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Applied and Environmental Microbiology, August 2004, p. 4532-4537, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4532-4537.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Purification, Cloning, and Sequencing of a 3,5-Dichlorophenol Reductive Dehalogenase from Desulfitobacterium frappieri PCP-1

Jacinthe Thibodeau, Annie Gauthier, Marie Duguay, Richard Villemur, François Lépine, Pierre Juteau, and Réjean Beaudet^*

Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada H7V 1B7

Received 7 January 2004/ Accepted 26 April 2004

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22°C, compared to 30 and 37°C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K_m for 3,5-dicholorophenol was 49.3 ± 3.1 µM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.

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* Corresponding author. Mailing address: Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Laval, QC H7V 1B7, Canada. Phone: (450) 687-5010. Fax: (450) 686 5501. E-mail: rejean.beaudet{at}inrs-iaf.uquebec.ca.

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Applied and Environmental Microbiology, August 2004, p. 4532-4537, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4532-4537.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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