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, 80 (3), 808-18

Identification and Environmental Distribution of dcpA, Which Encodes the Reductive Dehalogenase Catalyzing the Dichloroelimination of 1,2-dichloropropane to Propene in Organohalide-Respiring Chloroflexi

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Identification and Environmental Distribution of dcpA, Which Encodes the Reductive Dehalogenase Catalyzing the Dichloroelimination of 1,2-dichloropropane to Propene in Organohalide-Respiring Chloroflexi

Elizabeth Padilla-Crespo et al. Appl Environ Microbiol.

Abstract

Dehalococcoides mccartyi strains KS and RC grow with 1,2-dichloropropane (1,2-D) as an electron acceptor in enrichment cultures derived from hydrocarbon-contaminated and pristine river sediments, respectively. Transcription, expression, enzymatic, and PCR analyses implicated the reductive dehalogenase gene dcpA in 1,2-D dichloroelimination to propene and inorganic chloride. Quantitative real-time PCR (qPCR) analyses demonstrated a D. mccartyi cell increase during growth with 1,2-D and suggested that both D. mccartyi strains carried a single dcpA gene copy per genome. D. mccartyi strain RC and strain KS produced 1.8 × 10(7) ± 0.1 × 10(7) and 1.4 × 10(7) ± 0.5 × 10(7) cells per μmol of propene formed, respectively. The dcpA gene was identified in 1,2-D-to-propene-dechlorinating microcosms established with sediment samples collected from different geographical locations in Europe and North and South America. Clone library analysis revealed two distinct dcpA phylogenetic clusters, both of which were captured by the dcpA gene-targeted qPCR assay, suggesting that the qPCR assay is useful for site assessment and bioremediation monitoring at 1,2-D-contaminated sites.

Figures

FIG 1
FIG 1
Arrangements of the dcpA gene and its corresponding dcpB gene in D. mccartyi strains RC and KS. Approximate binding sites for the degenerate primers RRF2 and B1R as well as the dcpA-specific primers designed in this study are indicated. Also shown are the characteristic dehalogenase features encoded by the dcpA gene, which include the conserved Tat signal peptide RRXFXK at the N terminus and two iron-sulfur clusters closer to the C terminus, in the form of FCXXCXXCXXXCP (or FCX2CX2CX3CP) and CXXCXXXC (or CX2CX3C). dcpB is located downstream of dcpA and encodes a small, highly hydrophobic protein with the conserved twin-arginine motif in the form WYXW. The dcpA and dcpB genes (Dehly_1524 and Dehly_1523) in D. lykanthroporepellens strain BL-DC-9 also encode a dehalogenase with these common features.
FIG 2
FIG 2
Activity assays following BN-PAGE separation of crude extracts of D. lykanthroporepellens BL-DC-9 cells grown with 1,2-D. (A) Coomassie-stained BN-PAGE showing the predominant proteins and the gel sections that were subjected to dechlorination activity testing with 1,2-D. For enzyme activity assays, gel slices from unstained lanes adjacent to the Coomassie-stained lanes were used. (B) Propene formation was observed only in gel slice 4, which was subjected to SDS-PAGE. Three bands were visualized by SDS-PAGE, and gel slices N1, N2, and N3 were further analyzed by LC-MS/MS. DcpA (Dehly_1524) was the only RDase detected in gel slice 4.
FIG 3
FIG 3
Relative transcript copy abundances in cells growing with 1,2-D. dcpA transcript levels were normalized to rpoB or to dcpA gene copy numbers. Triplicate samples were analyzed, and the reported values represent the averages for at least three independent biological cultures. Error bars depict standard errors. Negative numbers represent downregulated target genes, while positive numbers represented upregulated genes. A ratio near unity (close to 1) indicates an insignificant change in the number of dcpA transcripts per rpoB transcript or the ratio of dcpA to D. mccartyi (Dhc) gene copy numbers.
FIG 4
FIG 4
Phylogenetic tree of DcpA sequences. The neighbor-joining tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic relationships. All positions containing gaps and missing data were eliminated, and a total of 247 aa positions were included in the final data set. Evolutionary distances were computed using the number of differences method, and the scale bar indicates the number of amino acid differences per sequence. Samples that clustered together were grouped, and numbers in parentheses indicate the number of sequences of each group. The RDase DET1538 of D. mccartyi strain 195 served as an outgroup to root the tree. Cluster 1 shares highest aa sequence identity to DcpA of D. lykanthroporepellens strain BL-DC-9 (93 to 95%), while cluster 2 comprises sequences with higher sequence identity to DcpA of D. mccartyi strains KS and RC (95 to 99%).

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