United Kingdom.

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