Date of Award

8-2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Frank E. Loeffler

Committee Members

Steven W. Wilhelm, Alison Buchan, Erick Zinser, Qiang He

Abstract

Chlorinated solvents are among the most encountered groundwater pollutants. These toxic compounds cause harm to ecosystem functioning and human health. 1,2-Dichloropropane (1,2-D) was used in a variety of industrial and agricultural applications until it was banned in the U.S. in the 1970s. Only a handful of bacteria have been described to reductively dechlorinate 1,2-D to innocuous propene and inorganic chloride, among these the Dehalococcoides (Dhc) strains RC and KS. In order to shed light into the genetic basis of 1,2-D dechlorination, efforts focused in identifying the gene encoding the enzyme system (i.e., reductive dehalogenase) responsible for 1,2-D to propene transformation. To accomplish this goal, a multiple lines of evidence approach combining gene cloning, transcriptional studies, and enzyme activity assays implicated the dcpA gene in 1,2-D reductive dechlorination in Dhc strains RC and KS. This gene was also identified in Dehalogenimonas lykanthroporepellens (Dhgm) strain BL-DC-9, another member of the organohalide-respiring Chloroflexi group, and also capable of growth with 1,2-D as electron acceptor. Propene-producing enrichment cultures were derived from a variety of environments and the presence of dcpA correlated with 1,2-D reductive dechlorination observed in situ and/or in microcosms. Nested PCR and qPCR assays were designed and validated to detected and quantify the gene in laboratory cultures and in environmental samples. These surveys shed light into the distribution of this gene in diverse environments including pristine environments. Genomic and bioinformatics tools explored the gene neighborhood of dcpA and revealed a genomic island shared between Dhc and Dhgm indicative of a horizontal gene transfer event. Metagenome analysis of consortia RC and KS enabled the draft genome assemblies of these two Dhc strains. This analysis revealed that the Dhc strain RC and strain KS harbor at least 34 and 31 reductive dehalogenase genes, including genes implicated in PCB reductive dechlorination. These findings reveal broad reductive dechlorination potential and emphasize that such dedicated dechlorinators (i.e., Dhc strain FL2) occur in pristine environments and are members of natural microbial assemblages that have not been exposed to anthropogenic contamination.

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