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J. Biol. Chem., Vol. 279, Issue 48, 49910-49918, November 26, 2004

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Regulation of Anaerobic Dehalorespiration by the Transcriptional Activator CprK^*

Stelian M. Pop, Ryan J. Kolarik, and Stephen W. Ragsdale

From the Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664

Desulfomonile, Desulfitobacterium, and Dehalobacter are anaerobic microbes that can derive energy from the reductive dehalogenation of chlorinated organic compounds, many of which are environmental pollutants. There is very little information about how anaerobic dehalorespiration is regulated. An open reading frame within the Desulfitobacterium dehalogenans chlorophenol reductase (cpr) gene cluster (cprK) was proposed to be a transcriptional regulatory protein (Smidt, H., van Leest, M., van der Oost, J., and deVos, W. M. (2000) J. Bacteriol. 182, 5683–5691). We have cloned, actively overexpressed in Escherichia coli, and purified to homogeneity the D. dehalogenans CprK. The results of electrophoretic mobility shift assays, DNA footprinting studies, and promoter-lac fusion experiments indicate that CprK is a transcriptional activator of the cpr gene cluster. CprK binds 3-chloro-4-hydroxyphenylacetate (CHPA) with high affinity (K_d = 3.5 µM, determined by isothermal titration calorimetry), which promotes its specific interaction with a DNA sequence (TTAAT-N4-ACTAA) located upstream of the –35 and –10 promoter regions of several cpr genes and activates transcription of these genes. Binding to the upstream "box" sequence increases the affinity of CprK for CHPA by 10-fold (K_d = 0.4 µM, determined by electrophoretic mobility shift assays). Chlorophenylacetate, which lacks the ortho-hydroxy group, and hydroxyphenylacetate, lacking the chlorine group, do not activate transcription or promote DNA binding, even at millimolar concentrations, at least 1000-fold higher than the K_d value for CHPA. Lacking metals, CprK is oxygen-sensitive. Oxidation by diamide, which converts thiols to the disulfide, inactivates CprK, and reduction of the oxidized protein by dithiothreitol fully restores DNA binding, indicating that CprK is redox-regulated and is active only when reduced. This is the first reported characterization of a transcriptional regulator of anaerobic dehalorespiration.

Received for publication, August 17, 2004 , and in revised form, September 13, 2004.

^* The titration calorimeter was purchased with funds from National Institutes of Health Grant 1P20RR17675 to help support the Biophysics Core Instrumentation Core of the Redox Biology Center at the University of Nebraska, Lincoln. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Beadle Center, University of Nebraska, Lincoln, NE 68588-0664. Tel.: 402-472-2943; Fax: 402-472-7842; E-mail: sragsdale1{at}unl.edu.

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