Doctor of Philosophy (PhD)
Dehalogenimonas lykanthroporepellens BL-DC-9T is an anaerobic bacterial strain that can reductively dehalogenate a variety of vicinally chlorinated alkanes. Making use of the strain’s genome sequence, PCR primers were designed to uniquely target each of the 25 reductive dehalogenase homologous (rdhA) genes present in the genome. RNA extracted from D. lykanthroporepellens BL-DC-9T cultures that were dechlorinating three chlorinated solvents (1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane) were used in reverse transcriptase PCR (RT-PCR) to determine rdhA gene expression patterns. Transcripts from 19 rdhA genes were detected in the RT-PCR experiments, with identical gene expression patterns during growth with all three chlorinated electron acceptors employed in the study. The transcripts included thirteen full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having a cognate rdhB gene. Transcripts from four putative transcriptional regulators were also detected during organohalide respiration with 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane, providing insights in the regulatory controls on rdhA gene expression. A dehalogenase enzyme assay was developed, and attempts were made to heterologously express the rdhA gene with locus tag Dehly_1524 (recently identified as encoding a 1,2-dichloropropane reductive dehalogenase) in Escherichia coli. The majority of the heterologously expressed protein was found in the insoluble fraction and in a catalytically inactive form under all conditions tested; this was inferred to be due to the absence of de novo cobamide synthesis in E. coli and the requirement for this cofactor for enzymatic activity. Functional expression of the Dehly_1524 protein was also attempted under anoxic conditions using the cobamide producing species Shimwellia blattae that was recently identified as having a genetic system that allowed the functional expression of dehalogenases from another species (Desulfitobacterium hafniense strains Y51 and DCB-2). The Dehly_1524 protein was not catalytically active when expressed in Shimwellia blattae under the conditions tested, however, indicating that Shimwellia blattae may not contain a genetic system broadly suitable for functional expression of the growing number of putative reductive dehalogenase genes available in the public databases.
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Mukherjee, Kalpataru, "Insights into Chlorinated Aliphatic Alkane Reductive Dehalogenation by Dehalogenimonas Lykanthroporepellens BL-DC-9T and Development of Nucleic Acid-Based Techniques for Monitoring Gene Expression" (2015). LSU Doctoral Dissertations. 3938.
Rainey, Frederick A