The PceA-orthologous reductive dehalogenase of Dehalococcoides mccartyi CBDB1 is involved in 1,2,4-trichlorobenzene respiration

The reductive dehalogenation of halogenated benzenes by anaerobic bacteria is of great environmental and biotechnological importance; however, the role of specific reductive dehalogenases in the dehalogenation of different isomers has not been studied in detail. Here, we cultivated the obligate organohalide-respiring Dehalococcoides mccartyi strain CBDB1 with either 1,2,3- or 1,2,4-trichlorobenzene (TCB) as electron acceptor and investigated the transcription of its 32 reductive dehalogenase (rdhA) genes using RNA-sequencing. The chlorobenzene reductive dehalogenase gene cbrA, and rdhA cbdbA80 were the two most highly expressed rdhA genes with 1,2,3-TCB. In the presence of 1,2,4-TCB, cbrA was the most highly expressed rdhA followed by rdhA cbdbA1588, encoding an orthologue of the tetrachloroethene reductive dehalogenase PceA of D. mccartyi strain 195. RNA-sequencing also allowed for the detection of small RNAs and an unannotated protein. Proteomics confirmed the synthesis of RdhA CbdbA1588 during respiration with 1,2,4-TCB and also with hexachlorobenzene, which is dehalogenated via 1,2,4-TCB. Dehalogenase activity assays with cell extracts from 1,2,4-TCB-grown cultures indicated a higher activity towards 1,2,4-TCB and a ten-fold higher activity towards 2,3-dichlorophenol compared to that in extracts from 1,2,3-TCB-grown cultures. These findings demonstrate the functionality of RdhA CbdbA1588 and further support a role in 1,2,4-TCB dechlorination by strain CBDB1.