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. 2006 Apr;188(7):2604-13.
doi: 10.1128/JB.188.7.2604-2613.2006.

Characterization of CprK1, a CRP/FNR-type transcriptional regulator of halorespiration from Desulfitobacterium hafniense

Krisztina Gábor  1 , Carla S Veríssimo, Barbara C Cyran, Paul Ter Horst, Nienke P Meijer, Hauke Smidt, Willem M de Vos, John van der Oost
Affiliations

Characterization of CprK1, a CRP/FNR-type transcriptional regulator of halorespiration from Desulfitobacterium hafniense

Krisztina Gábor et al. J Bacteriol. 2006 Apr.

Abstract

The recently identified CprK branch of the CRP (cyclic AMP receptor protein)-FNR (fumarate and nitrate reduction regulator) family of transcriptional regulators includes proteins that activate the transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. Here we report the characterization of the CprK1 protein from Desulfitobacterium hafniense, an anaerobic low-G+C gram-positive bacterium that is capable of reductive dechlorination of 3-chloro-4-hydroxyphenylacetic acid (Cl-OHPA). The gene encoding CprK1 was cloned and functionally overexpressed in Escherichia coli, and the protein was subsequently purified to homogeneity. To investigate the interaction of CprK1 with three of its predicted binding sequences (dehaloboxes), we performed in vitro DNA-binding assays (electrophoretic mobility shift assays) as well as in vivo promoter probe assays. Our results show that CprK1 binds its target dehaloboxes with high affinity (dissociation constant, 90 nM) in the presence of Cl-OHPA and that transcriptional initiation by CprK1 is influenced by deviations in the dehaloboxes from the consensus TTAAT----ATTAA sequence. A mutant CprK1 protein was created by a Val-->Glu substitution at a conserved position in the recognition alpha-helix that gained FNR-type DNA-binding specificity, recognizing the TTGAT----ATCAA sequence (FNR box) instead of the dehaloboxes. CprK1 was subject to oxidative inactivation in vitro, most likely caused by the formation of an intermolecular disulfide bridge between Cys11 and Cys200. The possibility of redox regulation of CprK1 by a thiol-disulfide exchange reaction was investigated by using two Cys-->Ser mutants. Our results indicate that a Cys11-Cys200 disulfide bridge does not appear to play a physiological role in the regulation of CprK1.

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Figures

FIG. 1.
FIG. 1.
(A) Desulfitobacterium hafniense chlorophenol reductive dehalogenase gene cluster. (B) Alignment of three D. hafniense intergenic regions containing 14-bp inverted repeats termed “dehaloboxes” (DB1 to DB3) with the corresponding cprE-cprB intergenic region from D. dehalogenans. Positions of dehaloboxes (boldfaced), putative Pribnow boxes (underlined), and the transcription start site previously mapped in D. dehalogenans are indicated with arrows above the DNA sequence alignment. The proposed consensus sequence for dehaloboxes is given below the sequence alignment.
FIG. 2.
FIG. 2.
Purification of CprK1. The SDS-PAGE gel shows consecutive steps of CprK1 purification: Lane 1, cell extract (35 μg); lane 2, heparin affinity chromatography (35 μg); lane 3, gel filtration chromatography (12 μg).
FIG. 3.
FIG. 3.
Interaction of purified CprK1 with the cprE-cprB intergenic region containing dehalobox DB3. An electrophoretic mobility shift assay was performed in the presence (+) or absence (−) of 25 μM Cl-OHPA or 250 μM 4-OHPA using approximately 1 nM 32P-labeled DNA and increasing amounts of CprK1 protein. Concentrations of CprK1 in lanes are as follows, from left to right: 0, 10, 20, 50, 100, 200, 300, 500, 1,500, 1,500, and 1,500 nM.
FIG. 4.
FIG. 4.
Interaction of CprK1 with dehaloboxes DB1, DB2, and DB3. An electrophoretic mobility shift assay was carried out in the presence (+) or absence (−) of 100 nM CprK1 protein, 25 μM Cl-OHPA, and one of the three 32P-labeled DNA-containing dehaloboxes DB1, DB2, and DB3 as indicated above the gel.
FIG. 5.
FIG. 5.
In vivo DNA-binding properties of CprK1. In E. coli cells CprK1 was overproduced in the presence of one of the three D. hafniense promoters containing dehalobox DB1, DB2, or DB3 fused with the lacLM genes. Aerobic E. coli cultures were grown under two different conditions: in the presence of 20 mM Cl-OHPA (solid bars) or in the absence of the effector (shaded bars). Promoter activity was expressed by measuring β-galactosidase activity (Miller units).
FIG. 6.
FIG. 6.
Sequence alignment of E. coli CRP and FNR proteins with D. dehalogenans CprK and five D. hafniense CprK homologues (CprK1 to CprK5). Essential FNR/CRP residues (AR) previously identified to be involved in RNA polymerase contact are indicated above the alignment as follows: rectangles, AR1; circles, AR2; diamonds, AR3. The secondary structure of E. coli CRP is given below the sequence alignment (∼, α-helix; >, β-sheet). Vertical arrows indicate amino acids that are conserved in most CprK homologues and correspond to AR1, AR2, or AR3. In boxes, amino acids are highlighted that correspond to the position of the nonconserved Cys11 and Cys200 residues in D. hafniense CprK1, capable of intermolecular disulfide bridge formation, and to the position of Val192 in the recognition α-helix of CprK1, which is conserved in all CprK homologues and corresponds to the Glu181 and Glu209 residues, essential for specific DNA binding in CRP and FNR, respectively. Protein accession numbers (based on the D. hafniense genome sequence, version of 24 June 2005) are as follows: ZP_00558871.1 (CprK1), ZP_00558892.1 (CprK2), ZP_00558887.1 (CprK3), ZP_00560926.1 (CprK4), and ZP_00559167.1 (CprK5).
FIG. 7.
FIG. 7.
Transcriptional activation by the FNR-type mutant CprK1(V192E). In vivo promoter probe assays were carried out with aerobically grown E. coli JM109(DE3) cells overproducing wild-type CprK1 (solid bars) or CprK1(V192E) (dark shaded bars) or carrying an empty pET24d expression vector (light shaded bars). All experiments were done in the presence of 20 mM Cl-OHPA with cells carrying either a DB3::lacLM promoter fusion or a DB3FNR::lacLM promoter fusion.
FIG. 8.
FIG. 8.
Effect of redox state of CprK1 on its activity. (A) SDS-PAGE gel of purified wild-type CprK1, CprK1(C11S), and CprK1(C200S) proteins (5.8 μg) that were heat denatured in the presence (+) or absence (−) of 5% β-mercaptoethanol (β-ME). CprK1(C11S) and CprK1(C200S) proteins each contain a C-terminal six-His tag, causing a slight difference in their mobilities compared to that of wild-type CprK1 on SDS-PAGE gels but leaving other properties of the proteins unaffected. (B) In vivo promoter probe assay using E. coli JM109(DE3) cells harboring plasmid pWUR166 with a DB3::lacLM promoter fusion and a pET24d plasmid derivative that overproduces either wild-type CprK1, CprK1(C11S), or CprK1(C200S) protein. Cells were grown either in the presence of 20 mM Cl-OHPA (solid bars) or in the absence of the effector (shaded bars).
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