Applications of recombinant bacteria for remediation and detection of heavy metals

Heavy metals come from natural and anthropogenic sources. Several well-known mercury and cadmium poisoning incidences have increased our understanding of the metals' extreme toxicity. Therefore, it is important to have cost-effective and efficient cleanup methods. The main goal of this dissertation is to apply biotechnology towards developing a strategy to address heavy metal contamination. By nature, heavy metals are recalcitrant and cannot be degraded. Therefore, optimal treatment methods usually involve immobilization and sequestration of the metals. The use of microbes for biosorption is an attractive option because of the inexpensive production cost and high binding affinity. In this dissertation, microbial sorbents expressing different metal binding proteins are explored for the sequestration of mercury and cadmium.; An E. coli JM109 strain expressing surface-expressed MerR is constructed for removal of mercury from wastewater. The mercury binding protein is anchored on cell membrane using the ice nucleation protein (INP) from Pseudomonas syringae. The biosorbent is shown to have high affinity and selectivity towards mercury (II).; For removal of cadmium from soil, the symbiotic relationship between plant root and bacteria is used to sustain the biosorbent population in the environment. The rhizobacterium, Pseudomonas putida 06909, expressing synthetic phytochelatin (EC20) intracellularly is demonstrated to have increased cadmium binding capability and metal toxicity resistance in growing and resting cell culture experiments. In addition, hydroponically grown sunflower seedlings inoculated with recombinant P. putida bind more cadmium than non-inoculated, and exhibit enhanced cadmium resistance.; A P. putida 06909 whole-cell cadmium biosensor is constructed, using cadR a naturally occurring cadmium-resistant gene, for the specific detection of cadmium. The genetic circuit, in a broad-host-range vector, contains the cadR promoter fused to lacl q and gfp, and a divergently transcribed tac promoter fused to cadR. The toggle sensor demonstrates lowered background fluorescence, and improved cadmium detection sensitivity by 20-fold compared to a non-toggle sensor. In addition, the cadR promoter toggle sensor is highly specific to cadmium (II) and not to other cations.; Fluorescence in situ hybridization (FISH), targeting both rRNA and mRNA, is employed for increasing stringency in taxonomic differentiation and tracking of recombinant bacteria in the rhizosphere. A recombinant strain of P. putida 06909 expressing a chromosomally inserted toluene monooxygenase (tom) gene and EC20 is inoculated into wheat rhizosphere grown in sandy soil. Two oligonucleotide probes with different fluors hybridize to P. putida specific 16S rRNA and tom gene mRNA. Using confocal laser scanning microscopy, the recombinant strain is visualized and differentiated from native bacteria on root hair.; This dissertation provides a comprehensive approach in formulating a solution for heavy metal contamination. The research utilizes biotechnological tools for heavy metal sequestration, detection, and monitor of recombinant bacteria in the environment. The incidences of heavy metal contamination will most likely increase due to global industrialization. The strategy developed in this research provides a cost-effective and feasible remediation alternative for regulators around the world.