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Journal of Bacteriology, December 2002, p. 6602-6614, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6602-6614.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Arginine Operator Binding by Heterologous and Chimeric ArgR Repressors from Escherichia coli and Bacillus stearothermophilus

Anahit Ghochikyan,¹ Iovka Miltcheva Karaivanova,¹ Michèle Lecocq,¹ Patricia Vusio,² Marie-Claire Arnaud,¹ Marina Snapyan,¹ Pierre Weigel,¹ Laetitia Guével,¹ Malcolm Buckle,^3, and Vehary Sakanyan^1,4*

Laboratoire de Biotechnologie, FRE CNRS 2230, Unité Biocatalyse, Faculté des Sciences et des Techniques, Université de Nantes, 44322 Nantes,¹ IFR 26, INSERM, 44035 Nantes,² Laboratoire d'Enzymologie et Cinétique Structurale, UMR 8532 du CNRS, LBPA, Ecole Normale Supérieure de Cachan, 94235 Cachan,³ ProtNeteomix, Université de Nantes, 44322 Nantes, France⁴

Received 23 May 2002/ Accepted 27 August 2002

Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the 4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the 4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.

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* Corresponding author. Mailing address: Laboratoire de Biotechnologie, FRE CNRS 2230 Unité Biocatalyse, Faculté des Sciences et des Techniques, Université de Nantes, 2 rue de la Houssinière, 44322 Nantes, France. Phone: (33) 251125620. Fax: (33) 251125637. E-mail: Vehary.Sakanyan{at}chimbio.univ-nantes.fr.

Present address: Institut Gustave Roussy UMR 8532 du CNRS PR II, 94805 Villejuif, France.

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Journal of Bacteriology, December 2002, p. 6602-6614, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6602-6614.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.