Ute Lechner

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.

The strictly anaerobic bactGIerium Dehalococcoides mccartyi obligatorily depends on organohalide respiration for energy conservation and growth. The bacterium also plays an important role in bioremediation. Since there is no guarantee of a continuous supply of halogenated substrates in its natural environment, the question arises of how D. mccartyi maintains the synthesis and activity of dehalogenating enzymes under these conditions. Acetylation is a means by which energy-restricted microorganisms can modulate and maintain protein levels and their functionality. Here, we analyzed the proteome and Nε-lysine acetylome of D. mccartyi strain CBDB1 during growth with 1,2,3-trichlorobenzene as an electron acceptor. The high abundance of the membrane-localized organohalide respiration complex, consisting of the reductive dehalogenases CbrA and CbdbA80, the uptake hydrogenase HupLS, and the organohalide respiration-associated molybdoenzyme OmeA, was shown throughout growth. In addition, the number of acetylated proteins increased from 5% to 11% during the transition from the exponential to the stationary phase. Acetylation of the key proteins of central acetate metabolism and of CbrA, CbdbA80, and TatA, a component of the twin-arginine translocation machinery, suggests that acetylation might contribute to maintenance of the organohalide-respiring capacity of the bacterium during the stationary phase, thus providing a means of ensuring membrane protein integrity and a proton gradient.

The anaerobic biodegradation of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated using enrichment cultures from freshwater sediments at two different sites in the region of Halle, central Germany. 2,4,5-T and different organic acids or hydrogen were added as possible electron acceptor and electron donors, respectively. The primary enrichment cultures from Saale river sediment completely degraded 2,4,5-T to 3-chlorophenol (3-CP) (major product) and 3,4-dichlorophenol (3,4-DCP) during a 28-day incubation period. Subcultures showed ether cleavage of 2,4,5-T to 2,4,5-trichlorophenol and its stoichiometric dechlorination to 3-CP only in the presence of butyrate. In contrast, the primary enrichment culture from sediment of Posthorn pond dechlorinated 2,4,5-T to 2,5-dichlorophenoxyacetic acid (2,5-D), which, in the presence of butyrate, was degraded further to products such as 3,4-DCP, 2,5-DCP, and 3CP, indicating ether cleaving activities and subsequent dechlorination steps. Experiments with pure cultures of Dehalococcoides mccartyi and Desulfitobacterium hafniense demonstrated their specific dechlorination steps within the overall 2,4,5-T degradation pathways. The results indicate that the route and efficiency of anaerobic 2,4,5-T degradation in the environment depend heavily on the microorganisms present and the availability of slowly fermentable organic compounds.

Summary: The herbicide 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T) was a major component of Agent Orange, which was used as a defoliant in the Vietnam War. Little is known about its degradation under anoxic conditions. Established enrichment cultures using soil from an Agent Orange bioremediation plant in southern Vietnam with pyruvate as potential electron donor and carbon source were shown to degrade 2,4,5‐T via ether cleavage to 2,4,5‐trichlorophenol (2,4,5‐ TCP ), which was further dechlorinated to 3,4‐dichlorophenol. Pyruvate was initially fermented to hydrogen, acetate and propionate. Hydrogen was then used as the direct electron donor for ether cleavage of 2,4,5‐T and subsequent dechlorination of 2,4,5‐ TCP . 16S rRNA gene amplicon sequencing indicated the presence of bacteria and archaea mainly belonging to the Firmicutes , Bacteroidetes , Spirochaetes, Chloroflexi and Euryarchaeota . Desulfitobacterium hafniense was identified as the dechlorinating bacterium. Metaproteomics of the enrichment culture indicated higher protein abundances of 60 protein groups in the presence of 2,4,5‐T. A reductive dehalogenase related to RdhA3 of D. hafniense showed the highest fold change, supporting its function in reductive dehalogenation of 2,4,5‐ TCP . Despite an ether‐cleaving enzyme not being detected, the inhibition of ether cleavage but not of dechlorination, by 2‐bromoethane sulphonate, suggested that the two reactions are catalysed by different organisms.

