title: Regulation of anaerobic dehalorespiration by the transcriptional activator CprK.
abstract: 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 mum, 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 approximately 10-fold (K(d) = 0.4 mum, 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.
authors.affiliation: Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
authors.forename: Stelian M
authors.initials: SM
authors.lastname: Pop
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Kolarik
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Ragsdale
mesh_terms: D001483:Base Sequence; D002733:Chlorophenols / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D045858:Desulfitobacterium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015533:Transcriptional Activation
chemical_list: D002733:Chlorophenols; D004247:DNA; D010088:Oxidoreductases