Characterization of CprK1, a CRP/FNR-type transcriptional regulator of halorespiration from Desulfitobacterium hafniense.
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.
Laboratory of Microbiology, Wageningen University and Research Centre, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. krisztina.gabor@wur.nl
Krisztina
K
Gábor
Carla S
CS
Veríssimo
Barbara C
BC
Cyran
Paul
P
Ter Horst
Nienke P
NP
Meijer
Hauke
H
Smidt
Willem M
WM
de Vos
John
J
van der Oost
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045858:Desulfitobacterium / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D010084:Oxidation-Reduction; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D014157:Transcription Factors