PCB dechlorinases revealed at last.

It has taken three decades from the first report of microbial polychlorinated biphenyl (PCB) dechlorination to identify even one of the enzymes responsible. By combining conventional techniques with their own ingenuity, the latest technologies, and a bit of luck, Wang et al., in PNAS, have identified not one, but three distinct enzymes that can reductively dechlorinate PCBs (1). This finding is important because, despite being banned in the 1970s, PCBs still contaminate the sediments of rivers, lakes, and harbors worldwide. PCBs are notorious for their ability to bioaccumulate and biomagnify in the food chain, and for their multiple suspected health effects. Commercial mixtures of PCBs, known in the United States as Aroclors, are complex mixtures of 60–90 types, congeners, of PCB molecule that differ in the number (1–10) and position of chlorines on the phenyl rings. PCBswere used for decades as dielectric fluids in capacitors and transformers, and as hydraulic fluids, heat transfer fluids, lubricants and cutting oils, and additives in a variety of products (2). For example, Aroclor 1260 is amixture of PCBswith five to eight chlorines that was used as transformer dielectric fluid. Thirty years ago it was discovered that PCBs in the anaerobic sediments of rivers were being dechlorinated by unknown agents, presumably anaerobic bacteria (3, 4). This discovery offered the best hope for an effective means of dealing with the notoriously persistent PCBs. PCB dechlorination helps to reduce the toxicity and bioaccumulation potential of PCBs and makes them more susceptible to oxidation and destruction by many organisms. However, despite years of research in multiple laboratories, the PCBdechlorinating agents were not identified until 2007. In that year two different laboratories identified Dehalococcoides mccartyi as the bacterium responsible for dechlorinating Aroclor 1260 in aquatic sediments (5, 6). The lifestyle of D. mccartyi explains why it was so hard to identify; it is a tiny, strictly anaerobic bacterium that must derive its energy for growth by removing chlorines from chlorinated organic molecules and using them as electron acceptors for respiration, a process known as organohalide respiration (7). These organisms have a tiny genome, yet each encodes a suite of 10–36 different reductive dehalogenase enzymes (RDases) to assist in its highly restricted way of life (7, 8). At this point, hundreds of different D. mccartyi RDases have been identified and sequenced. However, the difficulty of growing these organisms and low biomass yields have prevented researchers from identifying the substrates of all but a handful of these enzymes. Those that have been identified include several tetrachloroethene (PCE) dehalogenases, a trichloroethene (TCE) dehalogenase, two vinyl chloride dehalogenases, and a chlorinated benzene dehalogenase (8). The isolation of D. mccartyi strains that can grow using highly chlorinated PCBs for respiration has been severely hampered by the inability to grow these organisms to high cell density because of the extreme insolubility of PCBs. Wang et al., in Jianzhong He’s laboratory, have overcome this problem by using a more soluble alternative substrate, tetrachloroethene (PCE), to grow, isolate, and characterize the genome of three new strains of D. mccartyi that can respire the highly chlorinated commercial PCB mixture Aroclor 1260 (1). It has long been known that many strains of this species can respire PCE, and the authors reasoned that PCE might offer a more rapid means of growing and further enriching PCB-respiring bacteria from cultures that had already been enriched with PCBs. Indeed, the authors showed that the three new strains grew to a 12.5to 22fold higher cell density in 30 days with PCE as the electron acceptor, versus 150 days with PCBs in the Aroclor mixture as the electron acceptors. To avoid loss of PCB RDases while growing with PCE, the authors first used shotgun metagenomics to identify and then monitor all RDase genes during the enrichment process. Wang et al., in He’s laboratory, identified and sequenced the genes for three PCB RDases, pcbA1, pcbA4, and pcbA5, one in each of the three new strains (1). Each of the corresponding enzymes attacks dozens of PCB substrates, but each exhibits distinct specificity, removing different chlorines and leading to different terminal products (Fig. 1). All three PCB RDases also dechlorinate PCE to trichloroethene (TCE) and to both cisand trans-dichloroethene (DCE) (1). This finding was completely unexpected because: (i) several different PCE RDases have already been identified in D. mccartyi, and (ii) the PCE molecule looks nothing like a PCB (Fig. 1). However, the experimental evidence is undeniable. In each case the same RDase gene is the most highly transcribed, whether growing with PCE or PCBs, and each of the three gel-purified PCB RDases dechlorinates both PCE and PCBs (1). The discovery that PCE and PCB dechlorinase capabilities are linked on a single enzyme (1) has enormous implications for PCB remediation. The ability to grow these PCB dechlorinators with PCE as the electron acceptor suddenly makes the possibility of bioaugmentation for PCB remediation much more feasible. It should be possible to grow large amounts of PCB dechlorinators using PCE as the electron acceptor. The chlorinated ethene substrates and products can then be removed by purging before using the cells to treat a PCB-contaminated site. This process, called bioaugmentation, is already widely used with great success for remediation of chlorinated ethenes by D. mccartyi (9). So what do these three PCB RDases tell us? First, that each enzyme can catalyze the dechlorination of dozens of PCB congeners as Fig. 1. Dechlorination of PCE and several PCBs by three different PCB dechlorinases. Note the different specificities for PCB dechlorination.