A Metagenome-Based Examination of Dechlorinating Enrichment cultures: Dehalococcoides and the Role of the Non-Dechlorinating Microorganisms.

摘要

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms' functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.