Multiple nonidentical reductive-dehalogenase-homologous genes are common in Dehalococcoides

Abstract

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.

FIG. 1.

Schematic diagram of gene fragments amplified from Dehalococcoides sp. strain CBDB1 and strain FL2. (A) Sequences identified with the primer pair RRF2-B1R. (B) Sequence rdh9_CBDB1 obtained with primers mern2 and mern5 and extended by additional PCR amplification with the primer pair mintF-B1R (shaded area). (C) Sequences amplified with the primer pair fdehal-rdehal. orfA represents the open reading frame encoding the catalytically active reductive dehalogenase, and orfB is the open reading frame encoding a small hydrophobic B protein. Tat signal, twin-arginine translocation signal sequence including the RRXFXK consensus motif. The scale bar indicates nucleotide position (1 = start codon of orfA).

FIG. 2.

Phylogenetic analysis of RDH genes from Dehalococcoides sp. strain CBDB1 (rdhA1-14_CBDB1), Dehalococcoides sp. strain FL2 (rdhA1-11_FL2), Dehalococcoides sp. strain BAV1 (rdhA1-7_BAV1), and D. ethenogenes strain 195 (rdhA1-17_DE) (48). The neighbor-joining tree shown was generated from amino acid sequences of nearly complete orfA genes (see Fig. 1 and text for details). Branching points supported by 85 to 100% of 1,000 bootstrap sampling events are indicated by solid circles. Open circles indicate 50 to 84% support by bootstrap sampling. Branching points supported by the maximum parsimony treeing method are marked with (P). Additional clones from strain FL2 (rdhA*FL2, sequences not determined) are indicated together with the rdhA_CBDB1 genes that have identical restriction patterns. Dotted double, triple, and quadruple lines indicate subclusters of highly similar genes from two, three, or four different Dehalococcoides strains, respectively (see the text for details). tceA, trichloroethene reductive dehalogenase, pceA, tetrachloroethene reductive dehalogenase, cprA,chlorophenol reductive dehalogenase. The tceA cluster comprises the tceA genes of D. ethenogenes strain 195 (GenBank accession number AAF73916), strain FL2 (accession number AY165309), and three chloroethene-dechlorinating enrichment cultures (accession numbers AAN85590, AAN85592, and AAN85594). The scale bar represents 20% sequence divergence.

FIG.3.

Alignment of the C-terminal region of deduced amino acid sequences of RDH genes from Dehalococcoides strains (tceA DE refers to the tceA gene of D. ethenogenes strain 195; see the legend of Fig. 2 for other designations). Also included is sequence rdh63A, an RDH gene obtained from a bacterial consortium (39). ClustalW alignments were manually corrected to align putative cofactor binding sites. Conserved residues of the two ISB motifs and cobalamin-binding consensus sequences are highlighted in gray. Boxes mark cobalamin-binding motifs in rdhA3_CBDB1 and rdhA4_CBDB1, rdhA1-3_FL2, rdhA12_DE, and rdhA1_BAV1. In rdhA12_DE and rdhA1_BAV1, the SXL motif could not be unambiguously located (see the text for details).