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 M, 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 ϳ10-fold (K d ‫؍‬ 0.4 M, 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.

The restricted metabolism of the obligate organohalide respiring bacterium Dehalobacter restrictus: lessons from tiered functional genomics

“…Most of rdh gene clusters are also associated with one rdhK subunit in various orientations. The rdhK-encoded proteins clearly belong to the large family of CRP/Fnr regulatory proteins from which CprK members of Desulfitobacterium dehalogenans and Desulfitobacterium hafniense DCB-2 were extensively studied and represent the paradigmatic DNA-binding regulatory protein for the respective chlorophenol reductive dehalogenase (cpr) operons [48][49][50][51][52][53][54][55][56] figure 3). First, a strong correlation could be established between the level of sequence identity (see also electronic supplementary material, table S7) and the genetic organization of the predicted rdh operons.…”

Transcriptional Activation by CprK1 Is Regulated by Protein Structural Changes Induced by Effector Binding and Redox State

“…Upon binding of CHPA, CprK activates transcription of dehalobox-containing promoters in D. dehalogenans (11,12). DNA binding studies have shown that CprK1 and CprK are only capable of binding its target DNA when the protein is in its reduced state (10,12). Recently, the x-ray structures of oxidized CprK1 from D. hafniense in complex with CHPA and reduced CprK from D. dehalogenans in its unliganded form have been determined by Joyce et al (13).…”

“…Previously, a close homologue called CprK was identified in the related halorespiring bacterium, D. dehalogenans, which shows 91% sequence identity with CprK1 from D. hafniense. Upon binding of CHPA, CprK activates transcription of dehalobox-containing promoters in D. dehalogenans (11,12). DNA binding studies have shown that CprK1 and CprK are only capable of binding its target DNA when the protein is in its reduced state (10,12).…”

Retentive Memory of Bacteria: Long-Term Regulation of Dehalorespiration in Sulfurospirillum multivorans

“…When the cultures were grown with alternative electron acceptors, such as fumarate or nitrate, CprA transcription ceased within the first subcultivation and was induced within less than 30 min (21). A CRP/FNR-like transcriptional activator, CprK, encoded within the cpr operon, was shown to interact with the DNA upstream of the cpr promoter region only when Cl-OHPA was bound to the protein (18). A direct role of CprK in the induction of dehalogenase gene expression in D. dehalogenans was assumed, and a model for transcriptional regulation of cprA expression was developed (13).…”