ABSTRACT: 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 (sequential) 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 differences in the expression of its 32 reductive dehalogenase (rdhA) genes using RNA-sequencing. The gene of the known chlorobenzene reductive dehalogenase, 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 also the most highly expressed rdhA, whereas rdhA cbdbA80 was transcribed at a slightly lower level. Instead, a third rdhA gene, cbdbA1588, encoding an orthologue of the tetrachloroethene reductive dehalogenase PceA, was the second most highly expressed rdhA. The screening of the transcripts encoded in intergenic regions predicted three abundant sRNAs and a so far unannotated small protein. Proteome analyses of cultures respiring 1,2,3-TCB, 1,2,4-TCB, or hexachlorobenzene (HCB) confirmed the specific synthesis of the RdhA CbdbA1588 protein during respiration with 1,2,4-TCB. It was also up-regulated by HCB, suggesting its involvement in sequential dechlorination to dichlorobenzenes. 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.
Nico Jehmlich 
