Date of Award
Doctor of Philosophy
Molecular Biology, Microbiology and Biochemistry
SECTION 1 Iron is the fourth most abundant element on the earth crust as well as an essential nutrient for all living organisms. The cycling of iron between the environment and biological systems and the microbial-mediated transformation between Fe2+ and Fe3+ has a significant impact on the biogeochemistry of the environment. The recently discovered microbially-mediated anaerobic nitrate-dependent oxidation of Fe2+ has been shown to play an important role in global iron biogeochemical cycling. Furthermore, the formation of iron oxide from anaerobic nitrate-dependent Fe2+ oxidation results in the adsorption and precipitation of soluble toxic heavy metals and radionuclides from surrounding environment. Therefore, this metabolism has been attracting more and more attention because this process could serve as a cost-effective way to co-remediate nitrate, heavy metals and radionuclides at contaminated sites. Little is known about the molecular genetics of the anaerobic nitrate-dependent Fe2+ oxidation pathway so far. Previous studies in our lab using a microarray approach on Dechloromonas aromatica RCB uncovered the likely involvement of lipoproteins, transmembrane proteins in major operons, cytochromes and signal transduction enzymes in this metabolism. In an effort to further elucidate the metabolic process, a recently isolated bacterium strain Acidovorax ebreus strain TPSY capable of anaerobic nitrate-dependent Fe2+ oxidation was selected as a model organism in this study. By utilizing a 2-dimensional electrophoresis method, a list of candidate proteins which exhibited elevated levels of expression were identified by the comparison of whole cell protein profile between Fe2+-oxidizing strain TPSY cells and control cells. Conserved domain analysis of the protein candidates along with the locus analysis of their corresponding genes revealed two operons (Dtpsy_1460-1463 and Dtpsy_3433-3438) that could encode key components in the anaerobic nitrate-dependent Fe2+ oxidation pathway. An outer membrane efflux pump protein complex encoded by the Dtpsy_1460-1463 operon could play a role in the exportation of periplasmic-accumulated Fe3+ as a detoxification procedure. In addition, a putative ferric reductase protein Dtpsy_3433 and cytochrome reductase-like protein Dtpsy_3436 are likely critical electron transport chain components in this metabolism. Quantitative reverse transcription PCR provided further evidence for the involvement of this operon by demonstrating the transcriptional level up-regulation of the genes in the Dtpsy_3433-3438 operon. This study serves as the first attempt to identify the proteins and genes responsible for anaerobic nitrate-dependent iron oxidation in Acidovorax ebreus strain TPSY. This work has led to the successful identification of a few key proteins and genes responsible for anaerobic nitrate-dependent Fe2+ oxidation, thus providing information important for the elucidation of other components in this electron transport pathway. SECTION 2 Perchlorate is a wide-spread contaminant detected in drinking water and ground water systems in the United States. The current development of a highly sensitive enzymatic bioassay for in situ perchlorate concentration quantification created a need for high-quality and low-cost perchlorate reductase. Perchlorate reductase, originally isolated from DPRB (dissimilatory perchlorate reducing bacteria), is encoded by an operon containing four genes, pcrABCD. Enzymatically active perchlorate reductase purified by traditional methods is composed of two structural subunits, PcrA and PcrB, encoded by the pcrA and pcrB genes, respectively. The lengthy traditional protein purification process and the slow growth rate of DPRB hinder the industrial mass production of this enzyme. In this study, we report an attempt to use E. coli host to overexpress perchlorate reductase and use a polyhistidine tag to enable ease of the subsequent purification. The pcrAB genes encoding the structural subunits of perchlorate reductase were cloned into an expression vector in E. coli. The purification of the recombinant perchlorate reductase was performed under strict anaerobic and denaturing conditions and a highly purified form of the enzyme was obtained. Possible solutions to avoid the formation of inclusion bodies while still maintaining the enzyme activity were discussed. This work proved the feasibility of recombinant perchlorate reductase expression using an E.coli host and the usefulness of the histidine tag in the purification process. In addition, this work provided insights into factors that need to be taken into future consideration in order to obtain the recombinant enzyme with full enzymatic activity. As a final goal, this study will contribute to the development of enzyme-based bioassay for the detection of perchlorate in the environment by lowering the production and purification cost of its key component, the perchlorate reductase.
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