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Applied and Environmental Microbiology, September 2004, p. 5290-5297, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5290-5297.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides

Tina Hölscher,1 Rosa Krajmalnik-Brown,2 Kirsti M. Ritalahti,2 Friedrich von Wintzingerode,3 Helmut Görisch,1 Frank E. Löffler,2,4* and Lorenz Adrian1

Fachgebiet Technische Biochemie, Institut für Biotechnologie, Technische Universität Berlin, Berlin, Germany,1 School of Civil and Environmental Engineering,2 School of Biology, Georgia Institute of Technology, Atlanta, Georgia,4 Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Berlin, Germany3

Received 15 December 2003/ Accepted 11 May 2004

Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene- and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains.

* Corresponding author. Mailing address: School of Civil and Environmental Engineering, 311 Ferst Dr., 3228 ES&T Building, Georgia Institute of Technology, Atlanta, GA 30332-0512. Phone: (404) 894-0279. Fax: (404) 894-8266. E-mail: frank.loeffler{at}ce.gatech.edu.

Applied and Environmental Microbiology, September 2004, p. 5290-5297, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5290-5297.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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