Reductive dehalogenases are essential enzymes in organohalide respiration and consist of a catalytic subunit A and a membrane protein B, encoded by rdhAB genes. Thirty-two rdhAB genes exist in the genome of Dehalococcoides mccartyi strain CBDB1. To gain a first insight into the regulation of rdh operons, the control of gene expression of two rdhAB genes (cbdbA1453/cbdbA1452 and cbdbA1455/cbdbA1454) by the MarR-type regulator Rdh2R (cbdbA1456) encoded directly upstream was studied using heterologous expression and in vitro studies. Promoter- lacZ reporter fusions were generated and integrated into the genome of the Escherichia coli host. The lacZ reporter activities of both rdhA promoters decreased upon transformation of the cells with a plasmid carrying the rdh2R gene, suggesting that Rdh2R acts as repressor, whereas the lacZ reporter activity of the rdh2R promoter was not affected. The transcriptional start sites of both rdhA genes in strain CBDB1 and/or the heterologous host mapped to a conserved direct repeat with 11- to 13-bp half-sites. DNase I footprinting revealed binding of Rdh2R to a ∼30-bp sequence covering the complete direct repeat in both promoters, including the transcriptional start sites. Equilibrium sedimentation ultracentrifugation revealed that Rdh2R binds as tetramer to the direct-repeat motif of the rdhA (cbdbA1455) promoter. Using electrophoretic mobility shift assays, a similar binding affinity was found for both rdhA promoters. In the presence of only one half-site of the direct repeat, the interaction was strongly reduced, suggesting a positive cooperativity of binding, for which unusual short palindromes within the direct-repeat half-sites might play an important role. IMPORTANCE Dehalococcoides mccartyi strains are obligate anaerobes that grow by organohalide respiration. They have an important bioremediation potential because they are capable of reducing a multitude of halogenated compounds to less toxic products. We are now beginning to understand how these organisms make use of this large catabolic potential, whereby D. mccartyi expresses dehalogenases in a compound-specific fashion. MarR-type regulators are often encoded in the vicinity of reductive dehalogenase genes. In this study, we made use of heterologous expression and in vitro studies to demonstrate that the MarR-type transcription factor Rdh2R acts as a negative regulator. We identify its binding site on the DNA, which suggests a mechanism by which it controls the expression of two adjacent reductive dehalogenase operons.

Polyhalogenated aromatic compounds are harmful environmental contaminants and tend to persist in anoxic soils and sediments. Dehalococcoides mccartyi strain DCMB5, a strain originating from dioxin-polluted river sediment, was examined for its capacity to dehalogenate diverse chloroaromatic compounds. Strain DCMB5 used hexachlorobenzenes, pentachlorobenzenes, all three tetrachlorobenzenes, and 1,2,3-trichlorobenzene as well as 1,2,3,4-tetra- and 1,2,4-trichlorodibenzo- p -dioxin as electron acceptors for organohalide respiration. In addition, 1,2,3-trichlorodibenzo- p -dioxin and 1,3-, 1,2-, and 1,4-dichlorodibenzo- p -dioxin were dechlorinated, the latter to the nonchlorinated congener with a remarkably short lag phase of 1 to 4 days following transfer. Strain DCMB5 also dechlorinated pentachlorophenol and almost all tetra- and trichlorophenols. Tetrachloroethene was dechlorinated to trichloroethene and served as an electron acceptor for growth. To relate selected dechlorination activities to the expression of specific reductive dehalogenase genes, the proteomes of 1,2,3-trichlorobenzene-, pentachlorobenzene-, and tetrachloroethene-dechlorinating cultures were analyzed. Dcmb_86, an ortholog of the chlorobenzene reductive dehalogenase CbrA, was the most abundant reductive dehalogenase during growth with each electron acceptor, suggesting its pivotal role in organohalide respiration of strain DCMB5. Dcmb_1041 was specifically induced, however, by both chlorobenzenes, whereas 3 putative reductive dehalogenases, Dcmb_1434, Dcmb_1339, and Dcmb_1383, were detected only in tetrachloroethene-grown cells. The proteomes also harbored a type IV pilus protein and the components for its assembly, disassembly, and secretion. In addition, transmission electron microscopy of DCMB5 revealed an irregular mode of cell division as well as the presence of pili, indicating that pilus formation is a feature of D. mccartyi during organohalide respiration.

