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

被引:35
作者
Gábor, K [1 ]
Veríssimo, CS [1 ]
Cyran, BC [1 ]
ter Horst, P [1 ]
Meijer, NP [1 ]
Smidt, H [1 ]
de Vos, WM [1 ]
van der Oost, J [1 ]
机构
[1] Univ Wageningen & Res Ctr, Microbiol Lab, NL-6703 CT Wageningen, Netherlands
关键词
D O I
10.1128/JB.188.7.2604-2613.2006
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 (CI-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 CI-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 a-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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页码:2604 / 2613
页数:10
相关论文
共 43 条
[1]   Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol -: disulfide status [J].
Åslund, F ;
Zheng, M ;
Beckwith, J ;
Storz, G .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (11) :6161-6165
[2]   Regulation at complex bacterial promoters: how bacteria use different promoter organizations to produce different regulatory outcomes [J].
Barnard, A ;
Wolfe, A ;
Busby, S .
CURRENT OPINION IN MICROBIOLOGY, 2004, 7 (02) :102-108
[3]   LOCATION AND ORIENTATION OF AN ACTIVATING REGION IN THE ESCHERICHIA-COLI TRANSCRIPTION FACTOR, FNR [J].
BELL, A ;
BUSBY, S .
MOLECULAR MICROBIOLOGY, 1994, 11 (02) :383-390
[4]   MOLECULAR GENETIC-ANALYSIS OF AN FNR-DEPENDENT ANAEROBICALLY INDUCIBLE ESCHERICHIA-COLI PROMOTER [J].
BELL, AI ;
COLE, JA ;
BUSBY, SJW .
MOLECULAR MICROBIOLOGY, 1990, 4 (10) :1753-1763
[5]   Structural basis of transcription activation:: the CAP-αCTD-DNA complex [J].
Benoff, B ;
Yang, HW ;
Lawson, CL ;
Parkinson, G ;
Liu, JS ;
Blatter, E ;
Ebright, YW ;
Berman, HM ;
Ebright, RH .
SCIENCE, 2002, 297 (5586) :1562-1566
[6]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[7]   Transcription activation by catabolite activator protein (CAP) [J].
Busby, S ;
Ebright, RH .
JOURNAL OF MOLECULAR BIOLOGY, 1999, 293 (02) :199-213
[8]   Transcription activation at class II CAP-dependent promoters [J].
Busby, S ;
Ebright, RH .
MOLECULAR MICROBIOLOGY, 1997, 23 (05) :853-859
[9]   Structural basis of the redox switch in the OxyR transcription factor [J].
Choi, HJ ;
Kim, SJ ;
Mukhopadhyay, P ;
Cho, S ;
Woo, JR ;
Storz, G ;
Ryu, SE .
CELL, 2001, 105 (01) :103-113
[10]   Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense [J].
Christiansen, N ;
Ahring, BK ;
Wohlfarth, G ;
Diekert, G .
FEBS LETTERS, 1998, 436 (02) :159-162