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Applied and Environmental Microbiology, April 2003, p. 2298-2305, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2298-2305.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of TsaR, an Oxygen-Sensitive LysR-Type Regulator for the Degradation of p-Toluenesulfonate in Comamonas testosteroni T-2

Tewes Tralau, Jörg Mampel, Alasdair M. Cook, and Jürgen Ruff^*

Department of Biology, The University of Konstanz, D-78457 Konstanz, Germany

Received 3 October 2002/ Accepted 23 January 2003

TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpressed as TsaR^His. TsaR^His was subject to reversible inactivation by oxygen, which markedly influenced the experimental approaches used. Gel filtration showed TsaR^His to be a monomer in solution. Overexpressed TsaR^His bound specifically to three regions within the promoter between the divergently transcribed tsaR and tsaMBCD. The dissociation constant (K_D) for the whole promoter region was about 0.9 µM, and the interaction was a function of the concentration of the ligand TSA. A regulatory model for this LysR-type regulator is proposed on the basis of these data.

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* Corresponding author. Mailing address: Department of Biology, The University of Konstanz, Universitätsstr. 10, D-78457 Konstanz, Germany. Phone: 49 7531 88 2100. Fax: 49 7531 88 2966. E-mail: juergen.ruff{at}uni-konstanz.de.

Present address: School of Biological Sciences, University of Manchester, Manchester, United Kingdom.

Present address: Institute of Biotechnology, Swiss Federal Institute of Technology, Zürich, Switzerland.

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Applied and Environmental Microbiology, April 2003, p. 2298-2305, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2298-2305.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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