The remarkable capacity of the genus Dehalococcoides to dechlorinate a multitude of different chlorinated organic compounds reflects the number and diversity of genes in the genomes of Dehalococcoides species encoding reductive dehalogenase homologues ( rdh ). Most of these genes are located in the vicinity of genes encoding multiple antibiotic resistance regulator (MarR)-type or two-component system regulators. Here, the transcriptional response of rdhA genes (coding for the catalytic subunit) to 2,3- and 1,3-dichlorodibenzo- p -dioxin (DCDD) was studied in Dehalococcoides mccartyi strain CBDB1. Almost all rdhA genes were transcribed in the presence of 2,3-DCDD, albeit at different levels as shown for the transcripts of cbrA , cbdbA1453, cbdbA1624 and cbdbA1588. By contrast, 1,3-DCDD did not induce rdhA transcription. The putative MarR CbdbA1625 was heterologously produced and its ability to bind in vitro to the overlapping promoter regions of the genes cbdbA1624 and cbdbA1625 was demonstrated. To analyse regulation in vivo , single-copy transcriptional promoter– lacZ fusions of different rdhA genes and of cbdbA1625 were constructed and introduced into the heterologous host Escherichia coli , and expression levels of the fusions were measured. The cbdbA1625 gene was cloned into a vector allowing a regulation of expression by arabinose and it was transformed into the strains containing the rdh -promoter– lacZ fusion derivatives. CbdbA1625 was shown to downregulate transcription from its own promoter resulting in a 40–50% reduction in the β-galactosidase activity, giving the first hint that it acts as a repressor.

Polychlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) are among the most harmful environmental contaminants. Their widespread distribution due to unintentional or unknown release coincides with environmental persistence, acute and chronic toxicity to living organisms, and long-term effects due to the compounds’ tendency for bioaccumulation and biomagnification. While microbial aerobic degradation of PCDD/Fs is mainly reported for the turnover of low chlorinated congeners, this review focuses on anaerobic reductive dehalogenation, which may constitute a potential remediation strategy for polychlorinated compounds in soils and sediments. Microorganisms in sediments and in microcosms or enrichment cultures have been shown to be involved in the reductive dechlorination of dioxins. Bacteria related to the genus Dehalococcoides are capable of the reductive transformation of dioxins leading to lower chlorinated dioxins including di- and monochlorinated congeners. Thus, reductive dehalogenation might be one of the very few mechanisms able to mediate the turnover of polychlorinated dioxins by reducing their toxicity and paving the way for a subsequent breakdown of the carbon skeleton.

The transcription of reductive dehalogenase homologous ( rdh ) genes of “ Dehalococcoides ” sp. strain CBDB1 was investigated during the growth and reductive dechlorination of 1,2,3- and 1,2,4-trichlorobenzene (TCB). A method was developed to monitor the expression of all 32 rdhA genes present in the genome based on reverse transcription-PCR amplification with 13 degenerate primer pairs and terminal restriction fragment length polymorphism (t-RFLP) analysis. With this approach, the upregulation of the transcription of 29 rdhA genes was indicated in response to 1,2,3- and 1,2,4-TCB added after a substrate depletion period of 72 h. The transcription of the remaining three rdhA genes additionally was detected using specific primers. While most rdhA genes were upregulated similarly in cultures after induction with 1,2,3-TCB or 1,2,4-TCB, three rdhA genes responded differentially to 1,2,3- and 1,2,4-TCB, as revealed by the comparison of t-RFLP profiles. The enhanced transcription of cbdbA1453 and cbdbA187 was observed in the presence of 1,2,3-TCB, while the transcription of cbdbA1624 was strongly induced by 1,2,4-TCB. Comparison of t-RFLP profiles obtained from cDNA and genomic DNA indicated a particularly high induction of the transcription of cbrA (=cbdbA84) by both TCBs. As indicated by reverse transcription-quantitative PCR, the transcription of these plus two other rdhA genes (cbdbA1588 and cbdbA1618) increased within 48 to 72 h by one or two orders of magnitude. Subsequently, transcript levels slowly decreased and approached initial transcript levels several days after complete dehalogenation. Finally, cbrA was transcribed to a level of 22 transcripts per cbrA gene, suggesting that cbrA mRNA could be an appropriate biomarker for the investigation of the natural dechlorination potential at chlorobenzene-contaminated sites.