Development of primers for amplifying genes encoding CprA- and PceA-like reductive dehalogenases in anaerobic microbial consortia, dechlorinating trichlorobenzene and 1,2-dichloropropane

Abstract

Gene sequence alignments of the reductive dehalogenases PceA (Dehalospirillum multivorans) and CprA (Desulfitobacterium dehalogenans) were used to develop specific PCR primers binding to conserved regions of these sequences. These primers enabled us to amplify and subsequently sequence cprA-like gene fragments from the chlororespiring species Dehalobacter restrictus, Desulfitobacterium sp. strain PCE1, and D. hafniense. No specific amplicons were obtained from the chlororespiring species D. frappieri, D. chlororespirans, and Desulfomonile tiedjei. Furthermore, we were able to amplify and sequence cprA/pceA-like gene fragments from both trichlorobenzene (TCB)- and 1,2-dichloropropane (DCP)-dechlorinating microbial consortia using the novel primers. Subsequent sequence analysis of the fragments obtained from the microbial consortia revealed a group of four clusters (I–IV). Of these, clusters I and II showed the highest similarities to the cprA-like gene of Dehalobacter restrictus (79.0 and 96.2%, respectively). Cluster III comprised cprA-like sequences found in both the TCB- and the DCP-dechlorinating consortia, whereas sequences of cluster IV were most similar to the pceA gene of Dehalospirillum multivorans (97.8%). Our detection of genes encoding reductive dehalogenases, the key enzymes of chlororespiration, supports the hypothesis that reductive dechlorination of TCB and DCP occurs via a respiratory pathway.