Metagenomic investigation of Dehalococcoidia used for bioremediation of groundwater and soil contaminated with chlorinated ethenes and ethanes

Chlorinated aliphatic hydrocarbons (CAHs) such as vinyl chloride (VC) and perchloroethene (PCE) are persistent, toxic groundwater and soil pollutants. Bioremediation using mixed microbial cultures such as KB-1 and WBC-2 has proven to be a technically viable and economically feasible remediation strategy. These cultures include members of the Dehalococcoides and Dehalogenimonas, genera from the class Dehalococcoidia, who use reductive dehalogenase enzymes (RDases) to dechlorinate CAHs in an obligatory respiratory process. Metagenomic DNA sequencing was conducted on the KB-1 and WBC-2 enrichment cultures to describe the diversity of microorganisms living in the cultures, and to close genomes of key organisms. Ten new complete genomes of Dehalococcoidia are presented here. Their reductive dehalogenase genes and their evolutionary history are described. Dehalococcoides mccartyi in KB-1 was used as an example to study mobile DNA, with a particular focus on the possible exchange of reductive dehalogenase genes. Extrachromosomal circular elements were discovered in D. mccartyi, and found to replicate in a circular intermediate state separate from the genome. Simultaneously, the VC reductase gene (vcrA)-containing genomic island was also found to periodically replicate in a circular intermediate state. Twelve new prophages and their interaction with CRISPR systems carried by D. mccartyi were described. Additionally, a reductive dehalogenase from Dehalogenimonas was identified and biochemically characterized using a combination of blue-native polyacrylamide gel electrophoresis followed by enzymatic assays and peptide sequencing. In summary, this work has nearly doubled the number of Dehalococcoidia genomes available in NCBI, has led to the discovery of new dehalogenases, new types of mobile DNA, and has begun to elucidate a mechanism for lateral gene transfer in D. mccartyi. Together this knowledge will help better manage contaminated sites and design more effective bioremediation approaches.Ph.D