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Journal of Bacteriology, March 2004, p. 1879-1889, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1879-1889.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

DNA Interaction and Phosphotransfer of the C4-Dicarboxylate- Responsive DcuS-DcuR Two-Component Regulatory System from Escherichia coli

Aly E. Abo-Amer,1 Jonathan Munn,1 Kerry Jackson,1 Murat Aktas,1,{dagger} Paul Golby,1,{ddagger} David J. Kelly,2 and Simon C. Andrews1*

The School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6AJ,1 Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom2

Received 18 September 2003/ Accepted 17 November 2003

The DcuS-DcuR system of Escherichia coli is a two-component sensor-regulator that controls gene expression in response to external C4-dicarboxylates and citrate. The DcuS protein is particularly interesting since it contains two PAS domains, namely a periplasmic C4-dicarboxylate-sensing PAS domain (PASp) and a cytosolic PAS domain (PASc) of uncertain function. For a study of the role of the PASc domain, three different fragments of DcuS were overproduced and examined: they were PASc-kinase, PASc, and kinase. The two kinase-domain-containing fragments were autophosphorylated by [{gamma}-32P]ATP. The rate was not affected by fumarate or succinate, supporting the role of the PASp domain in C4-dicarboxylate sensing. Both of the phosphorylated DcuS constructs were able to rapidly pass their phosphoryl groups to DcuR, and after phosphorylation, DcuR dephosphorylated rapidly. No prosthetic group or significant quantity of metal was found associated with either of the PASc-containing proteins. The DNA-binding specificity of DcuR was studied by use of the pure protein. It was found to be converted from a monomer to a dimer upon acetylphosphate treatment, and native polyacrylamide gel electrophoresis suggested that it can oligomerize. DcuR specifically bound to the promoters of the three known DcuSR-regulated genes (dctA, dcuB, and frdA), with apparent KDs of 6 to 32 µM for untreated DcuR and <=1 to 2 µM for the acetylphosphate-treated form. The binding sites were located by DNase I footprinting, allowing a putative DcuR-binding motif [tandemly repeated (T/A)(A/T)(T/C)(A/T)AA sequences] to be identified. The DcuR-binding sites of the dcuB, dctA, and frdA genes were located 27, 94, and 86 bp, respectively, upstream of the corresponding +1 sites, and a new promoter was identified for dcuB that responds to DcuR.

* Corresponding author. Mailing address: The School of Animal and Microbial Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading RG66AJ, United Kingdom. Phone: 44 118 378 8463. Fax: 44 118 931 0180 or 44 118 378 6537. E-mail: s.c.andrews{at}reading.ac.uk.

{dagger} Present address: Heinrich Heine University of Duesseldorf, Duesseldorf, Germany.

{ddagger} Present address: VLA, Weybridge, Surrey KT15 3NB, United Kingdom.

Journal of Bacteriology, March 2004, p. 1879-1889, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1879-1889.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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